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Effects of muscimol injections on conditioned eyeblink versus limb flexion responses in the rabbit cerebellar interpositus nucleus

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Effects of muscimol injections on conditioned eyeblink versus limb flexion responses in the rabbit cerebellar interpositus nucleus

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Content EFFECTS OF MUSCIMOL INJECTIONS ON CONDITIONED EYEBLINK
VERSUS LIMB FLEXION RESPONSES IN THE RABBIT CEREBELLAR
INTERPOSITU S NUCLEUS
Copyright 2004
by
Shahriar Mojtahedian
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(NEUROSCIENCE)
December 2004
Shahriar Mojtahedian
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UMI Number: 3145248
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TABLE OF CONTENTS
List of Figures.....................................................................................................................iii
List of Tables..................................................................................................... ix
Abstract.............................. x
Chapter 1
Introduction........................................................................ 1
Chapter 2
Materials and Methods...................................................................................................... 29
Chapter 3
Results................................ 38
Chapter 4
Discussion...........................................................................................................................92
References.........................................................................................................................102
ii
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LIST OF FIGURES
Figure 1. Generalized schematic of a typical classical or Pavlovian (delay)
conditioning paradigm in which the conditioned stimulus (CS) and
unconditioned stimulus (US or UCS) co-terminate. Note: the time
intervals may be variable depending on the conditions of the
experiment......................................................................................................... 3
Figure 2. Idealized schematic of progression of learning in a typical classical (delay)
conditioning paradigm. Normally, in the rabbit eyeblink-conditioning
paradigm, the animal begins to reliably elicit a learned or conditioned
response (CR) after approximately 100-150 conditioned stimulus (CS) and
unconditioned stimulus (US or UCS) pairings. Note: the time intervals
may be variable depending on the conditions of the experiment..................4
Figure 3. Putative eyeblink circuitry. The pontine nuclei and inferior olive send
projections via the mossy fibers and climbing fibers, respectively, to both
the cerebellar cortex and as collaterals to the interpositus nucleus. ( - )
represents inhibitory projections....................................................................10
Figure 4. Putative limb flexion circuitry. As in the eyeblink circuit, the pontine
nuclei and inferior olive send projections via mossy fibers and climbing
fibers, respectively, to both the cerebellar cortex and as collaterals to the
interpositus nucleus. ( - ) represents inhibitory projections....................... 12
Figure 5. Putative eyeblink versus limb flexion discrimination circuitry. The
conditioned stimulus (CS) and unconditioned stimulus (US) for the
eyeblink response (light-airpuff) converge in the lateral portion of the
interpositus nucleus (IP), whereas for the limb flexion response, the CS-US
(tone-shock) converge in the medial portion of the IP. ( - ) represents
inhibitory projections..................................................................................... 28
Figure 6. Rabbit #00-142. Comparison plot of the percent CRs, CR amplitude, and
UR amplitude for eyeblink acquisition, limb flexion acquisition, mixed
trials, and muscimol (M) infusion and recovery (R) trials for the lateral
(eye) and medial (limb) cannulas for paired trials......................................42
Figure 7. Rabbit #00-142. Comparison plot of the percent CRs, CR amplitude, and
UR amplitude for eyeblink acquisition, limb flexion acquisition, mixed
trials, and muscimol (M) infusion and recovery (R) trials for the lateral
(eye) and medial (limb) cannulas for unpaired trials................................. 43
m
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Figure 8. Histological profile of rabbit #00-142. The left and right arrows indicate
the positions of the tracks left in the tissue from the lateral (eye) and medial
(limb) cannulas, respectively. The muscimol injection sites are
approximately 2 mm below the base of the guide cannulas. Note: No
marking lesions were made before staining the brain of this animal. The
outline below the cannulas denotes the approximate borders of the
interpositus nucleus (IP)................................................................................. 44
Figure 9. Histological profile of rabbit #00-142 magnified IX. The left and right
arrows indicate the positions of the tracks left in the tissue from the lateral
(eye) and medial (limb) cannulas, respectively. The muscimol injection
sites are approximately 2 mm below the base of the guide cannulas.
Note: No marking lesions were made before staining the brain of this
animal. The outline below the cannulas denotes the approximate borders
of the interpositus nucleus (IP)......................... 45
Figure 10. Histological profile of rabbit #00-142 with suspected positions of the
lateral (left) and medial (right) cannulas. The ends of the dotted lines
represent the best estimation of the locations of the muscimol injection
sites for the two cannulas. The outline below the cannulas denotes the
approximate borders of the interpositus nucleus (IP)..................................46
Figure 11. Rabbit #00-162. Comparison plot of the percent CRs, CR amplitude, and
UR amplitude for eyeblink acquisition, limb flexion acquisition, mixed
trials, and muscimol (M) infusion and recovery (R) trials for the lateral
(eye) and medial (limb) cannulas for paired trials....................................... 50
Figure 12. Rabbit #00-162. Comparison plot of the percent CRs, CR amplitude, and
UR amplitude for eyeblink acquisition, limb flexion acquisition, mixed
trials, and muscimol (M) infusion and recovery (R) trials for the lateral
(eye) and medial (limb) cannulas for unpaired trials...................................51
Figure 13. Histological profile of rabbit #00-162. The left and right arrows indicate
the positions of the tracks left from the lateral (eye) and medial (limb)
cannulas, respectively. The muscimol injection sites are approximately 2
mm below the base of the guide cannulas. Note: No marking lesions were
made before staining the brain of this animal. The outline below the
cannulas denotes the approximate borders of the dentate nucleus (DN; left)
and interpositus nucleus (IP; right)...............................................................52
iv
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Figure 14. Histological profile of rabbit #00-162 magnified IX. The left and right
arrows indicate the positions of the tracks left from the lateral (eye) and
medial (limb) cannulas, respectively. The muscimol injection sites are
approximately 2 mm below the base of the guide cannulas. Note: No
marking lesions were made before staining the brain of this
animal.................................................................................... 53
Figure 15. Histological profile of rabbit #00-162 with suspected positions of the
lateral (left) and medial (right) cannulas. The ends of the dotted lines
represent the best estimation of the locations of the muscimol injection
sites for the two cannulas. The outline below the cannulas denotes the
approximate borders of the dentate nucleus (DN; left) and interpositus
nucleus (IP; right)............................................................................................54
Figure 16. Rabbit #02-025. Comparison plot of the percent CRs, CR amplitude,
and UR amplitude for eyeblink acquisition, limb flexion acquisition, mixed
trials, and muscimol (M) infusion and recovery (R) trials for the medial
(limb) cannula for paired trials. ...... 57
Figure 17. Rabbit #02-025. Comparison plot of the percent CRs, CR amplitude,
and UR amplitude for eyeblink acquisition, limb flexion acquisition, mixed
trials, and muscimol (M) infusion and recovery (R) trials for the medial
(limb) cannula for unpaired trials...................................................................58
Figure 18. Histological profile of rabbit #02-025. The left and right arrows indicate
the positions of the tracks left in the tissue from the lateral (eye) and medial
(limb) cannulas, respectively. The muscimol injection sites are
approximately 2 mm below the base of the guide cannulas. Note: No
marking lesions were made before staining the brain of this animal. The
outlines below the cannulas denote the approximate borders of the dentate
nucleus (DN; left) and interpositus nucleus (IP; right)................................59
Figure 19. Histological profile of rabbit #02-025 magnified IX. The left and right
arrows indicate the positions of the tracks left from the lateral (eye) and
medial (limb) cannulas, respectively. The muscimol injection sites are
approximately 2 mm below the base of the guide cannulas. Note: No
marking lesions were made before staining the brain of this animal ..60
Figure 20. Histological profile of rabbit #02-025 with suspected positions of the
lateral (left) and medial (right) cannulas. The ends of the dotted lines
represent the best estimation of the locations of the muscimol injection
sites for the two cannulas. The outlines below the cannulas denote the
approximate borders of the dentate nucleus (DN; left) and interpositus
nucleus (IP; right)............................................................................................61
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Figure 21. Rabbit #03-061. Comparison plot of the percent CRs, CR amplitude, and
UR amplitude for eyeblink acquisition, limb flexion acquisition, mixed
trials, and muscimol (M) infusion and recovery (R) trials for the lateral
(eye) and medial (limb) cannulas for paired trials....................................... 65
Figure 22. Rabbit #03-061. Comparison plot of the percent CRs, CR amplitude, and
UR amplitude for eyeblink acquisition, limb flexion acquisition, mixed
trials, and muscimol (M) infusion and recovery (R) trials for the lateral
(eye) and medial (limb) cannulas for unpaired trials...................................66
Figure 23. Histological profile of rabbit #03-061. The left and right arrows represent
the marking lesions from the end points (muscimol injection sites) of the
lateral (eye) and medial (limb) cannulas, respectively. The outline below
the cannulas denotes the approximate borders of the interpositus nucleus
(IP)................................................................................................................... 67
Figure 24. Histological profile of rabbit #03-061 magnified IX. The left and right
arrows represent the marking lesions from the end points (muscimol
injection sites) of the lateral (eye) and medial (limb) cannulas,
respectively..................................................................................................... 68
Figure 25. Histological profile of rabbit #03-061 with suspected positions of the
lateral (left) and medial (right) cannulas based on the end points of the
marking lesions. The outline below the cannulas denotes the approximate
borders of the interpositus nucleus (IP)........................................................ 69
Figure 26. Rabbit #04-029. Comparison plot of the percent CRs, CR amplitude,
and UR amplitude for eyeblink acquisition, limb flexion acquisition, mixed
trials, and muscimol (M) infusion and recovery (R) trials for the medial
(limb) cannula for paired trials.......................................................................73
Figure 27. Rabbit #04-029. Comparison plot of the percent CRs, CR amplitude,
and UR amplitude for eyeblink acquisition, limb flexion acquisition, mixed
trials, and muscimol (M) infusion and recovery (R) trials for the medial
(limb) cannula for unpaired trials...................................................................74
Figure 28. Histological profile of rabbit #04-029. The left and right arrows represent
the marking lesions from the end points (muscimol injection sites) of the
lateral (eye) and medial (limb) cannulas, respectively. As previously
mentioned, the positions of both cannulas appear to be shifted rather
medially. The outlines below the cannulas denote the approximate borders
of the dentate nucleus (DN; left) and interpositus nucleus (IP; right) 75
VI
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Figure 29. Histological profile of rabbit #04-029 magnified IX. The left and right
arrowheads represent the marking lesions from the end points (muscimol
injection sites) of the lateral (eye) and medial (limb) cannulas, respectively.
The outline below the cannulas denotes the approximate borders of the
interpositus nucleus (IP)................................................................................ 76
Figure 30. Histological profile of rabbit #04-029 with suspected positions of the
lateral (left) and medial (right) cannulas based on the end points of the
marking lesions. The outlines below the cannulas denote the approximate
borders of the dentate nucleus (DN; left) and interpositus nucleus (IP;
right).................................................................................................................77
Figure 31. Rabbit #04-049. Comparison plot of the percent CRs, CR amplitude,
and UR amplitude for eyeblink acquisition, limb flexion acquisition, mixed
trials, and muscimol (M) infusion and recovery (R) trials for the medial
(limb) cannula for paired trials.......................................................................81
Figure 32. Rabbit #04-049. Comparison plot of the percent CRs, CR amplitude,
and UR amplitude for eyeblink acquisition, limb flexion acquisition, mixed
trials, and muscimol (M) infusion and recovery (R) trials for the medial
(limb) cannula for unpaired trials...................................................................82
Figure 33. Histological profile of rabbit #04-049. The left and right arrows represent
the marking lesions from the end points (injection sites) of the lateral (eye)
and medial (limb) cannulas, respectively. The outlines below the cannulas
denote the approximate borders of the dentate nucleus (DN; left) and
interpositus nucleus (IP; right).......................................................................83
Figure 34. Histological profile of rabbit #04-049 magnified IX. The left and right
arrowheads represent the marking lesions from the end points (injection
sites) of the lateral (eye) and medial (limb) cannulas, respectively. The
outlines below the cannulas denote the approximate borders of the dentate
nucleus (DN; left) and interpositus nucleus (IP; right)................................84
Figure 35. Histological profile of rabbit #04-049 with suspected positions of the
lateral (left) and medial (right) cannulas based on the end points of the
marking lesions. The outlines below the cannulas denote the approximate
borders of the dentate nucleus (DN; left) and interpositus nucleus (IP;
right)..................................................... 85
VII
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Figure 36. Histological reconstructions of the rabbit cerebellum sectioned coronally at
three separate positions in relation to the stereotaxic landmark lambda (X)
in the anterior-posterior axis, namely, at X (top), 0.5 mm anterior (middle),
and 1.0 mm anterior (bottom). Filled circles (left) and unfilled circles
(right) over the left interpositus nucleus represent the approximate
muscimol injections sites for the lateral (eyeblink) and medial (hindlimb
flexion) cannulas, respectively, for all six animals tested in this
study ...................................................... 87
Vlll
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LIST OF TABLES
Table 1. Cumulative probability (p) values for the effects of muscimol injections on
the eyeblink (Eye) versus hindlimb (Limb) flexion conditioned responses for
paired trials for all six animals employed in this study. Effects are
considered to be significant if p<0.05. Commas separate the p values for
animals in which multiple days of inactivations with muscimol were
performed. IP=interpositus nucleus. NIP=No infusions performed. ...... ..88
Table 2. Cumulative probability (p) values for the effects of muscimol injections on
the eyeblink (Eye) versus hindlimb (Limb) flexion conditioned responses for
unpaired trials for all six animals employed in this study. Effects are
considered to be significant if p<0.05. Commas separate the p values for
