Cell-type specific distribution of chloride transporters in the rat suprachiasmatic nucleus.

The suprachiasmatic nucleus (SCN) is a circadian oscillator and biological clock. Cell-to-cell communication is important for synchronization among SCN neuronal oscillators and the great majority of SCN neurons use GABA as a neurotransmitter, the principal inhibitory neurotransmitter in the adult CNS. Acting via the ionotropic GABA(A) receptor, a chloride ion channel, GABA typically evokes inhibitory responses in neurons via Cl(-) influx. Within the SCN GABA evokes both inhibitory and excitatory responses although the mechanism underlying GABA-evoked excitation in the SCN is unknown. GABA-evoked depolarization in immature neurons in several regions of the brain is a function of intracellular chloride concentration, regulated largely by the cation-chloride cotransporters NKCC1 (sodium/potassium/chloride cotransporter for chloride entry) and KCC1-4 (potassium/chloride cotransporters for chloride egress). It is well established that changes in the expression of the cation-chloride cotransporters through development determines the polarity of the response to GABA. To understand the mechanisms underlying GABA-evoked excitation in the SCN, we examined the SCN expression of cation-chloride cotransporters. Previously we reported that the K(+)/Cl(-) cotransporter KCC2, a neuron-specific chloride extruder conferring GABA's more typical inhibitory effects, is expressed exclusively in vasoactive intestinal peptide (VIP) and gastrin-releasing peptide (GRP) neurons in the SCN. Here we report that the K(+)/Cl(-) cotransporter isoforms KCC4 and KCC3 are expressed solely in vasopressin (VP) neurons in the rat SCN whereas KCC1 is expressed in VIP neurons, similar to KCC2. NKCC1 is expressed in VIP, GRP and VP neurons in the SCN as is WNK3, a chloride-sensitive neuron-specific with no serine-threonine kinase which modulates intracellular chloride concentration via opposing actions on NKCC and KCC cotransporters. The heterogeneous distribution of cation-chloride cotransporters in the SCN suggests that Cl(-) levels are differentially regulated within VIP/GRP and VP neurons. We suggest that GABA's excitatory action is more likely to be evoked in VP neurons that express KCC4.

Ataxia and the olivo-cerebellar module.

Ataxia is a neurological symptom characterized by abnormal movement, due to cerebellar malfunction. Although the cause of the malfunction varies, from mutations in calcium or potassium channels to progressive degeneration of cerebellar tissue, the neurological symptoms of cerebellar-related ataxia are similar. In this short review we present a model that portrays the olivo-cerebellar system as a generator of temporal patterns. We then use the model to explain how mutations in different ionic channels located in different parts of the cerebellar system can result in similar neurological symptoms.

State-dependence of climbing fiber-driven calcium transients in Purkinje cells.

The recently described bi-stability of Purkinje cells and the state-dependence of the complex spike waveform suggest that calcium currents may play a pivotal role in both the complex spike waveform and the state of the membrane voltage. Here we used Ca2+ imaging to record the changes in intracellular [Ca2+] that are elicited by either spontaneous or climbing fiber-evoked activity in rat Purkinje cells. We show that a continuous somatic Ca2+ influx occurs during an "UP" state. Furthermore Ca2+ transients that are evoked by climbing fiber stimulation are state-dependent. Somatic transients are smaller following an "UP" state, while dendritic transients are smaller following a "DOWN" state. The state-dependence of these signals should affect the intrinsic firing of Purkinje cells as well as plastic processes that modulate synaptic strength.

Density is destiny--on [corrected] the relation between quantity of T-type Ca2+ channels and neuronal electrical behavior.

The electroresponsiveness fingerprint of a neuron reflects the types and distributions of the ionic channels that are embedded in the neuronal membrane as well as its morphology. Theoretical analysis shows that subtle changes in the density of channels can contribute substantially to the electroresponsive fingerprints of neurons. We have confirmed these predictions, using the dynamic clamp approach to emulate changes in channels' densities in neurons from the inferior olive. We demonstrate how the density of T-type channels determines the behavioral destiny of neurons. We argue that regulation of channel densities could be an efficient mechanism for controlling the electrical activity of single cells, as well as the output of neuronal networks.

Bidirectional multisite seizure propagation in the intact isolated hippocampus: the multifocality of the seizure "focus".

