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Volume 17, Number 1, Issue of January 1, 1997 pp. 70-82
Copyright ©1997 Society for NeurosciencePeptidergic Modulation of Intrathalamic Circuit Activity In Vitro: Actions of Cholecystokinin
Charles L. Cox, John R. Huguenard, and David A. Prince Department of Neurology and Neurological Sciences, Stanford University Medical Center, Stanford, California 94305
ABSTRACT
Cholecystokinin (CCK)-mediated actions on intrathalamic rhythmic activities were examined in an in vitro rat thalamic slice preparation. Single electrical stimuli in the thalamic reticular nucleus (nRt) evoked rhythmic activity (1-15 sec duration) in nRt and the adjacent ventrobasal nucleus (VB). Low CCK concentrations (20-50 nM) suppressed rhythmic oscillations in 43% of experiments but prolonged such activities in the remaining slices. Higher CCK concentrations (100-400 nM) had a predominantly antioscillatory effect.
Suppression of oscillations was associated with a relatively large membrane depolarization of nRt neurons that changed their firing mode from phasic (burst) to tonic (single-spike) output. This decreased burst discharge of nRt neurons during CCK application reduced inhibitory drive onto VB neurons from multiple peaked inhibitory postsynaptic currents (IPSCs) to single peaked inhibitory events. We hypothesize that suppression of inhibitory drive onto VB neurons decreases their probability of burst output, which, together with a reduction of nRt burst output, dampens the oscillatory activity.
Low CCK concentrations, which produced little or no depolarization of nRt neurons, did not alter the firing mode of the nRt neurons. However, the probability of burst output from nRt neurons in response to subthreshold stimuli was increased in low CCK concentrations, presumably leading to an increase in the number of nRt neurons participating in the rhythmic activity. Our findings suggest that the neuropeptide CCK, by altering the firing characteristics of nRt neurons, has powerful modulatory effects on intrathalamic rhythms; the ultimate action was dependent on CCK concentration and resting state of these cells.
Key words: cholecystokinin; thalamic reticular nucleus; oscillations; ventrobasal thalamus; neuropeptides; neuromodulation
INTRODUCTION
Rhythmic activities in thalamocortical circuits occur during sensory processing, slow-wave sleep, and pathophysiological conditions such as petit mal epilepsy (Williams, 1953
; Hubel, 1960
The firing mode of nRt neurons is important for the maintenance of thalamic rhythmic activity (Steriade and Deschênes, 1984
Cholecystokinin (CCK) is one of many neuropeptides localized within thalamic neurons (Hunt et al., 1987
Rat thalamic slices maintained in vitro possess sufficient connectivity to generate slow intrathalamic rhythms resembling those observed during slow-wave sleep and petit mal seizures (Huguenard and Prince, 1994
MATERIALS AND METHODS
General procedures were similar to those described previously (Cox et al., 1995a
Recording procedure. Whole-cell recordings were obtained using either blind (Blanton et al., 1989 when filled with the intracellular solutions below. The intracellular solution used in the majority of recordings contained (in mM): 117 K-gluconate, 13 KCl, 1.0 MgCl2, 0.07 CaCl2, 0.1 EGTA, 10.0 HEPES, 2.0 Na2-ATP, and 0.4 Na-GTP. In some experiments, Cs-gluconate and CsCl were substituted for K-gluconate and KCl, respectively. The pH of the solution was adjusted to 7.3 using KOH or CsOH, and osmolality was adjusted to 280 mOsm with distilled water.