animals in which multiple days of inactivations with muscimol were
performed. IP=interpositus nucleus. NIP=No infusions performed 89
Table 3. Contrast table displaying the constants that are multiplied by the
corresponding cumulative probabilities (p) and added together to yield a
number that represents an index of double dissociation. This double
dissociation index will be close to zero (0.0000) if there is no double
dissociation and becomes +2.0000 with a perfect double dissociation 90
Table 4. Double dissociation index values (total) for the paired and unpaired trials for
the two animals in this study that demonstrated an apparent double
dissociation between the conditioned eyeblink versus limb flexion responses.
As mentioned in the text, this index is close to zero (0.0000) when there is no
double dissociation and becomes +2.0000 when there is perfect double
dissociation..........................................................................................................91
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ABSTRACT
Thompson and colleagues have demonstrated that the interpositus nucleus of the
cerebellum is the essential locus for the classical conditioning of the somatic eyeblink
response (e.g., McCormick et al., 1981; McCormick and Thompson, 1984). In
preliminary findings, Donegan et al. (1983) reported that lesioning the cerebellar
interpositus nucleus ipsilateral to the side of training also appears to abolish conditioned
limb flexion responses. Previous studies (e.g., Chambers and Sprague, 1955) have
suggested that the interpositus nucleus is somatotopically organized with the eye being
represented laterally and the hindlimb medially. In the present study, we employed a
double dissociation paradigm to examine the effects of muscimol (a GABAa agonist)
injections on eyeblink versus limb flexion conditioned responses in the ipsilateral
cerebellar interpositus nucleus of New Zealand white rabbits. For eyeblink conditioning,
the conditioned stimulus (CS) was a 14-volt lamp bulb and the unconditioned stimulus
(US) was a 3 psi comeal airpuff to the left eye. For limb flexion conditioning, the CS
was a 1 KHz, 85-95 dB SPL tone and the US was a 3-5 mA shock to the upper left
hindlimb. Upon training on both responses to a 60-100% criterion, the rabbits were then
tested on eyeblink and limb flexion responses after injections of muscimol (0.1-0.3 M l of
a 0.01-1.0 M solution) into either the lateral (eyeblink) or medial (limb flexion)
interpositus nucleus. We have been able to successfully decrease or abolish the percent
conditioned responses (CRs) of both the eyeblink and limb flexion conditioning
selectively without affecting the other. These results thus lend further support for the
notion of the existence of a somatotopic map (i.e., eyeblink versus limb flexion) in the
interpositus nucleus for learning.
x
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CHAPTER 1
INTRODUCTION
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Classical, or Pavlovian, conditioning is commonly used to study the neuronal
substrates of basic associative learning (Pavlov, 1927; Schniederman et al., 1969;
Alkon, 1980; Kandel and Schwartz, 1982; McCormick and Thompson, 1984).
According to Rescorla (1988a; 1988b),
Basic associative learning, which results from exposure to relations among
events in the world, is the way organisms, including humans, learn about causal
relationships in the world. For both modem Pavlovian and cognitive views of
learning and memory, the individual learns a representation of the causal structure of
the world and, as a result of experience, then adjusts this representation to bring it in
tune with real causal structure of the world, thus striving to reduce any discrepancies
of errors between its internal representation and external reality.
Use of the rabbit classical nictitating membrane/eyelid conditioning
preparation, referred to as classical eyeblink conditioning, has arguably yielded more
data concerning brain structures and systems involved in associative learning than
any other paradigm or procedure. Classical eyeblink conditioning normally involves
paired presentations of a tone or light as a conditioned stimulus (CS) and a
periorbital shock or air puff unconditioned stimulus (US; see Figure 1). The US
consistently elicits eyelid closure and movement of the nictitating membrane in the
rabbit before training, known as the unconditioned response (UR), while presentation
of the light or tone CS initially produces no overt responses. Interstimulus intervals
of 150-2000ms are normally employed for training. In the rabbit, after about 100-
150 pairings of the CS and US, the CS eventually begins to reliably elicit an
eyeblink, referred to as the conditioned response (CR; reviewed in Steinmetz, 2000;
see Figure 2).
2
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CONDITIONED
STIMULUS
(CS)
UNCOWMTIONED
RESPONSE
(URorUCR)
COW3ITIONED1
RESPONSE I
(CR) / v !
UNCONDITIONED
STIMULUS
(US or UCS)
0 250 SO D 780
TIME (m sec)
Figure 1. Generalized schematic of atypical classical or Pavlovian (delay)
conditioning paradigm in which the conditioned stimulus (CS) and unconditioned
stimulus (US or UCS) co-terminate. Note: the time intervals may be variable
depending on the conditions of the experiment.
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coNomof®
STIMULUS
(CS)
0 260 500 750
TWIE (msec)
Figure 2. Idealized schematic of progression of learning in a typical classical (delay)
conditioning paradigm. Normally, in the rabbit eyeblink-conditioning paradigm, the
animal begins to reliably elicit a learned or conditioned response (CR) after
approximately 100-150 conditioned stimulus (CS) and unconditioned stimulus (US
or UCS) pairings. Note: the time intervals may be variable depending on the
conditions of the experiment.
4
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Approximately twenty years ago, Thompson and colleagues discovered an
interesting finding concerning how acquisition and performance of the classically
conditioned eyeblink response was encoded in the central nervous system (CNS).
They demonstrated that small lesions placed in the interpositus nucleus of the
cerebellum caused a permanent loss of conditioned responding in rabbits that had
been trained before lesions were placed (McCormick et al., 1981). In addition,
Lincoln et al. (1982) found that cerebellar lesions placed before conditioning began
prevented learning of the eyeblink CR. From these observations, McCormick and
Thompson (1984) thus hypothesized that the cerebellum was the critical structure
involved in encoding plasticity processes associated with learning and performance
of the conditioned eyeblink CR. These data also suggested to these investigators that
unlike previous arguments, the memory trace or “engram” for at least one form of
associative learning, the classically conditioned eyeblink response was rather highly
localized in the brain, that is, localized to one or more sites in the cerebellum (see
Figure 3).
Studies using kainic acid lesions, which destroy neuronal cell bodies while
leaving fibers of passage intact, have demonstrated that the neurons within the deep
cerebellar nuclei that are critical for eyeblink conditioning seem to be contained
within an area of about 1mm3 located in the dorsolateral portion of the anterior
interpositus nucleus (Katz and Steinmetz, 1997; Lavond et al., 1984). Contrary to
many other brain lesions effects, destruction of neurons in the interpositus nucleus of
the cerebellum resulted in a permanent loss of the conditioned eyeblink response in
5
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which rabbits trained daily for more than 12 months post-lesion, that is, given over
20,000 training trials, failed to demonstrate the reacquisition of the eyeblink CRs
(Steinmetz et al., 1993).
Unlike the deep cerebellar nuclei, lesions of the cerebellar cortex have not
produced as consistent effects on the conditioned eyeblink response. Two areas of
the cerebellar cortex are known to send axons of Purkinje cells to the interpositus
and dendate nuclei, namely, Larsell’s lobule HVI and regions of the anterior lobe of
the cerebellar cortex. Lesions placed in lobule HVI have demonstrated little or no
effect on conditioning (Woodruff-Pak et al., 1993), caused a reduction in the rate of
learning and eventual CR amplitude (Lavond and Steinmetz, 1989a), or caused a
complete abolition of CRs (Yeo et al., 1985). In addition, lesions placed in the
anterior lobe of the cerebellar cortex have been observed to generate a disruption in
the timing of CRs as well as to prevent extinction of CRs (Perrett et al., 1993).
Studies using reversible lesion (i.e., inactivation) methods during
conditioning have provided the most significant evidence for the essential nature of
the cerebellum as a primary location for neuronal alterations that underlie the
acquisition and maintenance of eyeblink CRs. For example, Clark et al. (1992) have
used cold probe techniques to reversibly lesion the interpositus nucleus region during
conditioning. They found that activation of the cold probe abolished CRs in
previously trained animals, but that the CRs immediately reappeared when the cold
probe was turned off. Furthermore, studies (Zhang and Lavond, 1991; Krupa et al.,
1996) have demonstrated that inactivation of the cranial motor nuclei (primarily the
6
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seventh and accessory sixth) and adjacent regions of the reticular formation, essential
for generating the UR and CR, with infusion of muscimol or cooling during standard
tone-airpuff training result in animals showing no CRs and URs (i.e., performance
was completely abolished). However, the animals exhibited asymptotic CR
performance and normal UR performance from the very beginning of
postinactivation training, which suggests that performance of the CR and UR are
completely unnecessary and make no contribution at to all to the formation of the
eyeblink memory trace. In fact, they are completely efferent from the trace.
Krupa et al. (1993) reported that inactivation by low doses of muscimol of
the magnocellular red nucleus for six days of training had no effect on the UR but
completely prevented expression of the CR. These animals demonstrated asymptotic
learned performance of the CR from the beginning of postinactivation training. In
addition, Clark and Lavond (1993) have revealed that training during cooling of the
magnocellular red nucleus gave identical results in which animals learned during
cooling, as evidenced in postinactivation training, but did not express CRs at all
during inactivation training. Still, cooling did seem to impair performance of the UR
in their study even though the animals learned normally, which is in support against
the performance argument. Taken together, these studies suggested that the red
nucleus must be efferent from the eyeblink memory trace.
Inactivation of the dorsal anterior interpositus nucleus and overlying cortex
with low doses of muscimol (Hardiman et al., 1996) and lidocaine (Nordholm et al.,
1993) have resulted in no expression of CRs during inactivation training and in no
7
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evidence of any learning during inactivation training. In subsequent postinactivation
training, animals learned normally as though completely naive, that is, they showed
no savings at all relative to noninactivated control animals. In fact, none of the
methods of inactivation seemed to have any effect at all on the performance of the
UR on US-alone trials (reviewed in Christian and Thompson, 2003). In addition,
Krupa and Thompson (1997) have shown that infusions of very low doses of
muscimol (1.0 nmole in 0.1 jiL of saline vehicle) limited to the anterior lateral
portion of the interpositus nucleus (with no significant radioactive muscimol label in
the cerebellar cortex) completely prevented learning of the eyeblink CR. Finally,
Krupa and Thompson (1995) infused tetrodotoxin (TTX) in the superior cerebellar
peduncle (scp) during eyeblink training to inactivate the output of the cerebellum.
This procedure inactivates both the descending and ascending efferent projections of
the cerebellar hemisphere. TTX infusion in the scp completely prevented expression
of the CR, with no effect on the UR, for the six days of training. On the seventh day,
TTX was not infused and the animals displayed asymptotic learned performance of
the CR. Control animals were also infused with TTX in the scp for six days but not
trained, and then trained, and revealed normal learning of the eyeblink response.
Taken together, the results from the abovementioned studies strongly support
the notion that the memory trace for the eyeblink response is formed and stored in
the cerebellum, particularly the interpositus nucleus. Inactivation of the interpositus
nucleus during eyeblink training completely prevents learning, but inactivation of the
output pathway from the region and its necessary (for the CR) efferent target,
8
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namely, the red nucleus, does not prevent learning at all. In fact, in no case do the
drug inactivations have any remote effect on the performance of the eyeblink
response on the US-alone trials. Even if part of the essential memory trace was
formed prior to the cerebellum in the essential memory circuit, then the animals
would have displayed savings following cerebellar inactivation training, which they
show none at all. Along the same lines, if part of the essential memory trace was
formed in the red nucleus or other efferent targets of the interpositus nucleus, such as
the brainstem, then animals could not demonstrate asymptotic CR performance
following scp or red nucleus inactivation training, but in fact they do. Therefore, the
argument for the essential role of the cerebellar interpositus nucleus in classical
conditioning of the eyeblink response appears beyond question (reviewed in
Christian and Thompson, 2003).
9
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H
Cerebellar Cortex
Climbing
Fibers
Mossy
Fibers
Interpositus Nucleus ^f.
Pontine Nuclei
Reticular
Formation
Red Nucleus
Abducens
Nuclei
(Light)
U S 1
(Airpuff) I
CR/UR (Eye)
Figure 3. Putative eyeblink circuitry. The pontine nuclei and inferior olive send
projections via the mossy fibers and climbing fibers, respectively, to both the
cerebellar cortex and as collaterals to the interpositus nucleus. ( - ) represents
inhibitory projections.
10
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The limb flexion withdrawal response appears to be one of the most
elementary cutaneomuscular reflexes. It is one of the first cutaneous reflexes of the
limb to appear during ontogeny and can be observed well before there is substantial
innervation of the spinal cord from higher levels of the nervous system (Bradley et
al., 1983). Initially, Fanardjian (1961) and Gambaryan (1960) reported that
permanent lesions of the cerebellar cortex and nuclei did not abolish the acquisition
and retention of classically conditioned and signaled avoidance limb withdrawal
reflexes in the dog. In addition, Marchetti-Gauthier et al. (1990) have shown that
restricted lesions of the cerebellar interpositus nucleus did not affect the capacity to
retain previously acquired conditioned limb withdrawal responses in mice. Similar
lesions, however, blocked acquisition of new responses (Marchetti-Gauthier et al.,
1990). In contrast, Donegan and Thompson (1991) have reported that lesions of the
interpositus nucleus disrupted retention of classically conditioned limb withdrawal
responses in the rabbit. Collectively, these studies are difficult to interpret because
they were performed in several animal species using different lesions and
conditioning protocols. Therefore, further understanding of cerebellar participation
in conditioned responses (CRs) in multiple effector systems requires additional,
carefully controlled experiments.
The basic circuitry for the limb withdrawal response lies mainly within the
spinal cord, and the spinal circuits demonstrate substantial adaptive capabilities
(Patterson et al., 1973; see Figure 4). Eccles and Lundberg (1959) first defined the
characteristics of spinal reflex circuits responsible for the organization of the limb
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Mossy
Fibers
1 Pontine Nuclei
Cochlear Nuclei
Interpositus Nucleus
Climbing
Fibers
(-)
Qnferior^^wJ
Red Nucleus
Spinal Cord
Spinal Cord
|^CS(Tone)J
CR/UR (Limb)
Figure 4. Putative limb flexion circuitry. As in the eyeblink circuit, the pontine
nuclei and inferior olive send projections via mossy fibers and climbing fibers,
respectively, to both the cerebellar cortex and as collaterals to the interpositus
nucleus. (- ) represents inhibitory projections.
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withdrawal response based on intracellular responses of flexor motoneurons to
