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Department of Biomedical Sciences, Iowa State University, Ames, Iowa 50011
Submitted 3 September 2003; accepted in final form 15 October 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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-aminobutyric acid-A (GABAA) agonists and antagonists affects the performance of classically conditioned eyeblinks, the effects of these drugs on IN neurons in vivo are not known. The purpose of the present study was to measure the effects of muscimol and picrotoxin on the expression of conditioned eyeblinks and the activity of IN cells simultaneously. Injections of muscimol abolished conditioned responses and either silenced or diminished the activity of IN cells. Two injections were administered in each picrotoxin experiment. The first injection of picrotoxin slightly modified the timing and amplitude of the eyeblink, produced mild tonic eyelid closure, increased tonic activity of IN cells, and reduced the amplitude of the neural responses. The second injection of picrotoxin abolished conditioned responses, further increased tonic eyelid closure, dramatically elevated the tonic activity of IN cells, and in most cases, abolished neuronal responses. These results demonstrate that both GABAA-mediated inactivation and tonic up-regulation of IN cells can interrupt the expression of conditioned eyeblinks and that this behavioral effect is accompanied by the suppression of the neuronal activity correlates of the conditioned stimulus and response. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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; Lincoln et al. 1982; Ramnani and Yeo 1996; Yeo et al. 1985) as well as the expression of previously learned responses (Bracha et al. 1994; Chapman et al. 1990; McCormick et al. 1982; Welsh and Harvey 1991). The precise role of the IN in conditioned response acquisition and retention is not clear. A number of recent studies examining IN involvement in eyeblink conditioning utilized microinjections of drugs to locally affect the functionality of the IN. Interpretations of these neuropharmacological studies, however, are limited, because the effects of the drugs on the activity of IN cells were not directly measured.
It is known that the IN receive two main inputs. The first is a relatively weak glutamatergic input from cerebellar afferents— collaterals of mossy and climbing fibers. Although this input plays a prominent role in some of the current hypotheses of cerebellar involvement in eyeblink conditioning (Medina et al. 2000; Steinmetz 2000; Thompson 1986; Thompson and Kim 1996), its precise function is unknown since it has not been thoroughly investigated. The second is the strong GABAergic input from the cerebellar cortex (Ito 1984). It is known that applying -aminobutyric acid-A (GABAA) agonists in the IN blocks the acquisition and expression of eyeblink CRs (Bracha et al. 1994; Krupa et al. 1993). This dramatic effect is most likely associated with the substantial inactivation of IN cells. On the other hand, injections of GABAA antagonists have varying reported effects including small effects on CR expression (Bracha et al. 2001), effects on CR timing and amplitude (Bao et al. 2002; Garcia and Mauk 1998), or abolishing CR expression (Bao et al. 2002; Mamounas et al. 1987). Based on the available behavioral data, several studies have made specific predictions about the expected effects of injected drugs on IN activity. For example, Garcia and Mauk (1998), who reported that relatively small doses of picrotoxin applied to the IN decrease the latency of CRs, predicted that the main effect of blocking the chloride channels of IN neurons would be shortening the latency of their response to the CS. Based on this assumption, it was proposed that the major role of the cerebellar cortex is to regulate the timing of the CR (Garcia and Mauk 1998). In our study which examined the effects of picrotoxin injections in the IN on tonic eyelid position, we predicted that manipulations of GABAA neurotransmission affect not only the modulation of IN neural activity but also its tonic level. This speculation was used in supporting the hybrid hypothesis of cerebellar function which proposes that both tonic and phasic components of IN activity are relevant to eyeblink circuits (Bracha et al. 2001).