Techniques used for obtaining recordings with the blind patch configuration were similar to those described earlier (Cox et al., 1995a
An Axoclamp2A amplifier (Axon Instruments, Foster City, CA) was used in bridge mode for voltage recordings and discontinuous single-electrode voltage-clamp mode for current recordings. In current-clamp recordings, an active bridge circuit was adjusted to balance the drop in potential produced by passing current through the recording electrode. In voltage-clamp recordings, switching frequencies ranged from 2.5 to 4 kHz with a gain of 150-800 pA/mV, and the headstage was monitored continually to ensure that the current transient had completely decayed before voltage measurements. Voltage-clamp recordings were limited to neurons that had a stable access resistance of <25 M
Extracellular multiple-unit activities were recorded using monopolar tungsten electrodes (0.2-2 M
Drugs. Concentrated stock solutions of CCK8S (1.0 mM) were prepared in 0.1 M NH4HCO3 and diluted in physiological saline to a final concentration of 0.01-10 µM. CCK8S was bath-applied in the majority of experiments. In some blind slice experiments, CCK8S was applied by injecting a bolus into the flow line of the chamber over 10-15 sec using a motorized pump. In these instances, based on the rate of CCK8S injection and the rate of chamber perfusion, the final bath concentration of CCK was estimated to be about one-fourth of the concentration introduced in the flow line (Cox et al., 1995a
Concentrated stock solutions of antagonists were diluted to a final concentration in physiological solution and applied by bath. CCK8S was purchased from Peninsula Laboratories (Belmont, CA), CNQX from Tocris Cookson (St. Louis, MO), and AP-5 from RBI (Natick, MA). CGP-35348 was a gift from CIBA-Geigy (Basel, Switzerland), and R( )-3-(2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid (d-CPPene) from Sandoz (Berne, Switzerland). All remaining compounds were purchased from Sigma (St. Louis, MO).
Analysis of intrathalamic oscillations. To quantify the degree of synchrony and the duration of the intrathalamic oscillations, autocorrelograms were constructed from the extracelluar multiple-unit data. A software Schmidt trigger was used to detect events, and the autocorrelograms were calculated over a period of 4-8 sec with a bin size of 8-10 ms. Autocorrelograms typically consisted of peaks of correlated phasic activity superimposed on random unit activity, as suggested by the nonzero counts in the interpeak valleys (Fig. 1A). We divided the functions into two components: the phasic oscillatory peaks (Fig. 1Bi) and the nonphasic random component that represented random unit activity (Fig. 1Bii). The random component was typically fit by a single exponential and could be subtracted from the overall auto correlogram. The oscillatory activity in the autocorrelograms was quantified by four measures: number of peaks; time constant of oscillation decay ( osc); oscillation amplitude (Amposc); and the period of oscillation. The
Fig. 1. Components of autocorrelograms. Schematic of representative autocorrelogram constructed from extracellular multiple-unit data. The plot consists of distinct peaks with nonzero valleys. The phasic synchronous oscillatory activity (Bi) was separated from the nonzero valleys, which represent nonphasic activity (Bii). The area under the valleys represents residual nonsynchronous discharge activity (Bii), and the decay of this activity (
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Fig. 9. CCK increases probability of burst discharge to subthreshold stimulation. A, Pre-drug, Extracelluar IC stimulation evokes a subthreshold EPSP in a nRt neuron. CCK, early, During the initial depolarization produced at the onset of CCK8S application (3 µM, 10 sec bolus), IC stimuli evoked burst discharge (truncated spikes). At the peak of the CCK depolarization (15 mV), the stimuli evoked single spike discharge (peak). After manual repolarization of the membrane potential to resting levels, the same stimuli that produced subthreshold responses in the control period (Pre-drug) evoked burst discharge (Vmatch). Dashed line, Vm =
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RESULTS
Intrathalamic oscillations
As shown previously, the reciprocal synaptic connectivity between nRt and VB is preserved in the in vitro thalamic preparation and supports intrathalamic rhythmic activities (Huguenard and Prince, 1994). A similar oscillatory activity that averaged 2.7 ± 0.3 Hz (n = 12 slices) could also be detected in extracellular multiple-unit recordings (e.g., Fig. 2C); these recordings were used to monitor rhythmic activities in large neuronal populations.