cutaneous afferents. These experimenters revealed that low and high threshold
cutaneous afferents and high-threshold muscle afferents impinge upon polysynaptic
spinal circuits that activate flexor motor neurons and inhibit extensor motor neurons,
a pattern consistent with the distribution of muscles required to execute the flexion
withdrawal reflex. These afferents were termed flexor reflex afferents or FRAs.
Even though subsequent examinations showed that the organization of reflexes
activated by specific low- and high-threshold spinal afferents is more complex,
generally, the notion of FRAs and the spinal circuits they activate remain highly
applicable to understanding the basis for this class of reflex behavior. Still, it is
necessary to realize that these afferents evoke a spectrum of behaviors that extend
considerably beyond a simple flexion movement of a distal extremity (McCrea,
1992). For instance, the pattern of muscle activation may not be restricted to motor
neurons innervating anatomical flexors but can consist of an extension movement or
movements requiring flexion at some joints and extension at others.
Lundberg (1966) has emphasized the spectrum of segmental actions that this
functional group of afferents (i.e., FRAs) can exert and how descending projections
can control these actions. Apparently, one of the most elegant features of this reflex
system is its flexibility since it is capable of generating multiple output patterns in a
highly task dependent manner. For example, during locomotion, even in spinal
animals, the same stimulus can evoke behaviorally appropriate flexion or extension
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depending on the phase of the step cycle in which it is generated (Rossignol and
Gauthier, 1980).
Several critical properties of the limb withdrawal reflex system have been
described even though the organization of the specific intemeurons mediating this
variety of responses in not known in detail. The related afferents can be viewed as
accessing three different patterns reflex pathways, namely, normal, alternative, and
private. McCrea (1992) has argued that normal and alternative FRA pathways refer
to dramatic alterations in the organization of motor neuron pools that can be
regulated by descending pathways as well as by other spinal circuits. These actions
can be as varied as a change from a predominately flexion movement to an extension
movement evoked by the same afferents. In addition, the private pathway refer to
those FRA-activated reflexes with a much more restricted distribution, distributed to
a much more confined set of motor neuron pools, than the normal FRA circuits.
Still, descending pathways can regulate even this subset of FRA projections. The
organizational features of this reflex system have been proposed as playing a critical
role in the organization of many types of movements (Schomburg, 1990).
Lundberg (1966; 1979) has further emphasized the functionally important
interactions between the spinal circuitry activated by the FRAs and the reflexes
evoked by proprioceptive afferents. The FRAs can modify the action of la and lb
afferents and affect the excitability of gamma motoneurons, which in turn regulate
the properties of the proprioceptors responsible for evoking the stretch reflex. In
addition, Lundberg argued that descending projections, in this situation the
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descending monoaminergic systems, controls this interaction between the FRA
system and the reflex projections of proprioceptive afferents. Thus, the reflex
system that underlies the withdrawal reflexes is a highly flexible, interactive system.
The organization of these segmental projections can be modified dramatically by the
action of central pathways. These modifications can include alterations in the
distribution of the affected motor neuron pools as well as changes in the
characteristics of the reflex itself, including complete reflex reversals.
The implementation of reflex circuits in the control and generation of
movements requires an integration of sensory information from the periphery, the
ongoing activity in central pathways (including reflex circuits) and the characteristics
of the activity in output pathways by which the movements are executed. The
organization of inputs to the cerebellum has provided one of the most comprehensive
substrates for this purpose. High- and low-threshold cutaneous and kinesthetic
afferents from the spinal and trigeminal systems activate a wide spectrum of
cerebellar afferents that project to regions receiving inputs from other sources
relevant to encoding properties of sensory stimuli, the nature of the execution signals
from descending pathways and the kinesthetic and cutaneous cues resulting from the
movement itself (reviewed in Bloedel and Bracha, 1995).
Generally speaking, both mossy fiber systems and climbing fiber projections
receive a substantial input from the group of peripheral afferents responsible for
evoking the flexion reflex, the flexor reflex afferents. Mossy fiber projections
activated by these afferents reach the cerebellum principally via two different routes:
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1) segmental inputs to the cells of origin of the specific spinocerebellar projections,
such as the dorsal and ventral spinocerebellar tracts; and 2) the reticulocerebellar
projections activated primarily via the bilateral ventral flexor reflex tract (bVFRT),
an ascending system in the ventral half of the spinal cord activated by flexor reflex
afferents and projecting to the lateral reticular nucleus (Brodal et al., 1967). Each of
these projections pre-process peripheral inputs in different ways.
It is known that spinal flexor reflex afferents activate several direct mossy
fiber projections from the spinal cord to the cerebellum. One of these pathways is
the exteroceptive or cutaneous subdivision of the dorsal spinocerebellar tract
(DSCT). Moreover, joint afferents access this spinocerebellar pathway. These
afferents can terminate directly on the cells of origin of these projections and
generate responses with properties comparable to those of the afferents themselves
(Mann, 1971). There are also polysynaptic projections that result in significant
spatial and temporal integration of these signals before they contact the
spinocerebellar projection (Osborn et al., 1993). A significant number of descending
pathways, including reticulospinal projections and the corticospinal pathway, modify
the processing of sensory information at the segmental level (Bloedel and Courville,
1981). In addition, mossy fiber projections originate from specific regions ofboth
dorsal column nuclei (DCN; Rinvik and Walberg, 1975). These projections can
hence provide information to the cerebellum regarding activity in low threshold
cutaneous afferents that also project to the medial lemniscus and most likely play an
important role in evoking the contact placing reaction (Cooke et al., 1971).
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A number of the mossy fiber inputs to the cerebellum activated by FRAs do
not merely convey information regarding the periphery. Instead, the modulation in
these pathways reflects the integration occurring in intemeuronal pools as a
consequence of inputs from these peripheral afferents as well as from descending
projections. The ventral spinocerebellar tract (VSCT), the projection of this type
from the lumbar region of the spinal cord, receives an input from flexor reflex
afferents mediated by an intemeuron pool that sends a parallel projection to alpha
motor neurons. Several descending projections modulate these same intemeurons
(Lundberg, 1971). The rostral spinocerebellar tract (RSCT) originating from this
spinal level is also organized similarly to the VSCT (reviewed in Bloedel and
Bracha, 1995).
Another set of projections involving the precerebellar reticular nuclei are
involved in processing of cutaneous inputs. The reticulocerebellar pathways have a
broader termination pattern in the cerebellar cortex (Bloedel and Burton, 1970) and
also respond to the activation of the peripheral afferents with very unique temporal
characteristics: patterns of burst-like activity that can only be evoked at relatively
low frequencies. The best studied of these projections originates from the lateral
reticular nucleus. Intemeurons receiving inputs from the FRAs project not only to
alpha motor neurons and cells of origin of the VSCT, but also to the bilaterally
organized ascending system in the ventral half of the spinal cord, the bilateral ventral
flexor reflex tract (bVFRT; Bloedel and Courville, 1981). This pathway activates
neurons in the lateral reticular nucleus that in turn project mainly to the vermis,
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paravermis, and the paramedian lobules of the cerebellum, considerably broader
termination pattern than any of the direct spinocerebellar pathways (Brodal, 1975).
As is the case for the VSCT and DSCT, several descending pathways,
including the pyramidal tract and reticulospinal projections, also can substantially
modulate the FRAs’ action at the level of the spinal input to the lateral reticular
nucleus (LRN). These interactions include inputs from the descending
vestibulospinal projection, which sends parallel projections to alpha motor neurons
and the spinal neurons projecting to the LRN (Corvaja et al., 1977). The
reticulocerebellar projections provide one additional site for integration between
ascending and descending projections: the reticular nuclei themselves. For instance,
the LRN receives descending inputs not only from the fastigial and interposed nuclei
(Qvist, 1989), but also from the red nucleus and corticospinal pathway (Courville,
1966). The projection from the magnocellular part of the red nucleus (RNm) is
known to be somatotopically organized, thus reflecting the same principle of
organization as the red nucleus projections to the spinal cord (Pompeiano and
Brodal, 1957). Taken together, these studies demonstrate that these
reticulocerebellar projections not only provide organizationally interesting substrates
for conveying peripheral information to the cerebellum, but they are likely
behaviorally significant, in as much as for the integration of reflexes and postural
adjustments evoked by vestibular stimuli.
Both high- and low-threshold cutaneous afferents as well as the other
afferents comprising the FRAs activate olivocerebellar afferents, the climbing fiber
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projection to the cerebellum. Historically, it was believed that only higher-threshold
cutaneous afferents activated cells in this unique cerebellar afferent system, it is now
evident that low-intensity cutaneous stimuli are very effective in activating
olivocerebellar neurons (Gellman et al., 1983). The importance of the FRA input to
the climbing fiber system is revealed by the observation that this information can
reach the cerebellum by at least 17 different spino-olivocerebellar projections.
Based on an electrophysiological assessment of the afferent fibers to which they
respond, the degree of their bilateral organization and the region of the cord through
which they pass for these pathways were described (Bloedel and Courville, 1981).
Similar to the mossy fiber projections, spinal projections to the olive itself as well as
projections from the dorsal column nuclei mediate the pathways responsible for
activating climbing fiber inputs.
The cerebellar efferent projections to the cells of origin of the rubrospinal
pathway are organized in a precise somatotopic manner. It is widely believed that
the spinal projections of this structure originate from its caudal part, the
magnocellular division (RNm). Phylogenetically speaking, there seems to be a
significant relationship between this part of the red nucleus and the intermediate
nuclei of the cerebellum, the interposed nuclei (Ten Donkelaar et al., 1991),
providing access of this cerebellar region to a directly projecting bulbospinal system.
The projection from the interposed nuclei is somatotopically organized, and partly
consists of collaterals from fibers projecting to the thalamus (Shinoda et al., 1988).
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Observations in the monkey (Kennedy et al., 1986) and the cat (Robinson et
al., 1987) have revealed a clear somatotopic interrelationship between components of
the anterior interpositus nucleus, RNm, the cervical and lumbar enlargements of the
spinal cord and even the cerebral cortex. This somatotopy involves a projection
from the facial region of this nucleus to the spinal trigeminal nucleus (Davis and
Dostrovsky, 1986), the somatotopically organized afferent nucleus receiving inputs
activated by airpuff corneal stimuli. Current anatomical findings support the notion
that the cerebellorubral projection originates exclusively from the interpositus
nucleus. In addition, this pattern of organization does not appear to be very species
dependent. Anatomical observations of the cerebellorubral projection failed to show
a projection to the RNm from the dendate nucleus in cats, monkeys, and rats (Daniel
et al., 1987). Still, electrophysiological studies demonstrating that stimulation of
dendate efferents can activate identified rubrospinal fibers (Hames et al., 1981)
support the existence of this pathway.
Even though further examination will be required to resolve the specific
question regarding a dentate-rubrospinal projection, there seems to be no question
that descending projections activated by this nucleus can affect interactions in the
spinal cord, in part via direct projections mediated by the descending limb of the
brachium conjunctivum. A number of investigations have revealed that dendate
stimulation activates descending pathways that can affect the excitability of
motoneurons and cutaneous reflex inputs to these cells in high decerebrate cats
and/or monkeys (Hames et al., 1981; Tolbert et al., 1980). Stimulation of the
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nucleus lateralis in the rat evoked movements that were affected minimally by
bilateral cortical ablation (Cicirata et al., 1989). In addition, lesions of the cerebral
cortex in monkeys did not qualitatively change movements evoked by dendate
stimulation, and stereotypic movements could still be evoked following transactions
caudal to the red nucleus (Schultz et al., 1979). It thus appears that both the
interpositus and dendate nuclei can modify spinal interactions via descending
projections originating in the brainstem.
As previously mentioned, several studies support the argument that the
cerebellorubral projections are involved in the regulation of reflex inputs activated
by cutaneous stimuli. The terminal pattern of the rubrospinal projection is highly
consistent with an action of this system on this class of reflexes. A significant
number of these terminals project to Rexed laminae V-VII depending on the specific
termination level (Robinson et al., 1987). In this area, these terminals contact
intemeurons that also receive inputs from high- and low-threshold cutaneous
afferents, in addition to muscle afferents, projecting to flexor motor neurons and/or
intemeurons that can evoke presynaptic inhibition of high-threshold cutaneous
afferents (Hongo et al., 1972). It seems clear that the mbrospinal system can also
affect the cutaneous inputs to extensor motor neurons, including those projecting
disynaptically in the lumbar cord (Fleshman et al., 1988). Furthermore, the
projection from the RNm can modify responses of ascending pathways, such as the
VSCT to peripheral inputs, including cutaneous afferents (reviewed in Bloedel and
Courville, 1981). Consistent with its role in regulating cutaneous reflexes, the RNm
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receives somatosensory projections from the dorsal column and trigeminal nuclei
(Robinson et al., 1987) as well as from a projection activated by peripheral afferents
(Padel et al., 1988). This ascending pathway projects in the ventromedial quadrant
of the spinal cord, terminates in the contralateral red nucleus and may relay
comparable information to the RN and the thalamocortical system (Padel and
Relova, 1988).
Relevant to its role in regulating motor system output, the rubrospinal
pathway likely projects directly to alpha motor neurons, presumably those associated
with distal musculature (Robinson et al., 1987). Still, monosynaptic activation of
motor neurons following red nucleus stimulation has been difficult to demonstrate, at
least in the cat spinal cord (Hongo et al., 1969). Similar to the organization of the
mbrospinal system, the rubrobulbar pathway terminates in motor nuclei controlling
the eyeblink reflex response (Holstege and Tan, 1988). Moreover, neurons in the red
nucleus project to the spinal trigeminal nucleus, the nucleus transmitting the sensory