To test the predictions of the timing and hybrid hypotheses, we designed an experiment in which we directly measured the effects of locally applied GABA antagonist picrotoxin on the expression of eyeblink CRs and IN neuronal activity simultaneously. For that purpose, we developed a micromanipulator/guide tube system that permits stable recording of extracellular single-unit activity close to the site of drug injection. Rabbits were implanted with guide tubes and electrodes and were classically conditioned. Trained animals were injected with muscimol to verify the functional relationship between the injection site and the expression of conditioned eyeblinks. Rabbits in which the inactivation of the IN abolished CR expression were then injected with picrotoxin, and its effects on the animal's behavior and IN activity were measured simultaneously.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Surgery
Animals were anesthetized with a mixture of ketamine (50 mg/kg), xylazine (6 mg/kg), and acepromazine (1.5 mg/kg). Skull anchor screws, together with an assembly of three guide tubes that was aimed 0.5 mm dorsal to the left cerebellar interposed nuclei, were implanted using sterile surgical procedures. The guide tube assembly consisted of three parallel 26-gauge stainless steel tubes arranged in an equilateral triangle with a distance of 0.8 mm between them. Two of the guide tubes contained a bundle of five microwires (a total of 10 microwires, stainless steel, 18 µm diameter, Formvar insulation). The microwire bundles were attached to a permanently implanted micromanipulator that permitted vertical adjustments of their position. The microwires were connected to a small 15-pin connector that was embedded in dental acrylic. The third guide tube was used for microinjections of drugs. The patency of the injection needle cannula was protected by a 33-gauge stainless steel stylet. During surgery, lambda was positioned 1.5 mm below bregma and the stereotaxic coordinates were as follows: AP = 0.2–1.2 mm rostral to lambda, ML = 5.2 mm from midline, and DV = 14.7 mm ventral to lambda. A small Delrin block designed to accommodate the air tube required for air-puff stimulation and the device used to measure the movement of the upper eyelid during experiments in addition to the rest of the implanted hardware was attached to the skull using anchoring screws and dental cement. All animals were treated with antibiotics for 1 week following surgery.
Training procedures
After surgery, rabbits were adapted to the restraining box and to the experimental environment for 3 days, 30 min per day. Following adaptation, rabbits were conditioned in the standard delay paradigm using a 100-ms air puff (210 kPa at the source) applied to the left eye as the unconditioned stimulus (US) and a 450-ms, 80-dB SPL, 1-kHz tone as the CS superimposed on 65 dB SPL of white noise. The interstimulus interval was 350 ms, and the intertrial interval varied pseudorandomly between 17 and 23 s. All animals were trained until they produced CRs in more than 90% of the trials for three consecutive days. The movement of the eyelids was not restricted. Although conditioned responses in this task could not affect the incidence of delivery of the US, they could modify the perception of the US by the animal. Strictly speaking, this paradigm does not conform to all of the common criteria of a classical conditioning paradigm (Gormezano et al. 1983). With this caveat in mind, the described task will still be referred to as a classical conditioning paradigm.
Injection and recording experiments
All trained rabbits were tested in experiments in which the cerebellar interposed nuclei was microinjected with solutions of either muscimol (ICN Biochemicals, 3.5 nmol/µl) or picrotoxin (PTX) (Sigma, 0.4 or 2.5 nmol/µl), and the effects of these drugs on eyeblinks as well as on the activity of IN neurons were monitored. Both drugs were dissolved in artificial cerebral spinal fluid (ACSF). All injections were delivered through a 33-gauge stainless steel needle inserted in the implanted guiding tube. The injection needle was connected to a 10-µl Hamilton syringe using transparent Tygon tubing. The injected volume was measured by observing the movement of a small air bubble relative to gradation marks on the tubing connecting the needle to the syringe. Most of the drug injections were performed with the injection needle tip flush with the end of the guide tube. Only one drug was injected on any given experimental day. The main objective of muscimol injections was to functionally test whether the guide tube for the injection needle was implanted in the IN region related to CR control. Based on previous studies (e.g., Bracha et al. 1994), it was expected that muscimol will abolish CRs in successfully implanted animals. During muscimol experiments, a single 0.5 µl injection was applied after 40 baseline trials. In each picrotoxin experiment, there were two 0.5 µl injections applied at a rate of 0.05 µl/min. These injections divided each experiment into three blocks of trials— 40 trials before any injections (baseline period), 80 trials following the first injection, and an additional 80 trials following the second injection. Equal volumes of vehicle (ACSF) were injected following the same procedure in control experiments.