Fig. 2. Intrathalamic oscillations recorded in nRt and VB. A, Schematic of intrathalamic circuit in thalamic slice preparation. nRt provides inhibitory innervation of VB that in turn forms excitatory synapses on nRt cells. Monopolar stimuli were delivered to either nRt (SnRt) or internal capsule (SIC). Ctx, Cortex. B, Intracellular current-clamp recordings from nRt and VB neurons illustrating the rhythmic synaptic activity evoked by single extracellular stimuli (S). nRt, Responses in an nRt cell consist of rhythmic EPSPs (3.8 Hz) that can lead to burst discharge (asterisks). VB, Similar stimuli evoke rhythmic IPSPs (2.9 Hz) in a VB neuron (bottom trace). The top trace corresponds to a recording from a different VB neuron and illustrates that a burst discharge (
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In most slices used for extracellular recordings, the oscillatory response evoked in normal physiological solution lasted 1-2 cycles but was quite variable between slices. Likely factors in slice-to-slice variations in rhythmic activity were differences in the extent of functional connectivity between nRt and VB and in the mean resting potentials of the neurons. The strength and duration of the rhythmic activities could be increased by the addition of the GABAA-receptor antagonist bicuculline methiodide (BMI, Huguenard and Prince, 1994 
Fig. 4. Low CCK concentrations either suppress or facilitate intrathalamic rhythms. A, Suppression. i, Extracellular multiple-unit recordings in nRt in control solution (Pre-drug), during 20 nM CCK8S application, and after washout of the peptide. Stimulation of nRt (0.05 Hz) in control solution containing 0.2 mM Mg2+ and 10 µM BMI evokes three cycles of rhythmic discharge (2.2 Hz). Addition of 20 nM CCK attenuates the phasic response and increases background discharge and the duration of the initial response [CCK (20 nM)]. After washout of the peptide, the phasic response returns (Wash). Aii, Contour plot of same experiment. The ordinate represents the time course throughout the experiment (i.e., Pre-drug, CCK, Wash). The abscissa reflects the ratemeter output; darker levels correspond to higher firing frequencies. The phasic activity evoked by nRt stimulation (S) lasts for three cycles in the control condition (open bar, left). In the presence of CCK8S (solid bar), there is increased spike discharge that continues throughout the sampled period and a reduction in the distinct phasic cycles. During washout of the peptide (gray bar), the phasic activity returns toward baseline levels. The contour interval for this plot was 10 spikes. Note the decrease in phasic activity and increased discharge rate during CCK application. Aiii, Autocorrelograms of same experiment illustrating the overall increase in activity but loss of synchronized discharge in CCK (thick line). The overall increased amplitude and lack of distinguishable peaks is indicative of a nonphasic increase in spike discharge. B, Facilitation. i, In a different slice, stimulation of nRt evoked a phasic rhythmic discharge (2.8 Hz) in nRt lasting many seconds (Pre-drug). In this experiment, the extracellular perfusate contained 1 µM BMI and 0.2 mM Mg2+. CCK (50 nM), The duration of the rhythmic activity doubled and returned to baseline conditions after washout of CCK (Wash). Bii, Contour plot of the same experiment. This plot was constructed similar to Aii. Note the phasic activity evoked by the stimulus. During CCK application, the number of cycles significantly increased and returned to near baseline on washout. Biii, Autocorrelogram of the same experiment illustrates the synchronicity of the rhythmic activity. CCK (thick line) increased the amplitude as well as the number of peaks, indicative of the enhanced rhythmic activity.
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Fig. 5. High CCK concentrations suppress intrathalamic rhythms. Ai, Bi, Stimulation of nRt evokes rhythmic discharge (2.5 Hz) in both nRt (Ai) and VB (Bi). In CCK (400 nM), the rhythmic discharge in suppressed in both structures. In addition, there is an increased baseline discharge in nRt, consistent with a prolonged suprathreshold depolarization produced by the high concentration of CCK. The extracellular solution contained 10 µM BMI and 0.2 mM Mg2+. Aii, Bii, Contour plots illustrating the time course of the CCK effect in nRt (Aii) and VB (Bii). Four cycles are evoked in the pre-drug and wash periods. During CCK perfusion, continuous discharge occurs in nRt and a decrease in the number of cycles in VB. The contour interval for both plots was 20 spikes. See legend to Figure 4B for further details of plot. Aiii, Biii, Autocorrelograms of unit activity in nRt (Aiii) and VB (Biii) in control conditions (thin line) and during application of 400 nM CCK8S (thick line). Although the period of the remaining weak rhythmic activity (2 peaks) in this slice was slightly increased in CCK, there was not a significant change in the period for the population of slices (see Fig. 6Bii).