information that triggers the unconditioned reflex (Holstege et al., 1986). The
activation of these fibers in turn results in the alterations in the response properties of
these trigeminal nuclei.
These extensive interactions with practically every class of spinal neuron are
consistent with the extensive ramification pattern of individual mbrospinal axons in
the spinal cord (Shinoda et al., 1988). These axons may provide collaterals over 2-3
cervical segments, have collaterals as far apart as 6mm, and end in individual
terminal patterns extending up to 2.3 mm in a rostrocaudal direction. This pattern of
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organization is thus well suited for regulating functional sets of interactions in the
spinal cord. In addition, activation of the rubrospinal system produces
predominately flexion movements of the extremities (Pompeiano, 1957), an
observation consistent with the distribution of excitatory and inhibitory inputs to
alpha motor neurons activated by stimulating these pathways (Hongo et al., 1972).
Yet, extensor movements evoked by RNm microstimulation in the monkey also have
been observed (Gibson et al., 1985).
In addition, there exists a cerebellothalamocortical projection system that has
a clear topographic organization. However, this organization includes a substantial
pattern of convergence of inputs from the cerebellar nuclei onto individual
thalamocortical neurons. In addition, the thalamocortical cells in turn project to
multiple sites within the motor cortex, predominately areas 4 and 6, even though the
cerebellothalamocortical projection certainly involves other cortical areas (Sasaki et
al., 1972). Other studies have further emphasized that specific regions of a given
cerebellar nucleus, at least within the dendate nucleus, project predominately to
different cortical regions (Strick et al., 1993). These specialized organizational
features are thus well suited for a system important for the execution and perhaps the
acquisition of certain reflexes evoked by cutaneous and kinesthetic stimuli,
particularly those characterized by a well-organized, seemingly complex motor
response to specific, behaviorally relevant inputs.
In an attempt to further define the role of the inferior olive in the performance
of an instrumentally and classically conditioned limb flexion response in cats,
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Voneida et al. (1990) placed lesions in various parts of the inferior olivary nucleus
and olivocerebellar tract. It was found that the degree of conditioned response (CR)
loss resulting from a given lesion was closely related to the precise locus of the
lesion. For example, rostromedial olivary lesions, which included the spino- and
cortico-olivary forelimb projection zones and the olivocerebellar projection area,
resulted in varying degrees of CR loss (from partial to near total), deregulation of
response latency, and a significant reduction of response amplitude. Moreover, the
CR deficit and degree of post-operative CR recovery were directly related to the
extent of damage to this area of the rostromedial olive.
Lesions restricted to the caudal olive or to caudal levels of the olivo
cerebellar tract resulted in no postoperative CR deficits. Yet, animals with caudal
lesions displayed more severe general motor deficits postoperatively than did those
with rostromedial lesions and loss of the CR. These investigators observed that
prolonged training of animals with the most complete CR deficits resulted in some
relearning, but response patterns were symbolized by long-latency, low-amplitude
CRs and a highly unstable response pattern. In addition, the animals seemed to show
extinction of the CR with repeated presentations of the tone alone CS.
To examine the role of the red nucleus in the mediation of the conditioned
limb response, Voneida (1999) placed stereotaxic lesions in the rubrospinal tract of
adult cats that had been trained to a criterion performance level of conditioned
forelimb flexion. The author found that tractotomy resulted in total or near-total loss
of the CR and that prolonged postoperative training resulted in no increase in CR
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performance levels. It is well known that the forelimb region of the magnocellular
red nucleus receives substantial inputs from both cerebellar interpositus nucleus and
those areas within ipsilateral sensorimotor cortex that have been observed to play a
critical role in the performance of a conditioned limb response. In addition, studies
have demonstrated that the pyramidal tracts are neither necessary nor sufficient for
transmission of signals related to conditioned limb flexion from cortex to spinal
motor neurons (reviewed in Voneida, 1999), which the author suggests reveals the
importance of the corticorubrospinal pathway in perhaps triggering the activation of
spinal motor neurons. The author contends that these results, along with findings
from a large number of other studies, thus implicate the sensorimotor cortex,
cerebellum (interpositus and lobule HVI), and the magnocellular red nucleus as
critical parts of the circuitry involved in associative learning.
Finally, Voneida (2000) carried out a series of sections of the brachium
conjunctivum (i.e., superior cerebellar peduncle) ipsilateral to the trained limb
following criterion CR performance. The brachium conjunctivum was observed to
have been sectioned in four of the seven subjects with each of these animals
demonstrating a total or near-total loss of the CR. Extended postoperative training
resulted in no increase in CR performance levels. It is well established that the
brachium conjunctivum represents the major efferent pathway from cerebellar
dendate and interpositus nuclei to the magnocellular red nucleus and the ventral
lateral/ventral anterior complex of the thalamus. Nonrubral and nonthalamic
projections from dentate and interpositus nuclei also exit the cerebellum via the
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brachium conjunctivum. These fibers terminate at two distinctive subcortical sites.
One is the principle inferior olivary nucleus, which is known to receive a
considerable projection from the contralateral dentate nucleus, and the other is the
dorsal and the medial accessory olivary nuclei, which receive input from the
contralateral nucleus interpositus anterior and posterior, respectively. The caudal
portion of the medial accessory olive does not appear to receive afferents from the
cerebellar nuclei. The contralateral pons is the second nonrubral, nonthalamic
projection from cerebellar dentate and interpositus nuclei via the brachium
conjunctivum. Both pons and inferior olive project back onto cerebellar cortex and
interpositus nucleus via the middle and inferior cerebellar peduncles. The inferior
olive is a structure that is well known to be part of the subcortical circuitry involved
in limb flexion conditioning (reviewed in Voneida, 2000). Therefore, based on these
results, the author concludes that the brachium conjunctivum plays a critical role in
the subcortical circuitry underlying the performance of the limb flexion response.
In the present study, we examined the effects of muscimol injections in the
interpositus nucleus of the cerebellum on eyeblink versus limb flexion conditioned
responses. The interpositus nucleus is somatotopically organized with the eye
represented laterally and the leg represented medially (Chambers and Sprague, 1955;
reviewed in Lockard, 1999; see Figure 5). Using a double dissociation paradigm
between eyeblink, hindlimb, and forelimb, Bracha et al. (1999) observed evidence
that suggested a somatotopic organization of the limb flexion and eyeblink response
in cats in the interpositus nucleus, but failed to reveal the corresponding
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topographical map for the learned response (Bracha et al., 1999). Presently, we will
attempt to employ a double dissociation procedure involving reversible inactivations
via injections of the GABAa agonist muscimol to demonstrate (in the same animal) a
somatotopic organization in the interpositus nucleus for learning. Our hypothesis is
that separate areas within the interpositus nucleus underlie the two distinct
conditioned responses examined in this study, namely, the eyeblink (lateral portion)
and the hindlimb flexion (medial portion) responses. In addition, we consider the
null hypothesis, which claims that a somatotopic map for learning does not exist for
the eyeblink and hindlimb flexion responses in the interpositus nucleus.
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I Pontine Nuclei
Mossy
Fibers
Limb '
Interpositus Nucleus
(medial) 1 (lateral)
1 Eye
j
Climbing
Fibers
Limb
jFnfarioToUvell
Cochlear Nuclei
Red Nucleus
Reticular
Formation
\
Abducens
Nuclei
Spinal Cord
^ |Trigem inalNucleus |
cs CS
(Light) (Tone)
| CR/UR (Limb) | | CR/UR (Eye) j
Spinal Cord- ' !
us
(A irp u ff)
US
(Shock)
Figure 5. Putative eyeblink versus limb flexion discrimination circuitry. The
conditioned stimulus (CS) and unconditioned stimulus (US) for the eyeblink
response (light-airpuff) converge in the lateral portion of the interpositus nucleus
(IP), whereas for the limb flexion response, the CS-US (tone-shock) converge in the
medial portion of the IP. ( - ) represents inhibitory projections.
28
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CHAPTER 2
MATERIALS AND METHODS
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Subjects
The present study employed six adult male New Zealand albino rabbits
(Oryctolagus cuniculus) with initial weights upon arrival being approximately 2.0 +/-
0.2 kg. The animals were housed individually with free access to food and water and
maintained on a 12 hr light/dark cycle. The experimenter, staff members, and
veterinarians of the vivarium were consistently caring for the animals. In addition,
the study followed the guidelines for the care and treatment of the animals as
established by the American Association of Accreditation of Laboratory Care
(AAALAC), the Federal Department of Agriculture, the Society for Neuroscience,
and the American Psychological Association.
Experimental Design
Rabbits were initially trained for 5-20 days on the eyeblink-conditioning
paradigm and then for approximately 10-100 days on the limb flexion paradigm. In
the limb flexion paradigm, an operant conditioning procedure was used in which the
CS (tone) and US (shock) were immediately turned off once the animal displayed a
certain level of limb flexion. Upon meeting criteria on both tasks, the animals were
subsequently trained on mixed trials of limb flexion and eyeblink conditioning for
roughly 10-30 days until they achieved criteria for performing both tasks in one
training session. After the rabbits met criteria on the mixed trials, the medial or
lateral interpositus nucleus was inactivated using the GABAa agonist muscimol
approximately one hour before being run on mixed trials. The drug muscimol is
known to be effective for approximately eight hours (Krupa, 1993). Thus, on
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separate days, with one day of recovery in between trials, each animal was injected
with muscimol in either the dorsal lateral or medial interpositus. Since muscimol
injections were administered to each nucleus on separate days, each animal served as
its control.
Surgery
Two metal guide cannulas were stereotaxically implanted into the left
cerebellar interpositus nucleus before any training was performed on the rabbits. All
surgeries were carried out under aseptic procedures. Initially, the animals were
anesthetized with subcutaneous Ketamine (Keta-ject; 60 mg/kg) mixed with
Xylazine (Xyla-ject; 5 mg/kg) and subsequently maintained on 2.0% halothane at 1
L/min for the duration of the surgery. Surgical procedures consisted of a midline
incision through the scalp, retraction of the periostium, and a small craniotomy
above the stereotaxic coordinates of the medial and lateral portions of the
interpositus nucleus (see below). Throughout the surgical procedure, the respiratory
rate and reflexes of the animal was carefully monitored. A 1 .O mm loop of 6-0
surgical suture (Ethilon) was also placed in the apex of the left nictitating membrane
(NM), which allowed for attachment of the eyeblink potentiometer. Upon
completion of the surgery, the animals were administered three injections of
Buprenex every 12 hours for pain relief. In addition, routine postoperative
procedures were carried out in a recovery room under constant supervision until the
animals began to recover from the anesthesia. For the next few hours, careful
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observations were made of the animal at 30 min intervals until it was sitting up and
alert and then returned to its cage.
The stereotaxic coordinates for the lateral interpositus cannula were 0.5 mm
anterior from lambda, 5.0 mm medial-lateral, and 14.0 mm dorsal-ventral. For the
medial interpositus cannula, the coordinates were 0.5 mm anterior from lambda, 3.0
mm medial-lateral, and 15.0 mm dorsal-ventral (Lavond et al., 1994; Lavond and
Steinmetz, 2003).
Apparatus
After approximately seven days, at which time the animal was fully
recovered from the surgery, it was adapted (i.e., habituated) in a standard rabbit
plexiglass restrainer for two days with the complete set of connections as in a normal
training session. For the limb flexion conditioning, a rectangular hole was cut at the
bottom, left side of the restrainer so that the animal’s left leg could pass through and
be extended. The modified restrainer was then placed on top of a wooden platform
with a corresponding rectangular hole cut in the same position, which allowed the
animal’s left leg to pass through. The platform was constructed highly enough so the
animal’s left hindlimb could suspend freely in the air without coming into contact
with the floor of the sound attenuating chamber. During the habituation and
subsequent training sessions, a potentiometer (attached to an air hose) was placed on
top of the animal’s head to measure the eye-blink response. Eye-clips were placed in
the left eye to keep the animal’s eye open during eye-blink conditioning and a light
bulb (serving as the light CS) was attached to the side of the restrainer. In addition, a
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leg cuff with metal plates used as electrodes to present shock (US) was attached to
the rabbit’s left hindlimb and a second potentiometer was attached to the animal’s
toe to measure the degree of limb flexion.
General Training
The eye-blink and limb flexion paradigm were performed on the same set of
runtime parameters. Each conditioning paradigm is run on a 122232222 block
sequence for 12 blocks with a total of 108 trials in which 1 signifies CS alone trial, 2
represents paired trials (i.e., CS and US), and 3 a US alone trial. In the delay
paradigm, the CS lasts for 1500 ms co-terminating with the 100 ms US. A
conditioned response (CR) is considered any movement of 0.5 mm or higher of the
nictitating membrane or limb flexion beginning 25 ms after the onset of the tone or
light in the CS period until US onset. The learning criterion is considered the first
time the animal performs CRs eight out of nine times consecutively. Moreover, the
performance criterion (approximately 70% or better for the eyeblink and 60% or
better for the hindlimb flexion) is determined by how consistently there are CRs after
meeting the learning criterion. The inter-trial interval varied on a pseudorandomized
schedule alternating between 20 and 40 sec. A small electric fan was also mounted
in the back of the chamber to provide ventilation and continuous background noise.
The response latency, amplitude of response, and a graphic analysis of each trial
were displayed on a monitor of an 8088 IBM computer clone. After completion of
each session, summary statistics, made possible by the Lavond and Steinmetz
interface card and Forth program (Lavond and Steinmetz, 1989b), were printed and
33
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
saved in the animal’s individual folder. The Lavond and Steinmetz apparatus was
capable of recording limb or eye responses of 0.1 mm or greater.
Specific Training
After the two-day habituation period, the training sessions began with the
eyeblink-conditioning paradigm. The CS (1500 ms) was a 14 V light source and the
US a 3 psi air puff. The light source was attached to the side of the restrainer within
full visibility of the animal. The eyelid nictitating membrane (NM) was attached to a
potentiometer via an eye suture and a lightweight metal wire. As mentioned
beforehand, the learning performance for the eyeblink-conditioning paradigm was
determined as approximately 70% or better.
In the limb flexion paradigm, the conditioned stimulus was a 1500 ms, 85-95
dB, and 1 kHz tone delivered through a loud speaker suspended directly in front of