Recording single-unit activity in the vicinity of a pressure-injected drug presents a technological problem since an experiment of this type requires long-term (approximately 2 h) maintenance of the same set of recorded units. The stability of the recorded units depends on the extent that the electrodes drift relative to the recorded neurons. To maximize signal stability, adjustments of the position of the electrodes were made 1 day prior to injection experiments. All injection experiments were performed only on days when the signal was acceptable and did not require re-positioning of electrodes. To further increase recording stability, only small volumes of the drugs (0.5 µl) were injected at a very slow rate to minimize tissue displacement caused by the injected volume of fluid. Despite these precautions, occasionally, the recorded set of cells could not be maintained throughout the experiment, as evidenced by a sudden disappearance of the signal or by a change in the shape of the recorded action potentials. In this study, we present data only from the experiments which yielded stable recording. The first injections in each animal were functional muscimol tests. In the muscimol injection experiments, no efforts were made to find and record cells that would respond in the CS-US interval. Picrotoxin injection experiments were initiated in animals in which 0.5 µl of muscimol completely blocked CR expression. Prior to picrotoxin experiments, the electrodes were advanced to positions yielding cells that responded in the CS-US interval. It should be noted that this sampling strategy in PTX experiments most likely resulted in over-representation of the CS- and CR-related neurons in the IN. Once a set of neurons was recorded successfully during picrotoxin and/or control experiments, the electrodes were re-positioned to search for a new set of IN cells. Consequently, several sets of cells were recorded in the vicinity of each injection site.
Data recording and analysis
The eyeblink was recorded by monitoring the upper eyelid position with an electro-magnetic lever system (Bracha et al. 2001). Eyeblink responses were considered CRs when they exceeded the mean of the signal before CS onset by more than seven SDs of the noise (approximately 0.25 mm). Spontaneous responses were defined as trials in which the difference between the maximum and minimum values in the baseline period before the onset of the CS exceeded 1 mm. Spontaneous responses were stored but were discarded from further data analysis. For each experiment, CR incidence, CR onset, and CR amplitude were calculated for the periods before and after the injection.
The multiple single-unit signals from the microwires were fed through a custom miniature 14-channel FET-based preamplifier to a multi-channel differential amplifier system (Grass-Telefactor model 12 Neurodata System). The amplified and band-pass-filtered (300 Hz-3 kHz) signal was digitized (25 kHz/channel) using a custom data acquisition system, displayed, and stored in 1400-ms epochs corresponding to individual trials. Unit discrimination was performed off-line using threshold detection followed by a cluster analysis of scatter plots of time and amplitude distances between the peak and valley of individual action potential wave shapes. Up to three units could be discriminated on each channel. The discriminated data were processed using custom software in addition to a commercial data analysis pack (NeuroExplorer, Nex Technologies). Raster and peri-event histograms were constructed for each unit and experiment. Separate histograms were constructed for baseline trials, trials after the first injection, and trials after the second injection. In each histogram, the baseline firing rate (250 ms before CS onset) and the time occurrence of significant excitatory and inhibitory changes were computed. Cell responses were considered significant if the modulation of the firing rate in the post-CS period exceeded the mean baseline ± tolerance limit for two consecutive 10-ms bins. The factor for the tolerance limit was computed based on the statistical assumption that 99% of observations should not exceed the tolerance limit with a probability of 0.95. Individual normalized cell histograms (spike frequency in Hz) were pooled together for each cell type, drug, and period of time to construct average population histograms. Baseline means of individual cell histograms were pooled together for each type of drug, concentration, and part of the experiment and statistically analyzed. Also, all histograms were binarized, with the logical one representing bins significantly exceeding the baseline rate. The binarized histograms were summarized to construct modulation frequency histograms depicting the relative number of cells showing significant excitatory and inhibitory modulation at each given time during the trial.
