Faculty Bibliography

New data on the organization of the intra-amygdaloid circuit is reviewed, beginning with the basolateral (BL) complex, the main input station of the amygdala for sensory afferents, and concluding with the central (CE) nucleus, an important source of projections to brain-stem structures mediating fear responses. The BL complex is endowed with a highly divergent system of intrinsic glutamatergic connections. Yet, BL projection cells have unusually low firing rates. This apparent contradiction is explained by the presence of powerful inhibitory pressures in the BL amygdala: (1) interneurons that generate large-amplitude inhibitory synaptic potentials and (2) projection cells that express a Ca(2+)-dependent K(+) current that can be activated by subthreshold synaptic inputs. Likewise, excitatory projections from the BL amygdala to the CE nucleus are controlled by clusters of GABAergic neurons, termed the intercalated (ITC) cell masses. In response to BL inputs, ITC cells generate feedforward inhibition in CE neurons. However, ITC neurons exhibit properties that allow them to modify the amount of inhibition they generate depending on the distribution of BL activity in space and time. Indeed, ITC cell masses can inhibit each other via lateromedial connections. Moreover, they express an unusual K(+) conductance that modifies their response to BL inputs depending on their recent firing history. Thus, inhibitory mechanisms of the amygdala allow for flexible, context-dependent gating of BL impulses to the CE nucleus

Purkinje cells generate simultaneous complex spikes as a result of olivocerebellar activity. This synchronization (to within 1 ms) is thought to result from electrotonic coupling of inferior olivary neurons. However, the distance from the inferior olive (IO) varies across the cerebellar cortex. Thus signals generated simultaneously at the IO should arrive asynchronously across the cerebellar cortex, unless the length differences are compensated for. Previously, it was shown that the conduction time from the IO to the cerebellar cortex remains nearly constant at approximately 4 ms in the rat, implying the existence of such compensatory mechanisms. Here, we examined the role of myelination in generating a constant olivocerebellar conduction time by investigating the latency of complex spikes evoked by IO stimulation during development in normal rats and myelin-deficient mutants. In normal rats, myelination not only reduced overall olivocerebellar conduction time, but also disproportionately reduced the conduction time to vermal lobules, which had the longest response latencies prior to myelination. The net result was a nearly uniform conduction time. In contrast, in myelin-deficient rats, conduction time differences to different parts of the cerebellum remained during the same developmental period. Thus myelination is the primary factor in generating a uniform olivocerebellar conduction time. To test the importance of a uniform conduction time for generating synchronous complex spike activity, multiple electrode recordings were obtained from normal and myelin-deficient rats. Average synchrony levels were higher in normal rats than mutants. Thus the uniform conduction time achieved through myelination of olivocerebellar fibers appears to be essential for the normal expression of complex spike synchrony

Inferior olivary neurons receive extensive glutamatergic and GABAergic innervation. Yet, because of the membrane properties of olivary neurons these neurotransmitters can produce only small changes in the firing rates of these cells. Moreover, olivary neurons can generate spontaneous spike activity in the absence of excitatory glutamatergic input. These facts suggest that glutamate and GABA have additional roles within the olivocerebellar system beyond simply modulating single cell firing probability. Indeed, one of the characteristics of the olivocerebellar system is its ability to generate synchronous complex spike activity across populations of Purkinje cells. The pattern of synchronous activity changes rapidly, and is thought to reflect the momentary distribution of effective electrotonic coupling between olivary neurons as shaped by afferent input to the inferior olive. However, it also possible that synchronous olivocerebellar activity is the result of synchrony inherent in the afferent activity itself. The issue of the origin of complex spike synchrony, and the role of glutamatergic olivary afferents in modulating its distribution were recently studied using multiple electrode recordings from Purkinje cells. The results of these studies, reviewed here, demonstrate that synchronous complex spike activity occurs in the absence of glutamatergic (and GABAergic) input to the inferior olive, and therefore indicate that synchronization of complex spike activity primarily results from the electrotonic coupling of olivary neurons, rather than from synchronization present within their afferents. Instead of triggering synchronous discharges directly, the results suggest that the function of tonic excitatory activity is to modulate the effective coupling of spike activity between olivary neurons. Blocking glutamate within the inferior olive causes an enhancement of the normal banding pattern of complex spike synchrony, with higher synchrony among parasagittally aligned Purkinje cells and less synchrony between non-aligned cells. This is in contrast to the more uniform synchrony distribution that follows block of GABAergic olivary afferents. Thus, GABA and glutamate play critical, and complementary, roles in determining the patterns of synchronous complex spike activity that are likely central to the functioning of the olivocerebellar system