the animal. A Velcro cuff with two silver electrodes was attached to the partly
shaved left hind limb, which delivered a 100 ms, 3-5 mA 60Hz AC shock (US)
generated by a generic shocker. The shock was adjusted to provide consistent
responses at the longest intensity. The performance criterion for the limb flexion
was determined by how consistently the animal performed the flexion response after
meeting the learning criteria. Ideally, as aforementioned, the performance criterion
would be generally 60% or better.
Upon completion of the training to the eyeblink (light and air puff) and limb
flexion (tone and shock) responses, the rabbits were trained on a combination of NM
eyeblink and limb flexion trials. These training sessions consisted of a series of six
34
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blocks and a total of 108 trials. Each block consisted of 1234224542624244, in
which 1 represented light only, 2 light and air, 3 tone only, 4 tone and shock, 5 shock
only, and 6 air only. The combined (i.e., mixed) paradigm was administered for 10-
20 days to allow the rabbit to over learn to the new combination.
On the day of testing for mixed trials, the drug muscimol was injected into
either the medial or lateral interpositus nucleus cannula. The guide cannulas for
injecting muscimol consisted of 23-guage stainless steel tubing cut to 25 mm in
length. Each cannula contained an internal stylet that was replaced by an injector
cannula during testing. Infusion procedures for all rabbits consisted of removal of
the internal stylet from the guide cannula and insertion of a stainless steel injector
cannula (31 guage, 021 mm o.d.) that extended 2.0 mm below the base of the outer
guide cannula. One hour prior to each of the testing sessions, the animals received
an infusion of muscimol (0.1-0.3 pL of 0.01-1.0 M solution diluted in isotonic
saline; Sigma) into the left medial or lateral interpositus nucleus. The ideal dosage
of muscimol infused was about 0.1 pL of a 0.01 M solution, which Krupa and
Thompson (1997) demonstrated that this very low dosage of muscimol inactivated
the interpositus nucleus without diffusing throughout the overlying cerebellar cortex
and thus inactivating that region of the cerebellum. After approximately one hour
that the muscimol was injected, the rabbit was run on mixed trials and its
performance on the particular learned response under examination (eyeblink or limb
flexion) was observed and recorded. The animal was then allowed one day of
35
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
recovery and ran again on mixed trials to see whether the learned response
reappeared. The data for this session was also recorded.
Histological Analysis
After completion of testing, each rabbit was sacrificed with an overdose of
intravenous sodium pentobarbital and perfused intra-aortically with saline followed
by 10% formalin. Marking lesions were subsequently made to better visualize the
location of the cannulas in the interpositus nucleus by placing an electrode through
the chronically implanted cannulas and passing a 100 pA current through the
electrode for 10 sec. The electrode used for making the marking lesions was equal in
length (27 mm) to the injection cannula and thereby extended 2.0 mm below the base
of the guide cannula. It is important to note that due to miscommunication between
the investigators, marking lesions were not made in the first three rabbit brains
examined in the study (i.e., rabbit #00-142, #00-162, and #02-025).
Subsequent to the placements of the marking lesions, the brain was then
removed and post-fixed in 10% formalin. In addition, each brain was blocked and
embedded in a gelatin-albumin matrix and allowed to sit in 10% formalin for at least
one week or until the block sank to the bottom of the jar. After ample time for
fixation, the embedded brains were frozen, cut on a microtome at an 80 pm
thickness, and mounted onto chrome alum subbed slides. The tissue sections were
subsequently stained with Prussian Blue and cresyl violet to visualize for marking
lesions and cells (i.e., neurons and glia), respectively. Finally, the stained sections
were analyzed under a light microscope to reconstruct the cannula placements.
36
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Data Analysis
The raw data for all animals examined were collected and comparison plots
of the percent CRs, CR amplitude, and UR amplitude for eyeblink acquisition, limb
flexion acquisition, mixed trials, and muscimol infusion and recovery trials for both
paired and unpaired trials were determined using the software program MATLAB
(version 6.1). In addition, we employed a binomial distribution to calculate the
cumulative probabilities (p) of muscimol effects using the recovery performance of
the eyeblink or hindlimb flexion conditioned responses as probabilities of CRs for
both paired and unpaired trials. The muscimol effects were considered to be
significant if p<0.05. Similar to the method of contrasts used to discriminate effects
in an analysis of variance, we then created a model to calculate the average of the
cumulative p values to determine the degree to which double dissociation was
achieved.
3 7
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CHAPTER 3
RESULTS
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
The results for this study are based on six different rabbits that completed all
the training and were injected with muscimol. Each animal is described separately
with the results obtained from paired and unpaired trials. The cumulative probability
(p) values for the effects of muscimol on the conditioned eyeblink versus hindlimb
flexion responses for all six animals employed in this study for both paired and
unpaired trials are summarized in Tables 1 and 2, respectively.
Our primary interest in this study was to see if the data show evidence for a
double dissociation between the conditioned eyeblink versus hindlimb flexion
responses depending on whether the muscimol injection was in the lateral or medial
portion of the interpositus nucleus. In cases where there appeared to be a double
dissociation (rabbit #00-142 and #00-162), we quantified this effect by creating a
contrast table similar in concept to the comparisons used in analysis of variance to
model double dissociation. Table 3 shows the constants that are multiplied by the
corresponding cumulative probabilities and added together to yield a number that
represents an index of double dissociation that we call the "double dissociation
index,” this index will be close to zero (0.0000) if there is no double dissociation and
becomes +2.0000 with a perfect double dissociation. Table 4 displays the double
dissociation index for the paired and unpaired trials for the two animals in this study
(#00-142 and #00-162) that seemed to demonstrate a double dissociation.
Finally, Figure 36 plots the approximate muscimol injection sites for the
lateral and medial cannulas for all six animals examined in this study at three
separate coronal sections relative to the stereotaxic landmark lambda (k).
39
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Rabbit #00-142
The results for this particular animal are summarized in Figures 6 and 7. In
general, this animal learned the eyeblink paradigm quickly, consistently performing
close to 100% with high UR amplitudes. On the other hand, the limb flexion
paradigm was learned rapidly, but the performance was inconsistent. An additional
five days of limb flexion conditioning only trials were done to improve the animal’s
performance. Unfortunately, the animal demonstrated approximately 23% CRs after
these additional five days of training. Nevertheless, the amplitude o f URs and CRs
remained more or less consistent throughout the limb flexion acquisition process.
Infusion of muscimol (0.1 pL at 1.0 M) into the lateral (eye) cannula resulted
in a significant difference in percent CRs for paired trials (p=0.0000; see Table 1),
going from 0% after about one hour of infusion to 100% after one day of recovery.
In addition, the eyeblink response displayed an apparent deficit in the UR and CR
amplitudes on the day of muscimol infusion that returned to normal on the day of
recovery. Infusion of muscimol (0.1 pL at 1.0 M) into the medial (limb) cannula
demonstrated a significant deficit in response (15.2%; p=0.0056) as compared to the
day of recovery. It is important to note that the eyeblink response did not seem to be
affected (p=0.1062) and remained at approximately 92.8% during muscimol infusion
and recovery testing in the medial cannula. There was also no apparent deficit in the
UR amplitude in either eyeblink or limb flexion response after muscimol infusion
into the medial cannula (see Figure 6). The calculated double dissociation index for
the paired trials for this animal was 0.6691 (see Table 4).
40
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
The results of impaired CS and US trials are summarized in Figure 7. In the
light (CS) alone trials of eyeblink conditioning, 100% CRs were observed for the
majority of the mixed trials. Muscimol testing in the lateral cannula displayed 0%
CRs for the eyeblink but 100% after recovery (p=0.0000; see Table 2). Infusion of
muscimol into the medial cannula demonstrated a slight deficit in limb flexion
performance as compared to the following day of recovery (p=0.3512), but no
overall difference in percent CRs as compared to the lateral cannula (p=0.7373).
There was no apparent reduction of the conditioned eyeblink response (p=0.6651)
when muscimol was injected into the medial cannula. Moreover, examination of the
CR and UR amplitude revealed a deficit in the eyeblink response for the lateral
cannula with no seemingly significant difference in the medial cannula. The
calculated double dissociation index for the unpaired trials for this animal was
1.0512 (see Table 4).
Finally, histological examination of the animal’s brain (see Figures 8 and 9)
showed that both cannulas were slightly too medial and posterior (approximately 1.0
mm) from where they should have been. The medial cannula seemed to be in the
lateral vermis and the lateral cannula in possibly the beginnings of the lower HVI
lobule. In addition, since marking lesions were not made in the brain of this animal
before staining, we made best approximations as to the locations of the muscimol
injections sites for the medial and lateral cannulas based on the fact that we designed
the injection cannula to extend 2 mm below the base of the guide cannulas (see
Figure 10).
41
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Rabbit 00-142 - EY E BU NK /LIM B FL E X IO N STUDY (Paired Trials)
100
80
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A cquisition
DAYS OF TRAINING
30 M R M R
L ateral (Eye) M edial (Limb)
Injection Injection
Figure 6. Rabbit #00-142. Comparison plot of the percent CRs, CR amplitude, and
UR amplitude for eyeblink acquisition, limb flexion acquisition, mixed trials, and
muscimol (M) infusion and recovery (R) trials for the lateral (eye) and medial (limb)
cannulas for paired trials.
42
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Rabbit 00-142 - EY E B L IN K /L IM B FLEXIO N STUDY (Unpaired Trials)
ce 60
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L ateral (Eye) M edial (Limb)
in jection in jection
Figure 7. Rabbit #00-142. Comparison plot of the percent CRs, CR amplitude, and
UR amplitude for eyeblink acquisition, limb flexion acquisition, mixed trials, and
muscimol (M) infusion and recovery (R) trials for the lateral (eye) and medial (limb)
cannulas for unpaired trials.
43
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Dursal
I aui.it - M i dial
\ cnl i n I
Figure 8. Histological profile of rabbit #00-142. The left and right arrows indicate
the positions of the tracks left in the tissue from the lateral (eye) and medial (limb)
cannulas, respectively. The muscimol injection sites are approximately 2 mm below
the base of the guide cannulas. Note: No marking lesions were made before staining
the brain of this animal. The outline below the cannulas denotes the approximate
borders of the interpositus nucleus (IP).
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
\
f
■
■
■
\
\
■
\
\
2 nun
\
Figure 9. Histological profile of rabbit #00-142 magnified IX. The left and right
arrows indicate the positions of the tracks left in the tissue from the lateral (eye) and
medial (limb) cannulas, respectively. The muscimol injection sites are
approximately 2 mm below the base of the guide cannulas. Note: No marking
lesions were made before staining the brain of this animal. The outline below the
cannulas denotes the approximate borders of the interpositus nucleus (IP).
45
Reproduced with permission of the copyright owner. Further reproduction prohibited without permission.
Figure 10. Histological profile of rabbit #00-142 with suspected positions of the
lateral (left) and medial (right) cannulas. The ends of the dotted lines represent the
best estimation of the locations of the muscimol injection sites for the two cannulas.
The outline below the cannulas denotes the approximate borders of the interpositus
nucleus (IP).
46
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.
Rabbit #00-162
The results for the paired trials for this animal are summarized in Figure 11.
This animal learned the eyeblink conditioning in six days, but once it learned this
paradigm, it rapidly learned the limb flexion paradigm in just five days. After eight
days of training in the mixed trials, the eyeblink performance reached 100% and the
limb flexion was mostly in the high 80th percentile.
Application of muscimol (0.1 pL at 0.5 M) to the lateral cannula resulted in
0% CRs in the mixed paired trials for eyeblink conditioning (p=0.0000; see Table 1).
The CR amplitude was also abolished while the UR amplitude remained the same
during the testing day versus the recovery testing the following day. Infusion of
muscimol (0.1 pL at 0.5 M) into the medial cannula resulted in 14.3% CRs in the
mixed paired trials for limb conditioning, which was a significant reduction
(p=0.0000) when compared to the 100% CRs recorded for the following day
recovery. In addition, the CR amplitude was observed to reduce significantly when
muscimol was applied to the medial cannula and returned to normal range after
recovery (0.1 mm recovering to 13.4 mm), whereas the UR amplitude remained
unaffected (see Figure 11).
Infusion of muscimol into the lateral cannula did not appear to affect the
paired limb flexion response at all (p=0.5989), which remained at 100% CRs on the
day of testing and recovery. However, injection of muscimol into the medial cannula
did seem to reduce the paired eyeblink response on the day of testing (p=0.0000).
The paired eyeblink response was 42.9% CRs on the day of testing recovering to
47
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.
100%, which may be considered a significant reduction in the learned performance.
Still, when we compare the amplitude CRs for the paired eyeblink response on the
day of testing versus recovery when muscimol was infused into the medial cannula,
we observe the response going from 1.0 mm on the day of muscimol testing to 1.8
mm on recovery, which does not appear to be a significant reduction in the learned
performance (see Figure 11). We calculated the double dissociation index for the
paired trials for this animal to be 0.5989 (see Table 4).
Comparison of the percent CRs for impaired trials presented in Figure 12 to
those of the paired trials in Figure 11 reveals a noticeable improvement in learning in
the unpaired mixed versus paired mixed trials. The unpaired tone (CS) alone trials in
the limb flexion paradigm appear very well learned when compared to the drops
observed in the paired trials of Figure 11. Infusion of muscimol into the lateral
cannula demonstrated 16.7% CRs for the unpaired light alone trials, which is a
significant reduction (p=0.0007; see Table 2) from 100% CRs observed in the
following day. Infusion of muscimol into the medial cannula also revealed a
significant reduction of CRs for the unpaired tone alone trials compared to the day of
recovery (16.7% compared to 100%; p=0.0007). Moreover, the CR amplitude for
unpaired light alone trials after muscimol infusion into the lateral cannula was 0.9
mm compared to 2.2 mm after recovery testing. For the US alone trials, the UR
amplitude was 0.9 mm after muscimol injections versus 1.7 mm for recovery.
Injection of muscimol into the lateral cannula appeared to slightly reduce the
unpaired limb flexion response on the day of testing (83.3%) to recovery (100%),
48
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.
which was not significant (p=0.6651). In addition, infusion of muscimol (0.1 jiL at
0.5 M) into the medial cannula revealed no significant deficit in learning (i.e.,
percent CRs) for the unpaired light alone trials (p=1.0000). The CRs remained at
100% after muscimol injection and the following day of recovery. The CS alone and