The statistical analysis of individual parameters of eyeblink responses, of the baseline firing of IN neurons, and of population histograms, was conducted using two factorial ANOVA with the following factors: drug (2 levels, PTX and ACSF) and time/dose (3 levels, before injection, after the first injection, and after the second injection). A separate analysis was performed on the results of the muscimol injection experiments which included only one injection. ANOVA was followed by Newman-Keuls post-hoc analysis. All of the significant data in RESULTS are based on the post-hoc analysis unless specified otherwise. The provided numerical results are group means ± SE. Also, all significant effects of PTX were significant when compared to both the preinjection level and the corresponding time period in the control experiment, unless specified otherwise. All statistical analyses were performed using Statsoft Statistica (a commercial analytical software package). The level for the rejection of the null hypotheses was set to P < 0.05 in all tests.
Histology
After completion of the experiments, animals were deeply anesthetized, and the injection sites were marked by injecting 1 µl of tissue marking dye. The location of electrodes was marked by passing 10 µA anodal DC current through each wire for 20 s. The animals were transcardially perfused with 1 L of phosphate-buffered saline followed by 1 L of fixative (10% buffered formalin) and 1 L 10% potassium ferrocyanide in 10% formalin. The perfused brains were removed from the skull and, following a postfixation in 30% sucrose formalin, sectioned coronally at 50 µm on a freezing microtome. Sections were mounted onto gelatin-coated slides, reacted with ferrocyanide hydrochloride to visualize electrode-recording sites, and then stained with neutral red. The location of the injection sites as well as electrodes were determined and transferred to a standardized set of 0.5-mm separated coronal sections of the rabbit cerebellum.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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All of the effects of the picrotoxin injections reported in this paper were observed following PTX infusion at sites at which a previous infusion of 0.5 µl of muscimol (1.75 nmol = 200 ng) abolished the expression of classically conditioned eyeblinks (Fig. 2A). In general, the effects of PTX on CR expression were dose-dependent. In preliminary experiments, we tested the effects of injecting two 0.5 µl injections of PTX, 0.2 nmol each. In these experiments, both the behavioral and the electrophysiological effects of this PTX dose were sporadic and marginal. More pronounced behavioral and electrophysiological effects were observed when 1.25 nmol (750 ng) of PTX in 0.5 µl ACSF was injected. The first microinjection (1.25 nmol of PTX) decreased CR latency, had the tendency to decrease the relative amplitude of CRs, slightly decreased CR incidence, and also slightly increased tonic eyelid closure. The second injection of PTX (an additional 1.25 nmol) increased CR latency to the preinjection level, increased tonic eyelid closure, and eventually abolished CRs (Fig. 3).
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The present experiments were performed in conditions which minimized head and eyelid restraint. This permitted the unobstructed observation of several behavioral side effects of the picrotoxin injections. Besides effects on the eyeblink reflex, picrotoxin infusions in the IN produced several types of behavioral anomalies in most of the rabbits. The following picrotoxin side effects were observed: asymmetry of the facial musculature that was mostly expressed as tonic flexion of the upper lip on the side ipsilateral to the air puff (47% of cases). This effect was often accompanied by an apparent protrusion of the contralateral eyeball (20% of cases). Picrotoxin infusions were followed by head rotation along the rostro-caudal axis, to the right side (away from the injected side) (100% of cases). These frequencies of behavioral PTX side effects were observed following the second PTX injection. Interestingly, most of the picrotoxin-induced behavioral changes were contingent on the restraint of the animal. The above behaviors, with the exception of the contraction of the ipsilateral lip, either diminished or could not be detected immediately on the removal of the rabbit from the restraining box.
Effects of muscimol and picrotoxin injections on the activity of cells in the IN
Eighty cells were recorded during functional muscimol tests. Activating GABAA receptors using muscimol abolished behavioral CRs and dramatically diminished the firing rate of IN cells (Fig. 2, B and C). The baseline firing rate of the recorded population of neurons was 20.8 ± 1.9 Hz (n = 80) before the injections and significantly decreased to 6.2 ± 1.1 Hz following the application of muscimol. This dramatic decrease of the firing rate was accompanied by almost the complete suppression of neuronal responses in the CS-US interval.