Localizing the seizure focus is difficult and frequently, multiple sites are found. This reflects our poor understanding of the fundamental mechanisms of seizure generation and propagation. We used multisite electrophysiological recordings in two seizure models and voltage-sensitive dye imaging, to spatiotemporally characterize the initiation and propagation of seizures in an intact epileptogenic brain region, the isolated hippocampus. In low-magnesium perfusate, seizures always originated in the temporal region, and propagated along the septotemporal axis to the septal region. After the seizure spread across the hippocampus, the bursts within a seizure became bidirectional, with different propagation patterns at different frequencies. When the intact hippocampus was separated along the septotemporal axis, independent bidirectional activity was observed in the two halves, and region-specific cuts to the tissue reveal that the CA3 region is critical for seizure generation and propagation. In a second seizure model, using focal tetanic stimulation of the septal and temporal CA3 region, seizures always originated at the stimulated site with bidirectionality later developing at different frequencies, as noted in the low magnesium model, behavior compatible with coupled neuronal network oscillators. These data provide novel insights into the dynamic multifocality of seizure onset and propagation, revealing that the current concept of a single seizure "focus" is complex.

Dynamic and spatial features of the inhibitory pallidal GABAergic synapses.

The globus pallidus, one of the basal ganglia nuclei, plays a major role in both basal ganglia physiology and pathophysiology. The globus pallidus is innervated mainly by striatal spiny neurons and globus pallidus collaterals. These GABAergic synapses constitute 90% of the input to globus pallidus cells. Despite the dominance of this inhibitory GABAergic input, globus pallidus cells are spontaneously active and most of them increase their firing rate in a task related manner. To explain this apparent inconsistency, we studied the dynamic and spatial effects of GABAergic inputs to globus pallidus neurons. To this end, we used intra-cellular recording from globus pallidus neurons in rat brain slices, investigating the effect of bath and local GABA application, as well as the responses to electrical stimulation of the striatum. We showed that the properties of the responses to either local or global GABA applications are similar to the responses of globus pallidus cells to GABA release from nerve terminals. Since the stimulus-evoked responses have been shown to be inhibitory in nature, we concluded that GABAergic inputs to globus pallidus both at soma and dendrite level are inhibitory. Furthermore, we showed that GABA can promote globus pallidus synchronization by affecting the timing of globus pallidus spiking, and that the globus pallidus GABAergic synapse undergoes rapid frequency-dependent depression. This prominent synaptic depression can account for the ability of globus pallidus neurons to fire in the presence of a majority of inhibitory inputs and might indicate that globus pallidus neurons are tuned to detect frequency changes. Furthermore, globus pallidus synaptic depression rules out the possibility of activation of GABAeregic afferents as the main mechanisms of high-frequency deep brain stimulation, used for treatment of severe parkinsonian patients.

Presynaptic and postsynaptic GABAA receptors in rat suprachiasmatic nucleus.

The mammalian suprachiasmatic nucleus (SCN), the brain's circadian clock, is composed mainly of GABAergic neurons, that are interconnected via synapses with GABA(A) receptors. Here we report on the subcellular localization of these receptors in the SCN, as revealed by an extensively characterized antibody to the alpha 3 subunit of GABA(A) receptors in conjunction with pre- and postembedding electron microscopic immunocytochemistry. GABA(A) receptor immunoreactivity was observed in neuronal perikarya, dendritic processes and axonal terminals. In perikarya and proximal dendrites, GABA(A) receptor immunoreactivity was expressed mainly in endoplasmic reticulum and Golgi complexes, while in the distal part of dendrites, immunoreaction product was associated with postsynaptic plasma membrane. Many GABAergic axonal terminals, as revealed by postembedding immunogold labeling, displayed GABA(A) receptor immunoreactivity, associated mainly with the extrasynaptic portion of their plasma membrane. The function of these receptors was studied in hypothalamic slices using whole-cell patch-clamp recording of the responses to minimal stimulation of an area dorsal to the SCN. Analysis of the evoked inhibitory postsynaptic currents showed that either bath or local application of 100 microM of GABA decreased GABAergic transmission, manifested as a two-fold increase in failure rate. This presynaptic effect, which was detected in the presence of the glutamate receptor blocker 6-cyano-7-nitroquinoxaline-2,3-dione and the selective GABA(B) receptor blocker CGP55845A, appears to be mediated via activation of GABA(A) receptors. Our results thus show that GABA(A) receptors are widely distributed in the SCN and may subserve both pre- and postsynaptic roles in controlling the mammalian circadian clock.

The olivocerebellar system as a generator of temporal patterns.