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Fig. 7. Pro- and antioscillatory actions of CCK8S on rhythmic EPSPs in nRt neurons. A, Intracellular recordings from an nRt neuron in which IC stimulation evokes rhythmic EPSPs (Pre-drug) that can lead to burst discharge (truncated spikes). The extracellular solution contained standard [Mg2+] (2 mM) and 10 µM BMI. After manual repolarization of the membrane potential during the peak of a CCK8S (25 nM, bath-applied)-induced depolarization (5 mV), the same stimulus evokes an increased number of rhythmic EPSPs [CCK (Vmatch)]. The enhanced rhythmic EPSPs persisted during washout of the peptide even as the membrane potential returned to within 2 mV of the control membrane potential (Post CCK). After complete washout of the peptide (5 min), the evoked response returned to baseline conditions (Wash). Vm (dashed line) =
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Low BMI concentrations (1-10 µM) enhanced the 2-5 Hz network oscillation (Figs. 2C, 3A; Huguenard and Prince, 1994 
Fig. 3. Duration and synchronicity of intrathalamic oscillations are increased by GABAA antagonists and suppressed by GABAB antagonists. A, Simultaneous extracellular recordings of oscillatory activity in response to single extracellular stimulus in nRt (). In 2 µM BMI, the evoked rhythmic discharge lasts 7-8 sec in nRt and ~5 sec in VB. With increased BMI concentration of 10 µM, the oscillation duration and number of units within each cycle are increased in both nuclei. Addition of the GABAB antagonist CGP 35348 (400 µM) blocks the rhythmic activity. B, Autocorrelograms constructed from five consecutive episodes in each experimental group above. Note the increased numbers and amplitudes of peaks in the higher BMI concentration [compare thin black (2 µM) and gray (10 µM) lines]. In CGP 35348 (thick line), the peaks are nearly completely suppressed.
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CCK modulates intrathalamic oscillations
In 6 of 14 experiments, low CCK concentrations (20-50 nM) suppressed the rhythmic activity (Fig. 4A). Typically, the baseline spike frequency was increased in nRt during CCK application, and subsequent stimulation evoked a robust discharge that lacked the phasic pattern observed in control conditions (Fig. 4Ai). Contour plots (Fig. 4Aii,Bii) illustrate the stability of the control response and the persistence and reversibility of the CCK-mediated effects. In this slice, three cycles of oscillation were obtained consistently. CCK (20 nM) reversibly increased spontaneous firing and suppressed the rhythmic discharge. The autocorrelograms of Figure 4Aiii illustrate the CCK-induced decrease in oscillatory activity that was associated with complete suppression of distinct peaks and an overall increase in spike counts, effects consistent with increased nonphasic spike discharge.
It was surprising that in the remaining experiments (8 of 14), CCK increased the duration of the rhythmic activity without altering the baseline discharge rate (Fig. 4Bi). The reversible increase in the duration of the oscillations is illustrated in the ratemeter output in Figure 4Bii (note different time bases in Figs. 4Ai,Bi). In this slice, the oscillation increased from 10 cycles lasting approximately 3 sec to 18 cycles lasting >6 sec, an 80% increase in oscillatory activity (Fig. 4B). The autocorrelograms of Figure 4Biii indicate the increased number and amplitude of peaks without alteration in the periodicity of the rhythm, suggesting an increase in the number of neurons participating in the phasic oscillatory activity.