During PTX experiments, 162 cells were recorded from the deep cerebellar nuclei. The locations of the recording sites are shown in Fig. 1. Based on the firing properties of these cells, they were subdivided into four groups. Among the responding cells, the most common were medium-frequency cells (MFC) with excitatory responses (n = 55, Fig. 5A). The MFC cells had baseline firing frequencies of approximately 20 Hz, and they exhibited large excitatory (frequently >100 Hz) responses in the CS-US interval. This excitatory response was often followed by an inhibition that started approximately 50 ms following US onset. In the second largest responding cell group were low frequency cells (LFC) that exhibited mostly excitatory responses (n = 32, Fig. 5D). LFC cells had low baseline firing rates (2–10 Hz), and their peak excitatory responses reached 
60 Hz. In the third group of significantly modulated neurons were high-frequency cells (HFC) with inhibitory responses (n = 14, Fig. 5G). HFC cells typically had a baseline activity of 25–55 Hz and exhibited a relatively small, short-latency excitatory response to the CS and a large inhibitory response to the US. The remainder of recorded cells did not exhibit significant firing modulation before PTX injections (n = 61, non-responding cells, NRC).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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Behavioral effects of muscimol and picrotoxin microinjections
As expected, inactivating the IN with muscimol abolished the expression of conditioned eyeblinks. This result confirms a number of previous reports (e.g., Bracha et al. 1994). Blocking chloride channels with picrotoxin had the three following major behavioral effects: 1) effects on eyeblink CR expression; 2) effects on the tonic position of the external eyelid; and 3) motor side effects on facial and head/neck musculature. The first two effects partly confirm previously reported results. The first picrotoxin injection decreased the latency of conditioned responses at the group level. Overall, the effects of PTX injections on CR latency resemble those previously reported (Bao et al. 2002; Garcia and Mauk 1998; Medina et al. 2001). Although muscimol injections in all cases included in this report abolished the expression of CRs, the effects of the first dose of picrotoxin injected at the same sites were surprisingly variable. In two rabbits, the decrease of CR latency was not observed. The source of this variability, that has not been reported previously, is not clear. It should be pointed out that the present study differs from previous experiments in several aspects. For example, we used a shorter inter-stimulus interval (350 vs. 450 ms). It cannot be excluded that the timing of CRs acquired with the shorter inter-stimulus interval is less sensitive to pharmacological manipulations of the IN. In the present study, the effects on CR latency were observed following injections of 1.25 nmol PTX. Although this amount of the drug is higher than in the study of Garcia and Mauk (1998) (who used 0.2–0.4 nmol PTX), it is lower than another study that reported more dramatic effects of PTX on CR latency (Medina et al. 2001; 2 nmol). Since the PTX dose used in the present study is in the range of previously reported effective amounts of the drug, it seems unlikely that the drug dose would be solely responsible for the relatively modest effects of PTX on CR latency in the present experiments. It seems more likely that the magnitude of the CR latency-decreasing effect of picrotoxin is related to a specific subpopulation of IN cells or to a specific PTX concentration at a relevant and so-far not precisely mapped part of the IN. The second injection of PTX initially increased CR latency to the preinjection level and eventually abolished CRs. This finding confirms previous reports of effects of high doses of PTX on CR expression (Bao et al. 2002; Mamounas et al. 1987). Besides the effects on conditioned eyeblinks, both PTX injections increased tonic eyelid closure. This finding supports our previous report (Bracha et al. 2001) and demonstrates that the output of the interposed nuclei controls both the phasic and the tonic components of eyelid movement. The several other side effects of PTX injections indicate that despite relatively small injected volumes, the drug spread to adjacent parts of the IN that are related to facial and head/neck effector systems.