The large number of diverse functions attributed to the cerebellum appears to be inconsistent with its simple, homogeneous and evolutionary preserved structure. A homogeneous structure that participates in a variety of functions implies that a common denominator underlies all of them. Since the concept of precise timing can be recognized in almost all cerebellar functions, it is likely, therefore, that the basic cerebellar circuit is capable of generating temporal patterns. Of the different mechanisms that can generate temporal patterns, two are suggested by the functional anatomy of the cerbellum: transmission lines or oscillators. Our recent experimental observations indicate that the olivary oscillatory property is more likely to serve this function. We propose that interactions between the cerebellum and the inferior olive endow the system with the ability to generate complex temporal patterns. These temporal patterns can be used for fine adjustment of motor output, sensory expectation, or shifting attentions.

GABA-induced current and circadian regulation of chloride in neurones of the rat suprachiasmatic nucleus.

1. We have shown previously that GABA, the main neurotransmitter in the suprachiasmatic nucleus (SCN), has dual effects on SCN neurones, excitatory during the day and inhibitory at night. This duality has been attributed to changes in [Cl(-)](i) during the circadian cycle. To unravel the processes underlying these changes we investigated the biophysical properties of the GABAergic receptors and the regulation of [Cl(-)](i) in SCN neurones. 2. We used voltage-clamp methodology in conjunction with local application of GABA to characterise the current induced by GABA in SCN neurones within acute brain slices. This current, mediated via GABA(A) receptors, shows moderate voltage dependence, does not desensitise and can significantly alter [Cl(-)](i). 3. Loading or depletion of intracellular Cl(-) was induced by a train of GABA pulses. The recovery of intracellular Cl(-) was deduced from the change in [Cl(-)](i) calculated from the response to a test GABA pulse presented at different intervals after the conditioning train of GABA application. The time course of recovery was described by an exponential curve. Recovery following Cl(-) depletion was slower than recovery from Cl(-) loading and was further delayed during the subjective night. 4. We concluded that: (a) SCN neurones express a large number of somatic GABA(A) receptors, which give rise to a modifiable, tonic Cl(-) conductance that modulates cell excitability; (b) two Cl(-) transport mechanisms operate in SCN neurones, one that replenishes the cell with Cl(-) following Cl(-) depletion and another that removes Cl(-) after Cl(-) loading; (c) the efficiency of the replenishing mechanism is reduced during the subjective night; and (d) this reduction explains a lower [Cl(-)](i) during the night phase of the circadian cycle.

The generation of oscillations in networks of electrically coupled cells.

In several biological systems, the electrical coupling of nonoscillating cells generates synchronized membrane potential oscillations. Because the isolated cell is nonoscillating and electrical coupling tends to equalize the membrane potentials of the coupled cells, the mechanism underlying these oscillations is unclear. Here we present a dynamic mechanism by which the electrical coupling of identical nonoscillating cells can generate synchronous membrane potential oscillations. We demonstrate this mechanism by constructing a biologically feasible model of electrically coupled cells, characterized by an excitable membrane and calcium dynamics. We show that strong electrical coupling in this network generates multiple oscillatory states with different spatio-temporal patterns and discuss their possible role in the cooperative computations performed by the system.

Spatial distribution and subunit composition of GABA(A) receptors in the inferior olivary nucleus.

GABAergic inhibitory feedback from the cerebellum onto the inferior olivary (IO) nucleus plays an important role in olivo-cerebellar function. In this study we characterized the physiology, subunit composition, and spatial distribution of gamma-aminobutyric acid-A (GABA(A)) receptors in the IO nucleus. Using brain stem slices, we identified two types of IO neuron response to local pressure application of GABA, depending on the site of application: a slow desensitizing response at the soma and a fast desensitizing response at the dendrites. The dendritic response had a more negative reversal potential than did the somatic response, which confirmed their spatial origin. Both responses showed voltage dependence characterized by an abrupt decrease in conductance at negative potentials. Interestingly, this change in conductance occurred in the range of potentials wherein subthreshold membrane potential oscillations usually occur in IO neurons. Immunostaining IO sections with antibodies for GABA(A) receptor subunits alpha 1, alpha 2, alpha 3, alpha 5, beta 2/3, and gamma 2 and against the postsynaptic anchoring protein gephyrin complemented the electrophysiological observation by showing a differential distribution of GABA(A) receptor subtypes in IO neurons. A receptor complex containing alpha 2 beta 2/3 gamma 2 subunits is clustered with gephyrin at presumptive synaptic sites, predominantly on distal dendrites. In addition, diffuse alpha 3, beta 2/3, and gamma 2 subunit staining on somata and in the neuropil presumably represents extrasynaptic receptors. Combining electrophysiology with immunocytochemistry, we concluded that alpha 2 beta 2/3 gamma 2 synaptic receptors generated the fast desensitizing (dendritic) response at synaptic sites whereas the slow desensitizing (somatic) response was generated by extrasynaptic alpha 3 beta 2/3 gamma 2 receptors.