In contrast to the anti- and pro-oscillatory actions observed in low CCK concentrations, the oscillatory activity was suppressed in nearly all experiments (11 of 12) when higher CCK concentrations were used (100-400 nM; Fig. 5). This effect was accompanied by an increase in baseline discharge in nRt (Fig. 5Ai; compare Pre-drug vs CCK) but not in VB (Fig. 5Bi), consistent with a selective CCK-induced depolarization of nRt neurons (Cox et al., 1995a
Data from a number of experiments are summarized in Figure 6A, which clearly illustrates the concentration-dependent biphasic effects of CCK on the oscillatory activity. Changes in the number of peaks and Amposc measurements obtained from the contour plots and autocorrelograms (see Materials and Methods) served as criteria for the overall action of CCK. The rhythmic activity was considered facilitated if both measures increased during CCK application (e.g., Fig. 4B), whereas reduction in both of these measures indicated a CCK-induced suppression (e.g., Figs. 4A, 5). At low concentrations (20-50 nM), CCK was equally likely to produce facilitation or suppression. However, with increasing CCK concentrations (100 nM), suppression of the rhythmic activities became the predominant effect. At the highest CCK concentration tested (400 nM), the intrathalamic oscillations were suppressed in all slices tested. There was no correlation between the overall CCK action on the oscillation (i.e., facilitation vs suppression) and the BMI concentration used to enhance the baseline oscillations. The characteristics calculated from the autocorrelograms (number of peaks, Amposc, 
Fig. 6. Summary of CCK actions on extracellularly recorded intrathalamic oscillations. A, Concentration dependence of CCK actions on thalamic rhythmic activity. The overall action of CCK (i.e., Facilitation, Suppression, No change) was determined by alterations of the number of peaks and Amposc. Mixed effects (i.e., facilitation and suppression) were observed at lower CCK concentrations (20 and 50 nM); however, with increasing CCK concentrations (100 and 400 nM), suppression became the predominant effect. The numbers in parentheses above each concentration indicate the number of experiments in each condition. B, Summary of effects of CCK on number of peaks (; # peaks), oscillation amplitude (
; Amposc), decay of oscillation (
;
; period) calculated from the autocorrelograms (see Materials and Methods for details). The values have been sorted by the overall effect and grouped by concentration. The histograms in Bi consist of only slices in which CCK produced a facilitation of the rhythmic activity, and have been grouped into <100 nM (20 and 50 nM) and
100 nM CCK (100 and 400 nM). Note the consistent increase in # peaks, Amposc, and
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In the next series of experiments, we tested the action of CCK on the rhythmic EPSPs observed from whole-cell recordings of nRt neurons. In many of these experiments, IC stimulation evoked rhythmic EPSPs in nRt neurons in standard physiological solution without the addition of BMI (e.g., Fig. 7B). As expected from extracellular recordings, low CCK concentrations (10-25 nM) increased the number of rhythmic EPSPs in three of five neurons (Fig. 7A), an effect accompanied by a small membrane depolarization (range 4-10 mV). The increased number of rhythmically occurring EPSPs was independent of CCK-induced depolarization of the individual neuron because it persisted after manual repolarization of the membrane potential during the peak of the CCK action [Fig. 7A, CCK (Vmatch)], and during early washout of the peptide, when the membrane potential had returned to within 2 mV of the resting level (Fig. 7A, Post CCK). The finding that altering membrane potential of individual neurons did not influence the occurrence of the repetitive EPSPs suggests that the modification of the rhythmic activity is a result of a network effect. The rhythmic activity recovered to baseline conditions with further washout of the peptide (Fig. 7A, Wash). In three neurons, in which CCK produced suppression of the rhythms at the peak CCK depolarization, there was a transient facilitation of the rhythms during the washout of the peptide, similar to that observed in the extracellular recordings.
High CCK concentrations (100-1000 nM) reversibly suppressed the rhythmic EPSPs (Fig. 7B; n = 12), and also produced a substantially larger membrane depolarization (range, 8-19 mV). As with the lower CCK concentrations, reduction of the rhythmic activity in individual neurons was independent of membrane depolarization because it persisted after manual return of the membrane potential to resting level [Fig. 7B, CCK (Vmatch)], and after the membrane potential returned to within 2 mV of the resting membrane potential after washout of the peptide (Fig. 7B, Post CCK). Rhythmic EPSPs recovered to baseline levels after longer CCK wash (Fig. 7B, Wash). CCK modifies discharge mode of nRt neurons
The generation and persistence of intrathalamic oscillations is dependent on the firing mode of nRt and relay thalamic neurons, which is in turn closely related to the membrane potential (Jahnsen and Llinás, 1984