US alone response amplitudes also remained the same for the eyeblink conditioning
on mixed trials. The double dissociation index for the unpaired trials for this animal
was calculated to be 1.6637 (see Table 4).
Histological examination of the animal’s brain (see Figure 13 and 14)
demonstrated that both the medial and lateral cannulas were situated rather high
above the coronal plane of the interpositus nucleus, which may explain why a rather
high concentration of muscimol (0.5 M) was required to abolish or significantly
reduce the eyeblink or hindlimb flexion conditioned responses on days of testing.
Also, as in rabbit #00-142, since no marking lesions were performed in the brain of
this animal before staining, we made best estimates as to the locations of the
muscimol injection sites (approximately 2 mm below the base of the medial and
lateral cannulas; see Figure 15).
4 9
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.
Rabbit 00-162 - EY E B L IN K /L IM B FLEX IO N STUDY (Paired Trials)
23 M R M R
Eye Blink
Limb Flexion
O 10
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Limb Flexion M ixed E ye Blink/Limb Flexion Lateral (Eye) M edial (Limb)
A cquisition A cquisition Injection Injection
DAYS OF TRAINING
Figure 11. Rabbit #00-162. Comparison plot of the percent CRs, CR amplitude, and
UR amplitude for eyeblink acquisition, limb flexion acquisition, mixed trials, and
muscimol (M) infusion and recovery (R) trials for the lateral (eye) and medial (limb)
cannulas for paired trials.
50
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.
Rabbit 00-162 - EYE B L IN K /L IM B FL E X IO N STUDY (Unpaired Trials)
100
80
60
40
20
0
23 M R M R
20 r
Eye Blink
Limb Flexion
1 11 16
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16 23
Eye Blink
A cquisition
Limb Flexion M ixed Eye Blink/Leg Flexion L ateral (Eye) M edial (Limb)
A cquisition A cquisition Injection injectio n
DAYS OF TRAINING
Figure 12. Rabbit #00-162. Comparison plot of the percent CRs, CR amplitude, and
UR amplitude for eyeblink acquisition, limb flexion acquisition, mixed trials, and
muscimol (M) infusion and recovery (R) trials for the lateral (eye) and medial (limb)
cannulas for unpaired trials.
51
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.
Dorsai
l.aterah---------- Medial
Ventral
Figure 13. Histological profile of rabbit #00-162. The left and right arrows indicate
the positions of the tracks left in the tissue from the lateral (eye) and medial (limb)
cannulas, respectively. The muscimol injection sites are approximately 2 mm below
the base of the guide cannulas. Note: No marking lesions were made before staining
the brain of this animal. The outlines below the cannulas denote the approximate
borders of the dentate nucleus (DN; left) and interpositus nucleus (IP; right).
2 mm
52
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.
Figure 14. Histological profile of rabbit #00-162 magnified IX. The left and right
arrows indicate the positions of the tracks left in the tissue from the lateral (eye) and
medial (limb) cannulas, respectively. The muscimol injection sites are
approximately 2 mm below the base of the guide cannulas. Note: No marking
lesions were made before staining the brain of this animal.
53
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.
M M 1 llllllllllllill llllllB M IlllllliM lllB liB lM l^B ^^^^^^^^^^^^M B ^^^^^^^^^B IiillilillMMiiMitiiii^^^^^^M
1 .at era!----------- Medial
Figure 15. Histological profile of rabbit #00-162 with suspected positions of the
lateral (left) and medial (right) cannulas. The ends of the dotted lines represent the
best estimation of the locations of the muscimol injection sites for the two cannulas.
The outlines below the cannulas denote the approximate borders of the dentate
nucleus (DN; left) and interpositus nucleus (IP; right).
5 4
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.
Rabbit #02-025
The results for the paired and unpaired trials for this animal are summarized
in Figures 16 and 17. This animal initially learned both the eyeblink and limb
flexion responses in 10 days. However, the animal needed to be trained an
additional 22 days on limb flexion to demonstrate consistent learning (i.e., CRs at or
above the 70th percentile). At the 16th day of training on mixed trials, the animal
demonstrated acceptable performance on both the eyeblink (100th percentile) and
limb flexion (approximately 80th percentile) responses.
Infusion of muscimol (0.01 M) into the medial cannula on the first session of
testing resulted in 0% CRs (paired trials; see Figure 16) for the limb flexion response
(p=0.0000, see Table 1) and no apparent effect on the eyeblink response (p=1.0000)
in the mixed trials. The limb flexion response returned to approximately 90% CRs
on the following day. However, on the second day of testing with muscimol, limb
flexion CRs (paired trials) were not completely abolished (approximately 10% CRs),
but was still considered a significant reduction (p=0.0000), and the eyeblink response
also appeared reduced (approximately 5% CRs; p=0.0000). On the next day, the
limb flexion and eyeblink responses returned to approximately 65% and 85%,
respectively. The unpaired trials demonstrated similar results with the limb flexion
response being 0% and 30% after muscimol infusion for the first and second sessions
(p=0.0000, 0.0087; see Table 2), respectively, and returning to 80% after one day of
recovery (see Figure 17). The eyeblink response, on the other hand, showed no
effect and approximately 15% CRs for the first and second infusion sessions
55
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.
(p=l .0000, 0.0033), respectively, and 80% and 100% CRs after one day of recovery.
It is important to note that we were unable to perform any injections into the lateral
cannula because during the infusion sessions, the insect pin inside the lateral
cannula, used to prevent the accumulation of brain tissue at the bottom of the
cannula, became jammed and when we attempted to firmly remove the pin, the entire
lateral cannula came out of the animal’s brain.
Histological analysis of the animal’s brain (see Figures 18 and 19) reveals the
approximate locations of the medial and lateral cannulas. It is important to note that
as in rabbit numbers 00-142 and 00-162, no marking lesions were made in the brain
of this rabbit before staining. Thus, the locations of the muscimol injection sites
should be approximately 2 mm below the base of the medial and lateral cannulas. In
addition, since some sections were discarded before histological analysis was
completed, the end points of the medial and lateral cannulas are most likely lower
than they actually appear in Figure 19. In Figure 20, we made our best
approximation as to the locations of the muscimol injection sites for the medial and
lateral cannulas.
56
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.
Rabbit 02-025 - EYE B L IN K /L IM B FL EX IO N STUDY (Paired Trials)
100
□: 60
63 MR MR
44 12 1
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A cquisition A cquisition A cquisition Injection Only
DAYS OF TRAINING
Figure 16. Rabbit #02-025. Comparison plot of the percent CRs, CR amplitude,
and UR amplitude for eyeblink acquisition, limb flexion acquisition, mixed trials,
and muscimol (M) infusion and recovery (R) trials for the medial (limb) cannula for
paired trials.
57
R eproduced with perm ission o f the copyright owner. Further reproduction prohibited without perm ission.
R abbit 02-025 - E Y E B L IN K /L IM B F L E X IO N STUDY (U npaired T rials)
100
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Acquisition Acquisition Acquisition Injection Only
DAYS OF TRAINING
Figure 17. Rabbit #02-025. Comparison plot of the percent CRs, CR amplitude,
and UR amplitude for eyeblink acquisition, limb flexion acquisition, mixed trials,
and muscimol (M) infusion and recovery (R) trials for the medial (limb) cannula for
unpaired trials.
58
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.
I.aterah “Med ial
2 mill
Ventral
Figure 18. Histological profile of rabbit #02-025. The left and right arrows indicate
the positions of the tracks left in the tissue from the lateral (eye) and medial (limb)
cannulas, respectively. The muscimol injection sites are approximately 2 mm below
the base of the guide cannulas. Note: No marking lesions were made before staining
the brain of this animal. The outlines below the cannulas denote the approximate
borders of the dentate nucleus (DN; left) and interpositus nucleus (IP; right).
59
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.
Figure 19. Histological profile of rabbit #02-025. The left and right arrows indicate
the positions of the tracks left in the tissue from the lateral (eye) and medial (limb)
cannulas, respectively. The muscimol injection sites are approximately 2 mm below
the base of the guide cannulas. Note: No marking lesions were made before staining
the brain of this animal.
60
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.
Lateral ■Medial
Ventral
2 mill
Figure 20. Histological profile of rabbit #02-025 with suspected positions of the
lateral (left) and medial (right) cannulas. The ends of the dotted lines represent the
best estimation of the locations of the muscimol injection sites for the two cannulas.
The outlines below the cannulas denote the approximate borders of the dentate
nucleus (DN; left) and interpositus nucleus (IP; right).
61
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.
Rabbit #03-061
The paired and unpaired results for this particular animal are summarized in
Figures 21 and 22, respectively. The animal seemed to learn the eyeblink response
rather rapidly demonstrating 91.4% CRs for paired trials (Figure 21) by the seventh
day of training. Ftowever, the limb flexion response took a significantly longer
period (98 days) for the animal to learn consistently. In fact, during the mixed
eyeblink and limb flexion training period, the animal was returned to limb flexion
alone training for a few times to ensure consistent limb flexion performance. The
animal met criterion for the limb flexion response on the last five days of limb
flexion training demonstrating 58.3% and 81.8% CRs for unpaired trials and 58.5%
and 45.8% CRs for paired trials on the 97th and 98th day of training, respectively (see
Figures 21 and 22).
After 25 days of mixed eyeblink and limb flexion training, muscimol
injections were performed separately in the eyeblink (three times) and limb flexion
(four times) regions of the interpositus nucleus. Injection of 0.15 pL of 0.01 M
solution of muscimol into the lateral (eye) cannula seemed to optimally eradicate the
eyeblink response with partial effect on the limb flexion response. For example,
injection of 0.15 pL of muscimol on the second and third session of infusion into the
lateral cannula (after approximately one hour) reduced the eyeblink response to 4.8%
and 16.7% CRs (p=0.0000, 0.0000; see Table 1) for paired trials recovering on the
following day to 100% and 97.3% CRs, respectively. The limb flexion response for
these two sessions were observed to be 15.4% and 14.3% CRs (p=0.0000, 0.0000)
62
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for paired trials after muscimol injections recovering to 59.5% and 56.8%,
respectively, on the following day (see figure 21).
For infusions into the medial (limb) cannula, 0.15 pL of 0.01 M solution of
muscimol was initially observed to significantly eradicate the limb flexion response.
However, upon further infusions, 0.3 pL of 0.01M solution of muscimol seemed to
give a more consistent and robust eradication of the limb flexion response without
any apparent effect on the eyeblink response. For example, injection 0.30 pL of
0.01 M muscimol into the medial cannula on the second infusion session
significantly reduced the limb flexion response to 26.2% CRs (p=0.0000) for paired
trials recovering to 78.0% on the following day. The eyeblink response for this
infusion session did not appear to be significantly affected at all demonstrating
100.0% CRs (p=1.0000) after muscimol injection recovering to 97.6% CRs for
paired trials on the following day. In addition, injection of 0.3 pL of 0.01 M
muscimol into the medial cannula on the third and fourth day of infusions
significantly reduced the limb flexion response to 0% and 2.4% CRs (p=0.0000,
0.0000) for paired trials approximately one hour after injection recovering to 35.7%
and 72.5% CRs, respectively, on the following day. The eyeblink responses for both
of these infusion sessions was completely unaffected demonstrating 100.0% CRs
(p=l .0000, 1.0000) for paired trials after one hour of muscimol injections and on the
following day of recovery (see Figure 21).
The results for the unpaired trials (Figure 22) generally support those from
the paired trials. Overall, the percent CRs and amplitude CRs for the unpaired trials
63
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correlate rather closely with the ones for the paired trials. Nevertheless, there are a
few observable differences between the two figures. For example, on all three days
of muscimol infusion into the lateral cannula, the limb flexion response on paired
trials appeared significantly reduced (p=0.0061, 0.0000, 0.0000; Table 1) on day of
testing compared to recovery, whereas for the unpaired trials, the reduction in the
conditioned limb flexion response seems insignificant between the day of testing and
recovery (p=0.0878, 0.9844, 0.0878; see Table 2). In addition, muscimol infusion
into the medial cannula did not appear to significantly affect the conditioned
eyeblink response in any of the four testing days (p=1.0000, 1.0000, 1.0000, 1.0000)
for unpaired trials, whereas the conditioned limb flexion response appeared
significantly reduced in all four testing sessions (p=0.0178, 0.0007, 0.0014, 0.0001).
Finally, in the fourth session of infusion into the medial cannula, the amplitude CRs
for the limb flexion response after muscimol injection versus recovery seems
significantly greater for the unpaired trials (0.2 versus 9.0 mm) than the paired trials
(0.4 versus 3.7 mm; see Figures 21 and 22).
Histological examination of the brain of rabbit #03-061 (Figure 23) showed
that the medial and lateral cannulas were seemingly positioned correctly above the
suspected limb flexion and eyeblink loci, respectively, of the interpositus nucleus.
Still, upon further examination (Figure 24), the lateral cannula could have been
placed a little lower (more ventral) for more optimal positioning. Figure 25 shows
where the medial and lateral cannulas were positioned in the brain based on the
locations of the marking lesions.
64
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R abbit 03-061 - EYE B L IN K /L IM B FL E X IO N STUDY (Paired Trials)
100
O 60
1 8 106
10
o :
o
I 2
Bye Blink
o -o Limb Flexion □
d o
130MR&RMR M R M R M R M R
Eye Blink Limb Flexion
A cquisition A cquisition
130 M R M R M R M R M R M R M R
M ixed Eye Blink/ Laterai(Eye) MediaI(Llmb)
Limb Flexion injection Injection
A c q u isitio n
DAYS OF TRAINING
Figure 21. Rabbit #03-061. Comparison plot of the percent CRs, CR amplitude, and
UR amplitude for eyeblink acquisition, limb flexion acquisition, mixed trials, and
muscimol (M) infusion and recovery (R) trials for the lateral (eye) and medial (limb)
cannulas for paired trials.
65
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Rabbit 03-061 - EYE B L IN K /L IM B FL E X IO N STUDY (Unpaired Trials)
100
O 60
106 1 8
10
8
If
E
» 6
cr
O
g 4
a
I 2
Eye Blink ■
o— o Limb Flexion □
i.iii
1 8 106
1 3 0 MR MR MR MR MR MR MR
25
20
U
O L
Z >
■a 1 0
f
1 8
Eye Blink Limb Flexion
A cquisition A cquisition
106 130
MR MR HiR MR MR MR MR
M ixed Eye Blink/ Lateral(E ye) M edial(Limb)
Limb Flexion Injection Injection
Acquisition
DAYS OF TRAINING
Figure 22. Rabbit #03-061. Comparison plot of the percent CRs, CR amplitude, and
UR amplitude for eyeblink acquisition, limb flexion acquisition, mixed trials, and
muscimol (M) infusion and recovery (R) trials for the lateral (eye) and medial (limb)
cannulas for unpaired trials.
66
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L a it 'r a f -
Ventral v ’ , 2 1 1 1 ill
1 1 1
/
Figure 23. Histological profile of rabbit #03-061. The left and right arrows
represent the marking lesions from the end points (muscimol injection sites) of the
lateral (eye) and medial (limb) cannulas, respectively. The outline below the
cannulas represents the approximate borders of the interpositus nucleus (IP).
6 7
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Figure 24. Histological profile of rabbit #03-061 magnified IX. The left and right
arrows represent the marking lesions from the end points (muscimol injection sites)
of the lateral (eye) and medial (limb) cannulas, respectively.
6 8
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i.aU'raf Vlt'dia! . . . .
Figure 25. Histological profile of rabbit #03-061 with suspected positions of the