The amygdaloid complex has long been implicated in seizure disorders. Yet, projection cells of the lateral amygdaloid nucleus (LA) display little spontaneous activity suggesting that this seizure prone structure is normally controlled by strong inhibitory mechanisms. This control is achieved in part by local interneurons; however, a synaptically activated, Ca(2+)-dependent K(+) (K(Ca)) conductance has recently been identified as a second major inhibitory mechanism. In the present study, we investigated which K(Ca) channels underlie this conductance, and their roles in the generation of the synaptic responses and spike adaptation of LA projection cells.Intracellular recordings were obtained from LA projection cells in barbiturate-anesthetized rats. In recordings with K-acetate pipettes, perirhinal stimulation evoked an initial excitatory postsynaptic potential (EPSP) followed by a prolonged monophasic hyperpolarization, similar to what was observed in cats under in vivo conditions, and distinct from the multiphasic hyperpolarization observed previously in rodents with in vitro recordings. This indicates that differences in the cellular environment, not interspecies differences, are responsible for the differing response profiles previously reported. In recordings with pipettes containing 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid or Cs-acetate the reversal potentials were significantly more positive than those recorded with K-acetate, consistent with a K(Ca) conductance contribution to the response. To investigate the K(Ca) channels underlying this conductance, intracellular recordings were obtained while perfusing the LA with Ringer's solution and then switching to a solution containing charybdotoxin, isoproterenol, or apamin. Charybdotoxin and isoproterenol produced positive shifts in the reversal potential, whereas apamin did not. By contrast, all three substances decreased adaptation during spike trains elicited by depolarizing current injections. These results suggest that intermediate (IK) and small (SK) conductance K(Ca) channels limit LA projection cell excitability, with IK channels involved in controlling both the synaptic response and intrinsic excitability of these neurons, and SK channels being involved only in the latter

Olivocerebellar activity is organized such that synchronous complex spikes occur primarily among Purkinje cells located within the same parasagittally oriented strip of cortex. Previous findings have shown that this synchrony distribution is modulated by the release of GABA and glutamate within the inferior olive, which probably act by controlling the efficacy of the electrotonic coupling between olivary neurons. The relative strengths of these two neurotransmitters in modulating the patterns of synchrony were compared by obtaining multiple electrode recordings of spontaneous crus 2a complex spike activity during intraolivary injection of solutions containing a GABA(A) (picrotoxin) and/or AMPA [1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide disodium (NBQX)] receptor antagonist. Injection of either antagonist led to increased synchrony between cells located within the same parasagittally oriented approximately 250-microm-wide cortical strip. Picrotoxin also increased complex spike synchrony among cells located in different cortical strips, leading to a less prominent banding pattern, whereas injections of NBQX tended to decrease complex spike synchrony among such cells, enhancing the banding pattern. The relative strength of these two classes of olivary afferents was assessed by first injecting one of the antagonists alone and then in combination with the other. The enhanced banding pattern of complex spike synchrony following injection of NBQX alone remained during the subsequent combined injection of both antagonists. Furthermore, the widespread synchronization of complex spike activity following injection of picrotoxin alone was partially or completely reversed by combined injection of picrotoxin and NBQX. Changes in the climbing fiber reflex induced by the intraolivary injections paralleled the changes observed for spontaneous complex spike activity, indicating that the effects of picrotoxin and NBQX on the synchrony distribution reflect changes in the pattern of effective coupling of inferior olivary neurons and demonstrating that synchronous complex spike activity does not require simultaneous excitatory input to olivary cells. Finally the pattern of synchrony during motor cortical stimulation was examined. It was found that the patterns of synchrony for motor-cortex-evoked complex spike activity were similar to those of spontaneous activity, indicating an important role for electrotonic coupling in determining the response of the olivocerebellar system to afferent input. Moreover, intraolivary injections of picrotoxin increased the spatial distribution of the evoked response. In sum, the results provide evidence for the hypothesis that electrotonic coupling of inferior olivary neurons via gap junctions is the mechanism underlying complex spike synchrony and that this coupling plays an important role in determining the responses of the olivocerebellar system to synaptic input

The olivocerebellar system has been proposed to function as a timing device for motor coordination in which inferior olivary neurons act as coupled oscillators that spontaneously generate rhythmic and synchronous activity. However, the inferior olive receives excitatory afferents, which can also drive the activity of these neurons. The extent to which the olivocerebellar system can intrinsically generate synchronous activity and olivary neurons act as neuronal oscillators has not been determined. To investigate this issue, multiple electrode recordings of complex spike (CS) activity were obtained from 236 crus 2a Purkinje cells in anesthetized rats. Intraolivary injections of the glutamate antagonists 6-cyano-7-nitroquinoxaline-2,3-dione or 1,2,3,4-tetrahydro-6-nitro-2,3-dioxo-benzo[f]quinoxaline-7-sulfonamide disodium were made, and the resulting changes in CS activity were determined. Loss of evoked CS responses to motor cortex stimulation or perioral tactile stimulation was used to measure the efficacy of the block. Block of glutamatergic input decreased the average CS firing rate by approximately 50% but did not abolish spontaneous CS activity. The remaining CS activity was significantly more rhythmic than that in control. The patterns of synchrony were similar to those found in control conditions (i.e., synchronous CSs primarily occurred among Purkinje cells located within the same approximately 250-microm-wide rostrocaudally oriented cortical strip); however, this normal banding pattern was enhanced. These changes in CS activity were not observed with vehicle injections. The results suggest that excitatory afferent activity disrupts olivary oscillations and support the hypotheses that olivary neurons are capable of acting as neuronal oscillators and that synchronous CS activity results from electrotonic coupling of olivary neurons

