Electrical coupling: novel mechanism for sleep-wake control

STUDY OBJECTIVES: Recent evidence suggests that certain anesthetic agents decrease electrical coupling, whereas the stimulant modafinil appears to increase electrical coupling. We investigated the potential role of electrical coupling in 2 reticular activating system sites, the subcoeruleus nucleus and in the pedunculopontine nucleus, which has been implicated in the modulation of arousal via ascending cholinergic activation of intralaminar thalamus and descending activation of the subcoeruleus nucleus to generate some of the signs of rapid eye movement sleep. DESIGN: We used 6- to 30-day-old rat pups to obtain brainstem slices to perform whole-cell patch-clamp recordings. MEASUREMENTS AND RESULTS: Recordings from single cells revealed the presence of spikelets, manifestations of action potentials in coupled cells, and of dye coupling of neurons in the pedunculopontine nucleus. Recordings in pairs of pedunculopontine nucleus and subcoeruleus nucleus neurons revealed that some of these were electrically coupled with coupling coefficients of approximately 2%. After blockade of fast synaptic transmission, the cholinergic agonist carbachol was found to induce rhythmic activity in pedunculopontine nucleus and subcoeruleus nucleus neurons, an effect eliminated by the gap junction blockers carbenoxolone or mefloquine. The stimulant modafinil was found to decrease resistance in neurons in the pedunculopontine nucleus and subcoeruleus nucleus after fast synaptic blockade, indicating that the effect may be due to increased coupling. CONCLUSIONS: The finding of electrical coupling in specific reticular activating system cell groups supports the concept that this underlying process behind specific neurotransmitter interactions modulates ensemble activity across cell populations to promote changes in sleep-wake state.     

Heterogeneous electrotonic coupling and synchronization of rhythmic bursting activity in mouse Hb9 interneurons

The neurons and mechanisms involved in mammalian spinal cord networks that produce rhythmic locomotor activity remain largely undefined. Hb9 interneurons, a small population of discretely localized interneurons in the mouse spinal cord, are conditionally bursting neurons. Here we applied potassium channel blockers with the aim of increasing neuronal excitability and observed that under these conditions, postnatal Hb9 interneurons exhibited bursts of action potentials with underlying voltage-independent spikelets. The bursts were insensitive to antagonists to fast chemical synaptic transmission, and the bursting and spikelets were blocked by tetrodotoxin. Calcium imaging studies using 2-photon excitation in spinal cord slices revealed that clustered Hb9 interneurons exhibited synchronous and occasional asynchronous, calcium transients that were also insensitive to fast synaptic transmission blockade. All transients were blocked by the gap junction blocker carbenoxolone. Paired whole cell patch-clamp recordings of Hb9 interneurons in the late postnatal mouse revealed common chemical synaptic inputs but no evidence of current transfer (i.e., electrotonic coupling) between the neurons. However, Hb9 and a previously defined population of non-Hb9 interneurons were electrotonically coupled. In the absence of fast chemical transmission in the whole spinal cord preparation, 2-photon excitation calcium imaging revealed bursting activity of Hb9 interneurons synchronous with rhythmic ventral root output. Thus Hb9 interneurons are both endogenous bursters and rhythmically active within a heterogeneous electrotonically coupled network. A network with these properties could produce the wide range of stable rhythms necessary for locomotor activity.     

Intracellular activity of cortical and thalamic neurones during high-voltage rhythmic spike discharge in Long-Evans rats in vivo

Spontaneous high-voltage rhythmic spike (HVRS) discharges at 6–12 Hz have been widely described in the electrocorticogram (EcoG) of Long-Evans rats. These ECoG oscillations have been proposed to reflect a state of attentive immobility allowing the optimization of sensory integration within the corticothalamic pathway. This hypothesis has been challenged by recent studies emphasizing similarities between HVRS discharges and spike-and-wave discharges (SWDs) in well-established rat genetic models of absence epilepsy. Here, we made in vivo intracellular recordings to determine, for the first time, the cellular mechanisms responsible for the synchronized oscillations in the corticothalamic loop during HVRS discharges in the Long-Evans rats. We show that HVRS discharges are associated in corticothalamic neurones with rhythmic suprathreshold synaptic depolarizations superimposed on a tonic hyperpolarization, likely due to a process of synaptic disfacilitation. Simultaneously, thalamocortical neurones exhibit a large-amplitude 'croissant'-shaped membrane hyperpolarization with a voltage sensitivity suggesting a potassium-dependent mechanism. This thalamic hyperpolarizing envelope was associated with a membrane oscillation resulting from interactions between excitatory synaptic inputs, a chloride-dependent inhibitory conductance and voltage-gated intrinsic currents. These cortical and thalamic cellular mechanisms underlying HVRS activity in Long-Evans rats are remarkably similar to those previously described in the thalamocortical networks during SWDs. Thus, the present study provides an additional support to the hypothesis that HVRS activity in Long-Evans rats is an absence-like seizure activity.     