lateral (left) and medial (right) cannulas based on the end points of the marking
lesions. The outline below the cannulas denotes the approximate borders of the
interpositus nucleus (IP).
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.
Rabbit #04-029
The results of this animal for the paired and unpaired trials are summarized in
Figures 26 and 27, respectively. Overall, this animal learned the eyeblink response
very rapidly and meeting criterion for learning for all five days of training. The
animal reached 88.1% and 96.4% CRs for paired trials on the 4th and 5th day of
eyeblink training, respectively. The animal did not seem to learn the limb flexion
response (37 days of training) as rapidly as the eyeblink response, but did show
several days in which it met criterion for learning, namely days 20, 23, 24, 26,27,
29, and 37 with the last day demonstrating 35.7% CRs for paired trials. After
completion of the limb flexion training, the animal underwent 10 days of mixed
eyeblink and limb flexion training in which it met criterion for learning in all 10 days
for the eyeblink response and 5 out of 10 days for the limb flexion response (see
Figure 26).
Upon completion of mixed eyeblink and limb flexion training, the animal
underwent repeated muscimol infusions. However, attempts to eradicate the
eyeblink response through muscimol injections into the lateral (eye) cannula were
entirely unsuccessful even at high dosages of muscimol, such as 1.0 pL of 0.01 M
solution. In fact, when 1.0 pL of 0.01 M solution of muscimol was injected into the
lateral cannula, no apparent reduction in the eyeblink response was observed after
about one hour whereas the limb flexion response was almost completely abolished.
Thus, data was collected for muscimol infusions into the medial (limb) cannula only
for four separate sessions. For each of the four infusion sessions, injection of 0.15
70
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pL of 0.01M muscimol into the medial cannula had a very little observable effect on
the eyeblink response compared with a very significant reduction of the limb flexion
response. For example, for infusion sessions two and three, injection of 0.15 pL of
0.01 M muscimol into the medial cannula significantly reduced the limb flexion
response to 0% and 2.4% CRs (p=0.0000, p=0.0000; see Table 1) for paired trials
recovering to 57.1% and 50.0%, respectively, on the following day compared to
100% CRs after injection and 97.6% and 92.9% CRs (p=1.0000, 1.0000; no
significance), respectively, upon recovery for the eyeblink response. The amplitude
URs for the pair trials remained roughly the same on day testing compared to
recovery for the conditioned eyeblink response, whereas the amplitude URs for the
conditioned limb flexion response was consistently higher on the day of testing than
recovery for all four testing sessions (see Figure 26).
The results of the unpaired trials generally correlate with those of the paired
trials with a few exceptions. For example, the peak of the limb flexion CRs for the
paired trials appears to be 56.6% (day 20 of limb flexion training) compared to
66.7% (days 26, 27, and 29 of limb flexion training) for the unpaired trials. In
addition, the limb flexion response following one day of recovery from muscimol
appears much more robust for infusion sessions one and two for the impaired trials
than the paired trials. The animal demonstrated 11.9% and 0% CRs after one hour
injection of muscimol into the medial cannula for infusion sessions one and two
recovering to 56.1% and 57.1%, respectively, on the following day for paired trials.
On the other hand, for the unpaired trials, the CRs were observed to be 16.7% and
71
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0% (p=0.0007, 0.0000; see Table 2), respectively, after muscimol injection
recovering to 83.3% on the following day for infusion sessions one and two. In
addition, the amplitude URs for both the conditioned eyeblink and limb flexion
responses appeared higher on the day of muscimol testing compared to recovery (see
Figure 27).
Histological examination of the animal’s brain (Figures 28 and 29) revealed
that the medial and lateral cannulas were positioned correctly in the dorsal-ventral
plane, but were shifted rather medially. Based on the locations of the marking
lesions, a depiction of the positions of the medial and lateral cannulas is given in
Figure 30.
72
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Rabbit 04-029 - EYE B L IN K /L IW IB FLEX IO N STUDY (Paired Trials)
100
(/)
01
o
c
O
9 >
CL
52 MR MR MR MR 1 6 43
10
Eye Blink
8
E
g
i n 6
01
0
i>
1 4
"o.
1 2
0
1 6 43 52 MR MR MR MR
3
■8 10
3
D .
I 5
0
in in i n i i
Eye Blink Limb Flexion
A cquisition A cquisition
43
52 MR MR MR MR
M ixed Bye Blink/
Limb Flexion
Acquisition
Medlal(Llmb)
injection Only
DAYS OF TRAINING
Figure 26. Rabbit #04-029. Comparison plot of the percent CRs, CR amplitude,
and UR amplitude for eyeblink acquisition, limb flexion acquisition, mixed trials,
and muscimol (M) infusion and recovery (R) trials for the medial (limb) cannula for
paired trials.
73
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Rabbit 04-029 - EY E B L IN K /L IM B FLEXIO N STUDY (Unpaired Trials)
100
40
Q_
52 MR MR MR MR
43 1 6
10
Eye Blink
8
o -o Limb Flexion
E
m 6
O L
O
V
? 4
a
2
0
52 MR MR MR MR
6 43 1
r~- liln.i.
Eye Slink Limb Flexion
A cquisition A cquisition
M ixed Eye Blink/
Limb Flexion
Acquisition
MR MR MR MR
M edlal(Llmb)
Injectio n Only
DAYS OF TRAINING
Figure 27. Rabbit #04-029. Comparison plot o f the percent CRs, CR amplitude,
and UR amplitude for eyeblink acquisition, limb flexion acquisition, mixed trials,
and muscimol (M) infusion and recovery (R) trials for the medial (limb) cannula for
unpaired trials.
7 4
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Latvrah -:M c.liul 2 mm
Figure 28. Histological profile of rabbit #04-029. The left and right arrows
represent the marking lesions from the end points (injection sites) of the lateral (eye)
and medial (limb) cannulas, respectively. As previously mentioned, the positions of
both cannulas appear to be shifted rather medially. The outlines below the cannulas
denote the approximate borders of the dentate nucleus (DN; left) and interpositus
nucleus (IP; right).
75
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Figure 29. Histological profile of rabbit #04-029 magnified IX. The left and right
arrowheads represent the marking lesions from the end points (injection sites) of the
lateral (eye) and medial (limb) cannulas, respectively. The outline below the
cannulas denotes the approximate borders of the interpositus nucleus (IP).
76
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Figure 30. Histological profile of rabbit #04-029 with suspected positions of the
lateral (left) and medial (right) cannulas based on the end points of the marking
lesions. As previously mentioned, the positions of both cannulas appear to be shifted
rather medially. The outlines below the cannulas denote the approximate borders of
the dentate nucleus (DN; left) and interpositus nucleus (IP; right).
77
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Rabbit #04-049
The results for the paired and impaired trials for this animal are summarized
in Figures 31 and 32, respectively. This animal seemed to take more days than
normally observed to learn the eyeblink response (21 days), but did meet criterion
for learning on the last three days of eyeblink training demonstrating 76.5% CRs on
paired trials on the last day (day 21) of eyeblink training. On the other hand, the
animal appeared to leam the limb flexion response very rapidly (7 days with high
UR amplitudes) meeting criterion for learning on the last two days of training
showing 91.4% CRs for paired trials on the 7th day of limb flexion training. In
addition, the animal was trained for 9 days on mixed trials demonstrating
approximately 50-70% CRs for the paired conditioned eyeblink and limb flexion
responses (see Figure 31).
The animal was tested for the effects of muscimol on the conditioned
eyeblink response (0.1-0.3 pL of a 0.01 M solution) on three separate days and for
the conditioned limb flexion response (0.25-0.3 pL of a 0.01 M solution) on four
separate days (see Figure 31). On each day of muscimol injection into the lateral
cannula, the eyeblink response was significantly reduced (p=0.0000, 0.0000, 0.0000;
see Table 1) compared to the day of recovery in paired trials. However, the limb
flexion response also seemed to be significantly reduced (p=0.0000, 0.0000, 0.0000)
when muscimol injections were performed in the lateral cannula for paired trials.
Muscimol injections into the medial cannula significantly reduced the limb flexion
response on all four days of testing (p=0.0000, 0.0000, 0.0000, 0.0000 for paired
78
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trials), but appeared to affect the eyeblink response only on days 1 (p=0.0211) and 3
(p=0.0012) when compared to recovery on the following day (see Table 1).
Furthermore, the amplitude URs for the paired trials seemed to be slightly reduced
on the day of testing compared to recovery for the conditioned eyeblink response,
whereas for the conditioned limb flexion response, the amplitude URs for the day of
recovery were consistently lower than those for the day of testing (see Figure 31).
The results for the unpaired trials (Figure 32) are similar to those of the
paired trials. As seen in the paired trials, muscimol injections into the lateral seemed
to significantly reduce the conditioned eyeblink (except for day 1) and limb flexion
responses for the unpaired trials (see Table 2). However, examination of the
amplitude CRs for unpaired trials in Figure 32 reveals that muscimol injection into
the lateral cannula on day 2 did not appear to reduce the limb flexion response when
compared to day of recovery (5.4 mm compared to 3.6 mm). In addition, based on
the percent CRs for unpaired trials, muscimol injections into the medial cannula
demonstrated significant reduction of the conditioned limb flexion response of
testing (p=0.0014, 0.0014, 0.0007, 0.0007) with no apparent significant effect on the
conditioned eyeblink response (p=0.8906, 0.7368, 0.9645, 0.1094) on all four days
of testing. Finally, the amplitude URs for unpaired trials seemed to slightly alter
from day of testing to recovery for the conditioned eyeblink response and no change
for the conditioned limb flexion response (see Figure 32).
79
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Histological analysis of the animal’s brain (Figure 33 and 34) revealed that
both the lateral and medial cannulas were shifted rather laterally and were positioned
more on the posterior portion of the interpositus nucleus. Figure 35 gives a depiction
of the suspected positions of the lateral and medial cannulas based on the locations
of the marking lesions.
80
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Rabbit 04-049 - EY E B L IN K /L IM B FL EX IO N STUDY (Paired Trials)
100
80
MR MR MR MR 37 M R MR MR
29 22 1
Eye Blink ■
o -o Limb FlexionO
03
22 29
37 M R MR MR
1
MR MR MR MR
20
15
3 10
O - 5
22 29 37 M R MR MR MR MR MR MR
Eye Blink
A cquisition
M ediaJ(Umb)
Injection
Limb Flexion M ixed Eye Blink/ L ateral (Eye)
A cquisition Limb Flexion injection
Acquisition
DAYS OF TRAINING
Figure 31. Rabbit #04-049. Comparison plot of the percent CRs, CR amplitude,
and UR amplitude for eyeblink acquisition, limb flexion acquisition, mixed trials,
and muscimol (M) infusion and recovery (R) trials for the medial (limb) cannula for
paired trials.
81
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Rabbit 04-049 - EYE BLINK/LIMB FLEXION STUDY (Unpaired Trials)
T T ?
22 29 37 M R MR MR MR MR MR MR
Eye Blink ■
o -o Limb FlexionO
q . 2
1 22 29
37 MR MR MR MR MR MR MR
20
15
§10
E 5
<
22 29 37 MR MR MR MR MR MR MR
Eye Blink
A cquisition
M edial(Llmb)
Injection
Limb Flexion M ixed Eye Blink/ Lateral(Eye)
A cquisition Limb Flexion Injection
Acquisition
DAYS OF TRAINING
Figure 32. Rabbit #04-049. Comparison plot of the percent CRs, CR amplitude,
and UR amplitude for eyeblink acquisition, limb flexion acquisition, mixed trials,
and muscimol (M) infusion and recovery (R) trials for the medial (limb) cannula for
unpaired trials.
82
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Dorsal
l.atc-ral-
Vt'Hlral
Figure 33. Histological profile of rabbit #04-049. The left and right arrows
represent the marking lesions from the end points (injection sites) of the lateral (eye)
and medial (limb) cannulas, respectively. As mentioned in the text, both the lateral
and medial cannulas are shifted rather laterally and are positioned over the posterior
portion of the interpositus nucleus. The outlines below the cannulas denote the
approximate borders of the dentate nucleus (DN; left) and interpositus nucleus (IP;
right).
83
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Figure 34. Histological profile of rabbit #04-049 magnified IX. The left and right
arrowheads represent the marking lesions from the end points (injection sites) of the
lateral (eye) and medial (limb) cannulas, respectively. The outlines below the
cannulas denote the approximate borders of the dentate nucleus (DN; left) and
interpositus nucleus (IP; right).
84
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Figure 35. Histological profile of rabbit #04-049 with suspected positions of the
lateral (left) and medial (right) cannulas based on the end points of the marking
lesions. As mentioned in the text, both the lateral and medial cannulas are shifted
rather laterally and are positioned over the posterior portion of the interpositus
nucleus. The outlines below the cannulas denote the approximate borders of the
dentate nucleus (DN; left) and interpositus nucleus (IP; right).
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.
Figure 36. Histological reconstructions of the rabbit cerebellum sectioned coronally
at three separate positions in relation to the stereotaxic landmark lambda (A ,) in the
anterior-posterior axis, namely, at A (top), 0.5 mm anterior (middle), and 1.0 mm
anterior (bottom). Filled and unfilled circles represent the approximate muscimol
injections sites for the lateral (eyeblink) and medial (hindlimb flexion) cannulas,
respectively, for all six animals tested in this study.
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.
Effects of Muscimol Injections (Paired Trials)
In Lateral IP In Medial IP
Rabbit #
00-142
Eye p=0.0000 p=0.5685
Limb p=0.1062 p=0.0Q56
00-162
Eye p=0.0000 p=0.0000
Limb p=0.5989 p=0.0000
02-025
Eye NIP p=1.0000,0.0000
Limb p=0.0000,0.0000
03-061
Eye p=0.0000, 0.0000, 0.0000 p=0.0008, 1.0000,
1.0000, 1.0000
Limb p=0.0061,0.0000,0.0000 p=0.0000,0.0000,
0.0000, 0.0000
04-029
Eye NIP p=1.0000, 1.0000,
1.0000, 0.5851
Limb p=0.0000,0.0000,
0.0000, 0.0005
04-049
Eye p-0.0000, 0.0000, 0.0000 p=0.0211, 0.0667,
0.0012, 0.8613
Limb p=0.0000,0.0000,0.0000 p=0.0000,0.0000,
0.0000, 0.0000
Table 1. Cumulative probability (p) values for the effects of muscimol injections on
the eyeblink (Eye) versus hindlimb (Limb) flexion conditioned responses for paired
trials for all six animals employed in this study. Effects are considered to be
significant if p<0.05. Commas separate the p values for animals in which multiple
days of inactivations with muscimol were performed. IP=interpositus nucleus.
NIP=No infusions performed.
88
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Effects of Muscimol Injections (Unpaired Trials)
In Lateral IP In Medial IP
Rabbit #
00-142
Eye p=0.0000 p=0.6651
Limb p=0.7373 p=0.3512
00-162
Eye p=0.0007 p=1.0000
Limb p=0.6651 p=0.0007
02-025
Eye NIP p=1.0000,0.0033
Limb p=0.0000,0.0087
03-061
Eye p=0.0002, 0.0000, 0.0087 p = l.0000, 1.0000,
1.0000, 1.0000
Limb p=0.0878,0.9844,0.0878 p=0.0178,0.0007,
0.0014, 0.0001
04-029
Eye NIP p=1.0000, 1.0000,
1.0000, 1.0000
Limb p=0.0007,0.0000,
0.0878, 0.0014
04-049
Eye p=0.3512, 0.0156, 0.0156 p-0.8906, 0.7368,
0.9645, 0.1094
Limb p=0.0178,0.0087,0.0000 p=0.0014,0.0014,
0.0007, 0.0007
Table 2. Cumulative probability (p) values for the effects o f muscimol injections on
the eyeblink (Eye) versus hindlimb (Limb) flexion conditioned responses for
unpaired trials for all six animals employed in this study. Effects are considered to
be significant if p<0.05. Commas separate the p values for animals in which
multiple days of inactivations with muscimol were performed. IP=interpositus
nucleus. NIP=No infusions performed.
89
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Contrast Table
Paired Trials
Hveblink Response Limb Flexion Response
Lateral Injection -1 1
Medial Injection 1 -1
Unpaired Trials
Lateral Injection -1 1
Medial Injection 1 -1
Table 3. Contrast table displaying the constants that are multiplied by the
corresponding cumulative probabilities (p) and added together to yield a number that
represents an index of double dissociation. This double dissociation index will be
close to zero (0.0000) if there is no double dissociation and becomes +2.0000 with a