The olivocerebellar system is known to generate periodic synchronous discharges that result in synchronous (to within 1 msec) climbing fiber activation of Purkinje cells (complex spikes) organized in parasagittally oriented strips. These results have been obtained primarily in anesthetized animals, and so the question remains whether the olivocerebellar system generates such patterns in the awake animal. To this end, multiple electrode recordings of crus 2a complex spike activity were obtained in awake rats conditioned to execute tongue movements in response to a tone. After removal of all movement- and tone-related activity, the remaining data were examined to characterize spontaneous complex spike activity in the alert animal. Spontaneous complex spikes occurred at an average firing rate of 1 Hz and a clear approximately 10 Hz rhythmicity. Analysis of the autocorrelograms using a rhythm index indicated that the large majority of Purkinje cells displayed rhythmicity, similar to that in the anesthetized preparation. In addition, the patterns of synchronous complex spike activity were also similar to those observed in the anesthetized preparation (i.e., simultaneous activity was found predominantly among Purkinje cells located within the same parasagittally oriented strip of cortex). The results provide unequivocal evidence that the olivocerebellar system is capable of generating periodic patterns of synchronous activity in the awake animal. These findings support the extrapolation of previous results obtained in the anesthetized preparation to the waking state and are consistent with the timing hypothesis concerning the role of the olivocerebellar system in motor coordination

The perirhinal cortex lies at the interface between the neocortex and allocortex. Whether the perirhinal cortex expresses spontaneous electroencephalographic rhythms that are characteristic of the allocortex and/or of the neocortex is unknown. Thus, the present investigation was undertaken to characterize the activity of the perirhinal cortex with respect to various electroencephalographic rhythms that are displayed by neocortical areas or the entorhino-hippocampal system during different behavioral states of vigilance in chronically-implanted cats. Although perirhinal and neocortical electroencephalograms underwent similar state-dependent changes in amplitude, the ubiquitous neocortical sleep spindles were absent from the perirhinal cortex. In addition, while the slow sleep oscillation (0.5-1 Hz), which is pervasive in the neocortex, was present in the perirhinal cortex, its temporal relation to the neocortical oscillation was highly variable. In contrast, a high degree of correlation was found between perirhinal and entorhinal electroencephalographic activities in all behavioral states. In particular, during waking and paradoxical sleep, multiple simultaneously recorded entorhinal and perirhinal sites displayed an oscillation in the theta range which was highly correlated. To rule out the possibility that the perirhinal theta oscillation reflected volume conduction from neighboring structures, single-unit recordings were performed. Spike-triggered averages and peri-event histograms revealed that perirhinal cells displayed a statistically significant theta-related modulation of their spontaneous activity, albeit weaker than that observed in the entorhinal cortex. Thus, from the standpoint of spontaneous electroencephalographic rhythms, the perirhinal cortex is more closely related to the entorhino-hippocampal system than to the neocortex

Much of what is known about Ca2+ electrogenesis in neocortical cells has been derived from in vitro studies. Since Ca2+ currents are controlled by various modulators, comparing these findings to in vivo data is essential. Here, we analysed tetrodotoxin (TTX)-resistant, presumably Ca2+-mediated potentials in intracellularly recorded neocortical neurons in vivo. TTX was applied locally to block Na+ channels. Its effectiveness was demonstrated by the elimination of fast spikes and orthodromic responses. In response to depolarizing current pulses bringing the membrane potential beyond approximately -33 mV, 71% of neurons generated high-threshold Ca2+ spikes averaging 17 mV. This is in contrast with in vitro findings, where high-threshold spikes could only be elicited following the blockade of K+ conductances. Consistent with this, neurons dialysed with K+ channel blockers in vivo generated high-threshold spikes that had a lower threshold (approximately -40 mV) and, with intracellular Cs+, a larger amplitude, indicating the presence of K+ currents opposing the activation of Ca2+ channels. Only 15% of cortical cells displayed low-threshold Ca2+ spikes. To compare high-threshold Ca2+ spikes evoked by synaptic stimuli or current injection, another group of cortical neurons was dialysed with QX-314 and Cs+, in the absence of extracellular TTX. Synaptic stimuli applied on a background of membrane depolarization elicited presumed Ca2+ spikes whose amplitude varied in a stepwise fashion. Thus, although there are numerous similarities between in vivo and in vitro data, some significant differences were found, which suggest that the high-voltage activated Ca2+ currents and/or the K+ conductances that oppose them are subjected to different modulatory influences in vivo than in vitro