Dissociated retinal neurons form periodically active synaptic circuits

Throughout the developing nervous system, immature circuits generate rhythmic activity patterns that influence the formation of adult networks. The cellular mechanisms underlying this spontaneous, correlated activity can be studied in dissociated neuronal cultures. Using calcium imaging and whole cell recording, we showed that cultured dissociated mammalian retinal neurons form networks that produce spontaneous, correlated, highly periodic activity. As the culture matures, the spatial correlations of the periodic calcium transients evolve from being highly synchronized across neighboring cells to propagating across the culture in a wavelike manner reminiscent of retinal waves recorded in vivo. Spontaneous calcium transients and synaptic currents were blocked either by cadmium, tetrodotoxin, or the glutamate receptor antagonist 6,7-dinitroquinoxaline, indicating that the periodic activity was driven primarily by synaptic transmission between retinal ganglion cells. Evoked responses between pairs of ganglion cells exhibited paired-pulse synaptic depression, and the time constant of recovery from this depression was similar to the interval between periodic events. These results suggest that synaptic depression may regulate the frequency of network activity. Together, these findings provide insight into how networks containing primarily excitatory connections generate highly correlated activity.     

Electrotonic coupling between stratum oriens interneurones in the intact in vitro mouse juvenile hippocampus

Using the isolated juvenile (7–14 days) mouse whole hippocampus preparation, which contains intact complex local circuitry, 145 dual whole cell recordings were made from stratum oriens (s.o.) interneurones under infrared microscopy. In 11.7% of paired recordings, evidence for direct electrotonic coupling between the s.o. interneurones was obtained from the response of one interneurone to a long (400–600 ms) constant current pulse passed into the coupled interneurone. When specifically orienting the dual recordings in the transectional plane of the hippocampus, 18.5% of paired recordings showed electrotonic coupling. The coupling coefficient, estimated from averaged data, was 6.9 ± 4.7%, ranging from 1.3 to 17.6%. The time constant of the electrotonically transmitted hyperpolarization was inversely related to the coupling coefficient between the two neurones. The electrotonic responses of one neurone to constant current pulses injected into the other coupled neurone were intermittent. Spikes in one of the coupled neurones were associated with small electrotonic EPSPs (spikelets) in the other coupled neurone, in those neuronal pairs with coupling coefficients greater than 10%. Failure of spikelet production following a spike in the coupled cell occurred 5–10% of the time. Electrotonic coupling and spikelets persisted in the presence of chemical synaptic transmission blockade by CNQX, APV and bicuculline, or in zero Ca²⁺ perfusate, but were abolished by carbenoxolone (100 μ m ), a gap junctional blocker. These data confirm the existence of electrotonic coupling between s.o. interneurones, presumably via gap junctions located in dendrites.     

Rhythmic activities in the rat solitary complex in vitro.

In adult rat brainstem slices, rhythmic discharge of action potentials occurred spontaneously in 10 out of 197 cells of the solitary complex. In 6 neurones, fast rhythms (2-6 per min) were characterized by volleys of synaptic activity presenting abrupt onset denoting synchronized discharge of presynaptic elements. Synchronizing signals may be generated by cells discharging bursts of high-frequency action potentials and presenting extensive axonal arborization, as observed in one cell. Slower rhythms (0.3-0.8 per min) monitored in three cells did not involve synchronizing processes and could be evoked in non-rhythmic cells by 15-30 min bath application of the cholecystokinin octapeptide (100 nM). These results suggest distinct operating mechanisms of fast and slow rhythms in the solitary complex in vitro.     

Synaptic dynamics control the timing of neuronal excitation in the activated neocortical microcircuit

It is well established that sensory stimulation results in the activity of multiple functional columns in the neocortex. The manner in which neurones within each column are active in relation to each other is, however, not known. Multiple whole-cell recordings in activated neocortical slices from rat revealed diverse correlation profiles of excitatory synaptic input to different types of neurones. The specific correlation profile between any two neurones could be predicted by the settings of synaptic depression and facilitation at the input synapses. Simulations further showed that patterned activity is essential for synaptic dynamics to impose the temporal dispersion of excitatory input. We propose that synaptic dynamics choreograph neuronal activity within the neocortical microcircuit in a context-dependent manner.     