perfect double dissociation.
90
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Paired Trials
Double Dissociation Index
Eveblink Response_________Limb Flexion Response
Rabbit #
00-142
Lateral Injection 0.0000 0.1062
Medial Injection 0.5685 0.0056
Total: 0.6691
00-162
Lateral Injection 0.0000 0.5989
Medial Injection 0.0000 0.0000
Total: 0.5989
Unpaired Trials
00-142
Lateral Injection 0.0000 0.7373
Medial Injection 0.6651 0.3512
Total: 1.0512
00-162
Lateral Injection 0.0007 0.6651
Medial Injection 1.0000 0.0007
Total: 1.6637
Table 4. Double dissociation index (i.e., total values) for the paired and unpaired
trials for the two animals in this study that demonstrated an apparent double
dissociation between the conditioned eyeblink versus limb flexion responses. As
mentioned in the text, this index is close to zero (0.0000) when there is no double
dissociation and becomes +2.0000 when there is perfect double dissociation.
91
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CHAPTER 4
DISCUSSION
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Generally speaking, the results gathered from this study suggest that the
conditioned eyeblink and limb flexion responses can be selectively abolished or
substantially impaired with discrete cerebellar interpositus injections of muscimol
and thereby lending further support for the existence of a somatotopic map for
learning for these two behaviors in the interpositus nucleus with the eyeblink
represented laterally and the hindlimb represented medially. In addition, the evidence
collected in the present study does not appear to support the null hypothesis, which
states that a somatotopic map for learning does not exist for the eyeblink and
hindlimb flexion responses in the interpositus nucleus.
Throughout the course of the study, it was found the animals had a greater
difficulty in consistently learning the limb flexion than the eyeblink response. In one
case (rabbit #03-061), over three months (98 days) of training was required for the
animal to demonstrate consistent limb flexion responses. Several adjustments were
made to the parameters of the limb flexion paradigm to improve the ability of the
animal in displaying consistent responses. For example, initially the interstimulus
interval (ISI) for both the eyeblink and limb flexion training trials was 250 msec that
is normally used in eyeblink conditioning studies. However, we realized that since
the distance in which the signal has to travel from the rabbit’s hindlimb muscles to
give a flexion after hearing the CS (tone) is significantly longer than that for the
eyeblink response, 250 msec is not a sufficient time for the rabbit to react to the CS
before receiving the US (hindlimb shock). Thus, we increased the ISI interval for
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both the eyeblink and limb flexion paradigms to 1500 msec (1.5 sec) and observed
improved performance for the limb flexion response.
In addition, in the beginning of the study, we employed a classical
conditioning paradigm for both eyeblink and limb flexion training. In the limb
flexion conditioning paradigm, the animal is continually administered an aversive
stimulus (shock) to the hindlimb even if it decides to flex its limb. Therefore, we
decided to modify the limb flexion training sessions to classical conditioning
paradigm with an operant contingency (Kamin, 1956) in which the CS (tone) and US
(hindlimb shock) would simultaneously terminate if the rabbit displayed a CR (limb
flexion) in the ISI. In this case, the animal is rewarded for showing a CR in the ISI,
but unlike a true operant conditioning paradigm, the animal still receives a shock to
the hindlimb if it does not show a CR in the ISI. We observed that altering the limb
flexion training sessions to a classical conditioning paradigm with an operant
contingency improved the performance of the animals in the limb flexion response.
It is not entirely clear why the animals had such greater difficulty in learning
the limb flexion response. One explanation for the discrepancy between the learning
of the two responses might be that it might just simply be physically easier for the
animal to display an eyeblink rather than flex its hindlimb. It could be that during
the course of limb flexion training, the animal is suffering from physical fatigue of
the hindlimb muscles and thus even though it has learned flexion response, the
muscular fatigue is preventing it from flexing the hindlimb. Moreover, there is the
possibility of the evolutionary argument. Since rabbits primarily use their hindlimbs
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together for hopping, which does not require tremendous dexterity or fine motor
control as in say arm or hand movements, it can be imagined that the projections
from the hindlimb to the interpositus may not be as elaborate and in high number as
another part of the body, such as the forelimb of the cat. This would mean that the
somatotopic representation of the rabbit hindlimb in the interpositus nucleus might
not be very expansive, which would hamper the potential creation of associations
between the CS (tone) and US (hindlimb shock) in the interpositus nucleus.
In fact, when we attempted to administer the shock US to the rabbit left
forelimb, we found that the animal displayed a small number of URs with low
amplitudes in the first few trials of the limb flexion training session, which quickly
reduced to very low levels of response after a small number of successive trials.
Even when a 5 mA shock was administered to the left forelimb, the amplitude of the
URs rapidly habituated to a very low level after approximately 20 trials of the
training session. In the initial stages of the study, the shock electrodes were placed
on the bottom of the footpad and later on the ankle to examine whether any
improvements in learning the flexion response would occur. Instead, we observed
that as in the forelimb shock administration, the animal displayed very few flexion
responses with relatively low amplitudes that quickly habituated to baseline levels.
The optimal placement for the shock electrodes seemed to be on the upper portion of
the rabbit left hindlimb.
In their study, Donegan et al. (1983) measured conditioned flexion responses
based on electromyography (EMG) activity and reported an increase in activity to
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the tone CS in both hindlimbs of the rabbit. We attempted to record EMG activity
from the rabbit left hindlimb as a measure of flexion responses in the present study,
but were unsuccessful in observing any significant increase in EMG activity to the
tone CS during the limb flexion training trials. It would be interesting to know in
future studies whether the conditioned flexion responses we observed in the rabbit
left hindlimb in the present study would in fact carry over to the right hindlimb
including the effects of reversible lesions with muscimol injections. In addition, in
the Donegan et al. (1983) study, lesions were carried out on the left cerebellar nuclei,
including the dentate and interpositus nucleus, and/or overlying cerebellar cortex,
which resulted in an abolition or substantial impairment of the limb flexion CRs in
both hindlimbs. However, the muscimol lesions performed in the present study were
reversible and thus allowed for lesions to be carried out a multiple number of times
in the same animal to abolish or significantly reduce the conditioned flexion
response repeatedly. Also, unlike the Donegan et al. (1983) study, we attempted to
prevent the cerebellar lesions to include the overlying cerebellar cortex (particularly
the HVI lobule) and therefore attempted to infuse the minimum amount of muscimol
(normally around 0.1 pL of a 0.01 M solution) into the interpositus nucleus to
eradicate the conditioned eyeblink or limb flexion response without spreading into
the overlying cerebellar cortex.
The findings of the present study are similar to those reported by Bracha et al.
(1999) in that conditioned eyeblink and hindlimb flexion responses appear to be
localized in the interpositus nucleus. In their study, Bracha et al. (1999) examined
R eproduced with perm ission of the copyright owner. Further reproduction prohibited without perm ission.
classically conditioned withdrawal responses in three separate effector systems,
namely, the eyelid, forelimb, and hindlimb, through reversible microinjections of
muscimol into the ipsilateral interpositus nucleus of the cat. However, unlike the
Bracha et al. (1999) study that did not suggest a somatotopic organization of the
conditioned limb flexion and eyeblink responses in the cat interpositus nucleus, we
presently reveal the corresponding topographical map for these two learned
responses in the interpositus nucleus of the rabbit. In other words, we were able to
demonstrate that infusions of low concentrations of muscimol into two distinct
locations of the interpositus nucleus have separate effects (i.e., eradication or
significant reduction) on the conditioned eyeblink versus hindlimb flexion responses
in the same animal.
To measure the degree in which muscimol injections were able to eradicate
or significantly reduce either the conditioned eyeblink or limb flexion response
without significant effect on the other learned response, we introduced the concept of
the double dissociation index. In instances where animals demonstrated clear
evidence of a double dissociation between the conditioned eyeblink versus limb
flexion responses, such as rabbit #00-142 and 00-162, we calculated the double
dissociation index for the paired and unpaired trials. This index appeared to be
considerably higher for the unpaired trials for both animals compared to the paired
trials. In fact, the index for the unpaired trials for rabbit #00-162 was 1.6637, which
suggests a very strong evidence of a double dissociation for this animal since an
index of 2.0000 represents a perfect double dissociation according to our scheme.
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The double dissociation index may therefore be employed in future studies as a
measure of the degree that two distinct behaviors are localized in two separate areas
of the brain.
Over the course of the study, we noticed that as the placements of the medial
and lateral cannulas became more accurate and better positioned in the appropriate
plane with respect to the interpositus nucleus, a lower dosage of muscimol would be
needed to eradicate a particular response (eyeblink or hindlimb flexion). Ideally, we
always began the infusions with the lowest possible dosage of muscimol (i.e., 0.1 jiL
of a 0.01 M solution), which Krupa and Thompson (1997) showed in their study to
be effective in eradicating the eyeblink response with minimal spread, particularly
into the overlying cerebellar cortex (e.g., HVI lobule). However, if this initial
dosage of muscimol was ineffective in eradicating or significantly reducing the CRs
in the targeted conditioned response, then we injected an increasingly higher dosage
of muscimol until a significant decrease in the conditioned response was observed.
Once we found that a particular dosage of muscimol was effective in abolishing the
targeted conditioned response, we continued to test the animal repeatedly at that
particular dosage, especially the animals examined in the latter part of this study (i.e.,
rabbit #03-061, #04-029, and #04-049).
For example, in the two preliminary animals examined, namely, rabbit #00-
142 and #00-162, it was necessary to inject a relatively high concentration of
muscimol, 1.0 M and 0.5 M, respectively, at the same volume (0.1 pL) to
significantly reduce either the eyeblink or limb flexion response. Histological
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examination of the brain of rabbit #00-142 revealed that both the medial and lateral
cannulas were positioned relatively high above their intended targets over the
interpositus nucleus and slightly skewed. In the case of rabbit #00-162, the histology
showed that again both cannulas, particularly the medial one, were positioned rather
high above their intended targets. With successive surgeries, the cannula placements
improved as seen in rabbit #02-025 in which infusion of 0.1 pL o f 0.01 M muscimol
into the medial cannula completely abolished eyeblink CRs with full recovery on the
following day. Histological examination of this animal’s brain demonstrated that the
medial and lateral cannulas were placed directly over their intended targets of the
interpositus nucleus and in the appropriate plane. In fact, animals after rabbit #02-
025 were injected with 0.1-0.3 pL of only a 0.01 M solution of muscimol with
relative success of abolishing one response without affecting the other, which
signified that the placements of the medial and lateral cannulas were becoming more
and more accurate.
Histological examination of the brains of rabbits #03-061 and #04-029
revealed the positions of the medial and lateral cannulas were relatively accurate.
Still, the lateral cannula for rabbit #04-029 appeared to be positioned too medially,
which may explain why even when a relatively high dosage of muscimol (1.0 pL of
a 0.01 M solution) was infused into the medial cannula, the eyeblink response was
not significantly reduced whereas the limb flexion response was. It is most likely the
case that the muscimol injected into the lateral cannula diffused into the limb flexion
region of the interpositus nucleus and thus did not affect the eyeblink response. In
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addition, in the case of rabbit #04-049, histological examination of the brain
demonstrated that the both the lateral and medial cannulas were positioned rather
laterally and over the posterior portion of the interpositus nucleus. Previous studies
(reviewed in Lockard, 1999) have suggested that a somatotopy for the limb flexion
response exists in the posterior portion of the interpositus nucleus. Thus, it would be
interesting to know in future experiments whether a somatotopy for the conditioned
limb flexion response exists in the posterior portion of the interpositus nucleus.
In future studies, additional animals are needed to further support the notion
for the existence of a somatotopic map for learning in the cerebellar interpositus
nucleus. With an increase in number of surgeries performed on the animals, it would
be expected that the positions of the medial and lateral cannulas would be expected
to be even closer to the respective limb flexion and eyeblink loci in the interpositus
nucleus. Simultaneous electrophysiological recording during implantation of the
medial and lateral cannulas during surgery would significantly aid the researcher in
identifying the exact location of the end of the cannula in relation to the brain, such
as the cerebellar cortex, underlying white matter, or the interpositus nucleus. In
addition, since during the surgeries the experimenter is relatively blind (other than
the knowledge of the stereotaxic coordinates) when inserting the cannulas into the
cerebellum, the use of imaging tools, such as functional magnetic resonance imaging
(fMRI), in future studies would greatly aid in the accurate placements of the medial
and lateral cannulas. In this situation, the experimenter would have a “live” image of
the inside of the brain during cannula insertions.
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The greater accuracy in the positions of the medial and lateral cannulas
would thus allow for smaller dosages (i.e., volume and concentration) of muscimol
to be injected into the interpositus nucleus without having the diffusion of this
compound into either the adjacent eyeblink or limb flexion loci or the overlying
cerebellar cortex, particularly the HVI lobule. Similar to the Krupa and Thompson
(1997) study, infusions of very small concentrations of muscimol, such as the 0.1 pL
of 0.01 M solution that was used on the latter animals in the present study, into the
interpositus nucleus would prevent the diffusion of the drug into the overlying
cerebellar cortex and therefore eliminate the involvement of this area in the eyeblink
and limb flexion response.
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Asset Metadata

Creator Mojtahedian, Shahriar (author)

Core Title Effects of muscimol injections on conditioned eyeblink versus limb flexion responses in the rabbit cerebellar interpositus nucleus

School Graduate School

Degree Doctor of Philosophy

Degree Program Neuroscience

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, neuroscience,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c16-410659

Unique identifier UC11340224

Identifier 3145248.pdf (filename),usctheses-c16-410659 (legacy record id)

Legacy Identifier 3145248.pdf

Dmrecord 410659

Document Type Dissertation

Rights Mojtahedian, Shahriar

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

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