Fig. 8. CCK alters the firing mode of nRt neurons. A, Pre-drug, At resting membrane potential, either internal capsule stimuli (·) or intracellular depolarizing current pulses (200 pA, 100 msec) evoke burst discharge from an nRt neuron. B, CCK, During peak of the depolarization induced by application of 400 nM CCK8S, same stimuli as in A evoked single spike discharge from the neuron (peak). Repolarization of the membrane potential to resting levels restored the burst firing mode (Vmatch). C, After washout of CCK8S, Vm returned to close to baseline level (dashed line) and both stimuli evoked burst discharge. Vm (dashed line) =
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We have shown previously that CCK reduces a Kleak conductance in nRt neurons, resulting in a robust membrane depolarization associated with an increased input resistance (Cox et al., 1995a
This response enhancement was explored further by testing the effect of low CCK concentrations (10 nM) on subthreshold EPSPs in seven nRt neurons. This concentration of CCK produced a small membrane depolarization (range, 2-8 mV) but increased the probability of suprathreshold spike discharges. As illustrated in Figure 9Bi (Pre-drug) and Bii, repeated stimuli (0.1 Hz) evoked a suprathreshold response in approximately 50% of trials. During CCK application, the stimuli evoked burst discharge in 100% of trials [Fig. 9Bi, CCK (10 nM)], and after several minutes of washout, the proportion of evoked subthreshold responses reverted to pre-CCK levels. The time course of the CCK-induced action on the subthreshold EPSP is illustrated in Figure 9Bii. These findings suggest that low CCK concentrations may increase the number of nRt neurons bursting and participating in the oscillatory activity, and thus increase the strength and duration of the evoked rhythmic activity, as illustrated by the extracellular data of Figure 4. CCK reduces inhibitory input to VB neurons
Conversion of the firing mode of nRt neurons from bursts to single spikes by CCK would be expected to decrease the inhibitory output of nRt neurons onto VB neurons. In the next series of experiments, we directly assessed evoked IPSCs in VB neurons as a measure of nRt output. Extracellular stimulation of nRt evoked a multipeaked IPSC in VB neurons (Figs. 10Ai,Aii). In most of these recordings, we substituted Cs+ for K+ in the intracellular solution and used a holding voltage near 0 mV to increase the driving force on the IPSCs. Similar findings were observed with K+-containing electrodes (Vhold =
Fig. 10. IPSCs evoked in VB neurons by nRt stimulation. Ai, Aii, Voltage-clamp recordings from VB neurons (Vhold = 0 mV) in an intact thalamic slice reveal that extracellular stimulation of nRt evokes a multipeaked IPSC. These recordings, as well as those in Figure 11, were obtained using Cs+-containing intracellular solution. Consecutive responses in Ai and Aii show similar timing of peaks in each IPSC burst. Aiii, After bath perfusion of ionotropic excitatory amino acid antagonists CNQX (20 µM) and dCPPene (10 µM), the IPSC evoked by the same stimulus as in Ai and Aii is reduced to a single peak. Bi, Bii, In a different slice in which nRt has been removed, two consecutive stimuli to the lateral edge of VB evoke single peaked IPSCs. Biii, The synaptic response is unchanged in the presence of CNQX and dCPPene.
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Because the multipeaked IPSCs in VB are reflective of the firing mode of the nRt neurons, and CCK was found to alter the firing mode of nRt neurons (Fig. 8), we tested several concentrations of CCK on the IPSCs. High CCK concentrations (>100 nM) altered the configuration of the IPSCs from multiple peaks to a single peak (Fig. 11B) and significantly reduced the integrated areas of IPSCs by an average of 62.7 ± 20.3% (n = 15; paired t test, p < 0.05). Although lower CCK concentrations also significantly reduced IPSC areas (average 27.8 ± 16.3%; n = 3), the multipeak configuration of the IPSCs was unchanged, suggesting that the firing mode of only a subpopulation of neurons was altered (Fig. 11A). The summarized data (Fig. 11C) indicate that CCK produced a significant dose-dependent suppression of the IPSC-integrated area. 
Fig. 11. CCK alters the multipeaked configuration of IPSCs in VB neurons. A, Pre-drug, In the intact thalamic slice, extracellular stimulation of nRt evokes a multipeaked IPSC in a VB neuron (Vhold = 0 mV). Each pair of responses illustrated is evoked by consecutive stimuli to illustrate the consistency of the configuration of the multiple peaks of the IPSC. CCK (25 nM), During application of CCK8S, the multipeaked configuration of the IPSC is unchanged, although the total charge of the IPSC (Integrated area) is reversibly attenuated by CCK8S. B, Pre-drug, In a different VB neuron, nRt stimulation evoked a multipeaked IPSC. CCK (400 nM), When the slice is exposed to 400 nM CCK, a concentration that suppresses oscillatory activity, the IPSC is reduced to a single peak. C, Summary of the effect of various CCK concentrations on mean charge of IPSCs. The ordinate is the ratio of mean integrated areas of IPSCs in CCK/control (Pre-drug). The effects of CCK and wash were calculated as ratios relative to pre-drug values. Data are plotted as mean ± SD (*p < 0.05).