Reconfiguration of respiratory-related population activity in a rostrally tilted transversal slice preparation following blockade of inhibitory neurotransmission in neonatal rats

Recent studies showed that respiratory rhythm generation depends on oscillators located in the pre-Bötzinger complex (pre-BötC) and the parafacial respiratory group (pFRG). To study inhibitory synaptic interactions between these two oscillators, we developed a rostrally tilted transversal slice preparation, which preserves these regions. The onset of rhythmic mass activity in the retrotrapezoid nucleus (RTN)/pFRG preceded that of the pre-BötC. Blockade of glycinergic and gamma-aminobutyric acidic inhibition synchronized pre-BötC and RTN/pFRG activity and significantly increased pre-BötC burst frequency, amplitude, and duration. Population imaging revealed recruitment of inspiratory-like neurones, while expiratory-like neurones lost their phasic activity. The reconfiguration after disinhibition reveals: (1) synaptic inhibition of the pre-BötC arising from the RTN/pFRG, (2) excitatory drive from the RTN/pFRG that triggers the pre-BötC burst. Our findings support the view that these synaptic interactions in vitro relate to the initiation of the inspiratory phase or to the steering of the expiratory–inspiratory phase transition in vivo.     

Gap junctions and inhibitory synapses modulate inspiratory motoneuron synchronization

Interneuronal electrical coupling via gap junctions and chemical synaptic inhibitory transmission are known to have roles in the generation and synchronization of activity in neuronal networks. Uncertainty exists regarding the roles of these two modes of interneuronal communication in the central respiratory rhythm-generating system. To assess their roles, we performed studies on both the neonatal mouse medullary slice and en bloc brain stem-spinal cord preparations where rhythmic inspiratory motor activity can readily be recorded from both hypoglossal and phrenic nerve roots. The rhythmic inspiratory activity observed had two temporal characteristics: the basic respiratory frequency occurring on a long time scale and the synchronous neuronal discharge within the inspiratory burst occurring on a short time scale. In both preparations, we observed that bath application of gap-junction blockers, including 18 alpha-glycyrrhetinic acid, 18 beta-glycyrrhetinic acid, and carbenoxolone, all caused a reduction in respiratory frequency. In contrast, peak integrated phrenic and hypoglossal inspiratory activity was not significantly changed by gap-junction blockade. On a short-time-scale, gap-junction blockade increased the degree of synchronization within an inspiratory burst observed in both nerves. In contrast, opposite results were observed with blockade of GABA(A) and glycine receptors. We found that respiratory frequency increased with receptor blockade, and simultaneous blockade of both receptors consistently resulted in a reduction in short-time-scale synchronized activity observed in phrenic and hypoglossal inspiratory bursts. These results support the concept that the central respiratory system has two components: a rhythm generator responsible for the production of respiratory cycle timing and an inspiratory pattern generator that is involved in short-time-scale synchronization. In the neonatal rodent, properties of both components can be regulated by interneuronal communication via gap junctions and inhibitory synaptic transmission.     

Sensory regulation of network components underlying ciliary locomotion in Hermissenda

Ciliary locomotion in the nudibranch mollusk Hermissenda is modulated by the visual and graviceptive systems. Components of the neural network mediating ciliary locomotion have been identified including aggregates of polysensory interneurons that receive monosynaptic input from identified photoreceptors and efferent neurons that activate cilia. Illumination produces an inhibition of type I(i) (off-cell) spike activity, excitation of type I(e) (on-cell) spike activity, decreased spike activity in type III(i) inhibitory interneurons, and increased spike activity of ciliary efferent neurons. Here we show that pairs of type I(i) interneurons and pairs of type I(e) interneurons are electrically coupled. Neither electrical coupling or synaptic connections were observed between I(e) and I(i) interneurons. Coupling is effective in synchronizing dark-adapted spontaneous firing between pairs of I(e) and pairs of I(i) interneurons. Out-of-phase burst activity, occasionally observed in dark-adapted and light-adapted pairs of I(e) and I(i) interneurons, suggests that they receive synaptic input from a common presynaptic source or sources. Rhythmic activity is typically not a characteristic of dark-adapted, light-adapted, or light-evoked firing of type I interneurons. However, burst activity in I(e) and I(i) interneurons may be elicited by electrical stimulation of pedal nerves or generated at the offset of light. Our results indicate that type I interneurons can support the generation of both rhythmic activity and changes in tonic firing depending on sensory input. This suggests that the neural network supporting ciliary locomotion may be multifunctional. However, consistent with the nonmuscular and nonrhythmic characteristics of visually modulated ciliary locomotion, type I interneurons exhibit changes in tonic activity evoked by illumination.     