Resonance, oscillation and the intrinsic frequency preferences of neurons.

The realization that different behavioural and perceptual states of the brain are associated with different brain rhythms has sparked growing interest in the oscillatory behaviours of neurons. Recent research has uncovered a close association between electrical oscillations and resonance in neurons. Resonance is an easily measurable property that describes the ability of neurons to respond selectively to inputs at preferred frequencies. A variety of ionic mechanisms support resonance and oscillation in neurons. Understanding the basic principles involved in the production of resonance allows for a simplified classification of these mechanisms. The characterization of resonance and frequency preference captures those essential properties of neurons that can serve as a substrate for coordinating network activity around a particular frequency in the brain.

Cerebellar on-beam and lateral inhibition: two functionally distinct circuits.

Optical imaging of voltage-sensitive dyes in an isolated cerebellum preparation was used to study the spatiotemporal functional organization of the inhibitory systems in the cerebellar cortex. Responses to surface stimulation of the cortex reveal two physiologically distinct inhibitory systems, which we refer to as lateral and on-beam inhibition following classical terminology. Lateral inhibition occurs throughout the area responding to a stimulus, whereas on-beam inhibition is confined to the area directly excited by parallel fibers. The time course of the lateral inhibition is twice as long as that of the on-beam inhibition. Both inhibitory responses increase with stimulus intensity, but the lateral inhibition has a lower threshold, and it saturates at lower stimulus intensity. The amplitude of the on-beam inhibition is linearly related to the excitation at the same location, whereas that of the lateral inhibition is linearly related to the excitation at the center of the beam. Repetitive stimulation is required to activate on-beam inhibition, whereas the same stimulus paradigm reveals prolonged depression of the lateral inhibition. We conclude that lateral inhibition reflects the activation of molecular layer interneurons, and its major role is to increase the excitability of the activated area by disinhibition. The on-beam inhibition most likely reflects Golgi cell inhibition of granule cells. However, Purkinje cell collateral inhibition of Golgi cells cannot be excluded. Both possibilities suggest that the role of the on-beam inhibition is to efficiently modulate, in time and space, the mossy fiber input to the cerebellar cortex.

To beat or not to beat: a decision taken at the network level.

The cells of the inferior olivary nucleus, the sole source of the cerebellar climbing fibers, form a network of electrically coupled neurons. Experimental observations show that these neurons produce a large repertoire of electrical signals, among which sub-threshold oscillations of the membrane potential. Simultaneous recordings from pairs of neurons and optical imaging of voltage sensitive dyes show that sub-threshold activity occurs in synchrony throughout the network. The mechanism underlying the generation of the sub-threshold oscillations is not fully understood. Experimental observations suggest that the electrical coupling is essential but insufficient for their generation. Several theoretical mechanisms have been suggested to explain these observations. Up-to-date, the most realistic model is the heterogeneity model, that assumes a certain degree of heterogeneity among olivary neurons. The heterogeneity model proposes that sub-threshold oscillations are produced by electrical coupling of neurons with the same types of ionic conductances, but with different densities. The variability in channel densities yield neurons of different functional types. The main prediction of the model is that different functional types of neurons should be found in the inferior olive. Dynamic clamp experiments support this prediction.

Optical measurements of synchronized activity in isolated mammalian cerebellum.

An experimental system that combines optical imaging of voltage-sensitive dyes with an in vitro isolated cerebellum preparation is described. The optical imaging system is based on a photodiode array and two rather simple amplification stages. The isolated cerebellum preparation preserves the integrity of the neuronal circuitry, thus allowing the exploration of the path of information flow. In this study, we characterize the nature and sources of the optical signal evoked in the cerebellar cortex by surface stimulation. We show that this signal reflects inhibitory and excitatory synaptic potentials generated by cell bodies and dendrites of cortical neurons, whereas action potentials of the parallel fibers are not detected by the system. The spatial distribution of the optical signals agrees with the classical view of cerebellar activity following surface stimulation. Hence, this experimental system provides a powerful means to explore the functional organization, in time and space, of the cerebellar cortex.