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DISCUSSION
Our findings indicate that the neuropeptide CCK has a strong influence on intrathalamic rhythmic activities. Neuropeptides have been shown to produce lasting alterations in neuronal network functions in various invertebrate and vertebrate preparations, which may be associated with behavioral modifications (for review, see Harris-Warrick and Marder, 1991
The effects of CCK on intrathalamic oscillations are concentration dependent. Low concentrations of CCK suppressed rhythmic interactions between nRt and VB in 43% of experiments, whereas rhythmic activities were facilitated in the remaining. We speculate that application of lower concentrations of exogenous CCK may more closely approximate the level of modulation that occurs in vivo during thalamocortical circuit activities. With increasing CCK concentrations, suppression of rhythmic activity became the dominant action. Our working hypothesis is that the predominant suppression of rhythmic activity at higher CCK concentrations results from a strong depolarization of nRt neurons, which alters their firing mode from a resting burst- to single-spike discharge mode. This explanation is supported by direct recordings of the effects of CCK on nRt neurons and the resulting change of inhibitory output onto VB cells. Bath-applied CCK induces a concentration-dependent, long-lasting depolarization of nRt neurons by suppressing a K+ leak conductance, without altering the membrane potential or input resistance of VB neurons (Cox et al., 1995a
Because GABA-containing neurons of nRt provide the major source of inhibitory innervation of VB in rodents, a decrease in spike output of nRt neurons would reduce the inhibitory drive onto VB neurons. Intracellular recordings from VB neurons showed that nRt stimulation evoked multipeaked IPSCs, consistent with the burst output of the nRt cells. CCK, at concentrations that suppressed the rhythmic activity, changed the configuration of the IPSCs from multiple peaks to a single peak, a finding consistent with the CCK-induced alteration of nRt firing modes from burst to single spike. This decreased inhibitory current would reduce the probability of burst output from VB, and thus dampen the rhythmic activity.
The pro-oscillatory actions of CCK are more difficult to account for on the basis of a similar action of CCK. The increased strength and duration of the rhythmic activity likely occurs as a consequence of recruitment of additional nRt neurons that were subthreshold for burst generation before CCK exposure (Fig. 9B). CCK could have a biphasic action that is dependent on the resting membrane potential of the aggregate of nRt neurons. If portions of the latter were significantly hyperpolarized at rest, synaptic inputs might be subthreshold for evoking bursts of spikes, even though the low threshold Ca2+ current (IT) underlying the burst behavior might be fully deinactivated (Coulter et al., 1989 Functional significance of CCK effects on intrathalamic oscillations
The intimate reciprocal synaptic connectivity between nRt and the adjacent thalamic relay nucleus, together with the intrinsic properties of the involved neuronal populations, is the basis of intrathalamic rhythmic activities that have been associated with sleep spindles, slow-wave sleep, and the 3 Hz rhythms observed during absence epileptic seizures (Steriade and Llinás, 1988
Although the fundamental mechanisms underlying the maintenance of thalamic oscillatory activity have been studied extensively, the mechanisms underlying the initiation, cessation and modulation of this rhythmic activity, have received less attention. Our data indicate that CCK can either enhance or dampen intrathalamic rhythmic activity. Other neuromodulators, such as ACh, NE, and 5-HT, alter the firing mode of thalamic neurons by modifying the membrane potential of these neurons (McCormick and Prince, 1987
These two different sites of origin for CCK-containing afferents both send axon collaterals into nRt (Jones, 1985
Release of CCK in nRt by corticothalamic neurons might have quite different consequences. A synchronous high-frequency discharge, such as occurs during focal cortical epileptiform events (Gutnick and Prince, 1974
FOOTNOTES
Received July 19, 1996; revised Oct. 3, 1996; accepted Oct. 8, 1996.
This research was supported by National Institutes of Health Grants NS06477 and NS07280 and the Morris and Pimley Research Funds.
Correspondence should be addressed to Dr. Charles L. Cox, Department of Neurology and Neurological Sciences, Room M016, Stanford University Medical Center, Stanford, CA 94305.
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ABSTRACT