Effects of muscimol and picrotoxin on IN neuronal activity
The deep cerebellar nuclei, including the interposed nuclei, receive massive GABAergic input from Purkinje cells of the cerebellar cortex (Ito 1984). Activation of this input using the GABAA agonist muscimol blocks the expression of classically conditioned eyeblinks in the rabbit (e.g., Bracha et al. 1994). Interestingly, blocking this input with the appropriate doses of the GABAA antagonists bicuculline (Mamounas et al. 1987) or picrotoxin (Bao et al. 2002; Mamounas et al. 1987; this paper) can block the expression of conditioned eyeblinks as well. In this study, we demonstrated that the expression of CRs can be abolished either by silencing IN neurons or by their excessive activation. As expected, microinjections of muscimol suppressed the activity of IN cells and abolished neuronal responses in the CS-US interval, and these effects were associated with the disappearance of behavioral CRs. On the other hand, picrotoxin injections increased the tonic activity of interposed nuclear neurons in a dose-dependent manner and diminished the depth of their modulation, and this effect was accompanied by the eventual disappearance of behavioral CRs. Importantly, we did not observe the inactivation of IN cells (Freeman 1973) that was proposed as a putative mechanism for PTX-induced suppression of CRs (Garcia and Mauk 1998). It cannot be excluded that higher amounts of PTX, for example such as used by Bao et al. (2002) (10 nmol), would inactivate IN cells. This possibility is difficult to test, since the concentration of PTX utilized in the present study seems to represent the maximum solubility of PTX in ACSF. To deliver higher amounts of PTX, we would have to inject higher volumes of the drug. This, however, is impractical since injecting larger volumes destabilizes units recorded in the vicinity of the injection site. Pertinent to the mechanisms of CR abolition by PTX, the present amounts of PTX suppressed CRs without inactivating IN cells. These findings indicate that the behavioral expression of eyeblink CRs requires an optimal level of activity of eyeblink-related IN neurons. Extremely low or high levels of IN activity seem to block CR performance. Interestingly, the high spontaneous activity of IN cells observed following the second PTX injection correlated with significant diminishing or abolition of the firing modulation. Since changes in CR performance were associated with both the baseline firing rate and the firing pattern changes, it remains unknown which of the two or whether both of these parameters of IN activity caused the observed behavioral effects. It is possible that the modulation of IN activity participates in the generation of behavioral CRs (Berthier and Moore 1990; Jimenez-Diaz et al. 2002). If this is so, then the absence of the modulation following both muscimol and PTX would explain the suppression of conditioned eyeblinks. It is also possible that the tonic dysfunction of IN activity following both drugs rendered the extracerebellar circuits incapable of supporting CR expression. Dissociating between these two possibilities will require development of experimental approaches that can independently manipulate spontaneous activity and firing patterns of IN cells.
The mechanisms of the observed reduction of neural responding to the CS are not clear. Modulation of IN cell activity could conceivably be driven by GABAergic cerebellar cortical input or by presumably glutamatergic collaterals of mossy and climbing fibers. The disruption of IN activity modulation following picrotoxin injections is consistent with the notion that IN activity is driven by the cerebellar cortex, since blocking chloride channels would prevent IN cells from responding normally to GABA-mediated inputs. It is equally likely, however, that the reduced responding of IN cells to the CS was a consequence of picrotoxin-induced saturation of IN activity and that this rendered IN neurons unresponsive to extra-cerebellar inputs. The emergence of excitatory responses to the US (see Fig. 5, F and L) indicates that at least some of cells were not completely saturated and could respond to stimulation even after the second picrotoxin injection.
The picrotoxin-induced disinhibition of IN cells was dose dependent. Although the first injection of picrotoxin significantly elevated the spontaneous firing of IN neurons, their modulation was in most cases preserved, albeit diminished. Surprisingly, the latency of cellular responses did not change in parallel or in proportion with the decreased latency of CRs that was observed in these experiments. Consequently, changes in the timing of behavioral responses were not caused by corresponding shifts in timing of IN neuronal responses. This finding contradicts previous proposals that freeing the IN from cerebellar cortical inhibition uncovers short-latency IN responses to direct inputs from cerebellar afferents (Bao et al. 2002; Garcia and Mauk 1998). The preserved timing of the onset of IN responses following the first PTX injection indicates that the decrease of CR latency is more likely related to the general increase of the spontaneous firing rate of IN neurons. It is possible that the sustained increase of IN output produces excitability changes in cerebellar efferent targets and that this change in extracerebellar circuits shortens CR latency. If this process indeed takes place, it cannot be linear, since an additional increase in IN firing (following the second PTX injection) did not produce an additional decrease in CR latency. Instead, CRs were abolished.
The mechanisms leading to the dramatic elevation of tonic firing of IN cells following PTX injections is not clear. It is possible that blocking chloride channels reveals IN activation by presumably glutamatergic inputs from collaterals of mossy and climbing fibers. Another possibility could be that following PTX injections, IN cells express unopposed intrinsic pacemaker-like activity as suggested by Mouginot and Gahwiler (1996). The increase of tonic IN activity was closely associated with the tonic closure of the upper eyelid. This observation supports our previous suggestion (Bracha et al. 2001) that the output of the intermediate cerebellum plays a dual function when regulating brain stem eyeblink circuits. While the tonic component of IN activity is involved in the regulation of the tonic eyelid position, the phasic component contributes to the generation of CRs.
In summary, the present study demonstrated that the cerebellar cortex exerts a tonic inhibitory influence on IN cells. Activating GABAA receptors using muscimol silenced IN cells in the vicinity of the injection needle. Blocking chloride channels using PTX elevated IN baseline activity in a dose-dependent manner and at the same time, reduced and abolished CS-related modulation of IN cells. At its extremes, both inactivating and disinhibiting eyeblink-related parts of the IN resulted in the suppression of CR expression and produced changes in the tonic position of the eyelid. These findings support the notion that the IN participates in the control of both tonic and the phasic components of eyelid movements. These results also suggest that the behavioral expression of CRs depends on an optimal level of spontaneous IN activity and on the appropriate amplitude of IN modulation. The relative contribution of the tonic and phasic components of IN firing in CR expression remains to be resolved.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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This research was supported by National Institute of Neurological Disorders and Stroke Grants R01 NS-36210 and R01 NS-21958.
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FOOTNOTES |
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Address for reprint requests and other correspondence: V. Bracha, Department of Biomedical Sciences, 2032 Vet. Med., Iowa State University, Ames, IA 50011 (E-mail: vbracha{at}iastate.edu).
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REFERENCES |
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T. Ohyama, W. L. Nores, J. F. Medina, F. A. Riusech, and M. D. Mauk Learning-Induced Plasticity in Deep Cerebellar Nucleus J. Neurosci., December 6, 2006; 26(49): 12656 - 12663. [Abstract] [Full Text] [PDF]
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F.-P. Chen and C. Evinger Cerebellar Modulation of Trigeminal Reflex Blinks: Interpositus Neurons J. Neurosci., October 11, 2006; 26(41): 10569 - 10576. [Abstract] [Full Text] [PDF]
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M. Gerwig, K. Hajjar, A. Dimitrova, M. Maschke, F. P. Kolb, M. Frings, A. F. Thilmann, M. Forsting, H. C. Diener, and D. Timmann Timing of Conditioned Eyeblink Responses Is Impaired in Cerebellar Patients J. Neurosci., April 13, 2005; 25(15): 3919 - 3931. [Abstract] [Full Text] [PDF]
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D. P. Aksenov, N. A. Serdyukova, J. R. Bloedel, and V. Bracha Glutamate Neurotransmission in the Cerebellar Interposed Nuclei: Involvement in Classically Conditioned Eyeblinks and Neuronal Activity J Neurophysiol, January 1, 2005; 93(1): 44 - 52. [Abstract] [Full Text] [PDF]
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