Effects of synchrony between primate corticomotoneuronal cells on post-spike facilitation of muscles and motor units

Cross-correlating the activity of neighboring motor cortex neurons recorded with independent electrodes in behaving monkeys has revealed synchronization peaks, largely due to common synaptic input. Corticomotoneuronal (CM) cells produced post-spike facilitation (PSF) of rectified forearm electromyograms (EMG); 15 cells synchronized with CM cells showed no PSF. Five pairs of CM cells with overlapping muscle fields exhibited similar synchrony peaks. The contribution of this synchrony to facilitation of common target muscles was assessed by two new methods: selective spike-triggered averaging and convolution. They showed that the PSF is composed predominantly of effects mediated by output of the triggering cell, but may include a broad, shallow component mediated by synchrony with other CM cells.     

Electrical coupling can prevent expression of adult-like properties in an embryonic neural circuit.

Electrical coupling is widespread in developing nervous systems and plays a major role in circuit formation and patterning of activity. In most reported cases, such coupling between rhythmogenic neurons tends to synchronize and enhance their oscillatory behavior, thereby producing monophasic rhythmic output. However, in many adult networks, such as those responsible for rhythmic motor behavior, oscillatory neurons are linked by synaptic inhibition to produce rhythmic output with multiple phases. The question then arises whether such networks are still able to generate multiphasic output in the early stage of development when electrical coupling is abundant. A suitable model for addressing this issue is the lobster stomatogastric nervous system (STNS). In the adult animal, the STNS consists of three discrete neural networks that are comprised of oscillatory neurons interconnected by reciprocal inhibition. These networks generate three distinct rhythmic motor patterns with large amplitude neuronal oscillations. By contrast, in the embryo the same neuronal population expresses a single multiphasic rhythm with small-amplitude oscillations. Recent findings have revealed that adult-like network properties are already present early in the embryonic system but are masked by an as yet unknown mechanism. Here we use computer simulation to test whether extensive electrical coupling may be involved in masking adult-like properties in the embryonic STNS. Our basic model consists of three different adult-like STNS networks that are built of relaxation oscillators interconnected by reciprocal synaptic inhibition. Individual model cells generate slow membrane potential oscillations without action potentials. The introduction of widespread electrical coupling between members of these networks dampens oscillation amplitudes and, at moderate coupling strengths, may coordinate neuronal activity into a single rhythm with different phases, which is strongly reminiscent of embryonic STNS output. With a further increase in coupling strength, the system reaches one of two final states depending on the relative contribution of inhibition and inherent oscillatory properties within the networks: either fully synchronized and dampened oscillations, or a complete collapse of activity. Our simulations indicate that, beginning from either of these two states, the emergence of distinct adult networks during maturation may arise from a developmental decrease in electrical coupling that unmasks preexisting adult-like network properties.     

Evidence for bursting pacemaker neurones in cultured spinal cord cells

Intracellular recordings were made from dissociated mouse spinal cord cells in primary culture. One type of spinal cord neurone, with a large cell body (40-50 micron), 3-5 short neurites, and a mean resting potential of -65 mV, was found to fire rhythmic bursts of action potentials with a phase duration of approximately 1s when the membrane potential was depolarized to -55 mV. These bursts did not arise from spontaneous synaptic input, but appeared to result from endogenous ionic conductance properties of the membrane resembling those observed in molluscan bursting pacemaker neurones. Ionic conductances underlying this bursting activity were studied pharmacologically by local application of ionic conductance blockers. Pacemaker potentials depended on Na+ conductance, since tetrodotoxin and Na-free medium were the most potent agents for blocking spontaneous rhythmic activity. However, a Ca2+ conductance was involved in the depolarizing phase of membrane potential oscillations, since Ba2+ application increased oscillation amplitude. Action potentials observed during the bursts were Na+- and Ca2+-dependent. They did not differ significantly from those observed in other spinal cord neurones in culture. Application of tetraethylammonium, CoCl2, BaCl2 and 4-aminopyridine revealed at least three different potassium conductances which controlled this bursting pacemaker activity. A delayed potassium conductance controlled spike duration, a Ca-dependent potassium conductance controlled the duration of the burst and underlay the hyperpolarizing phase terminating the burst, and finally, a transient potassium conductance appeared to be involved in the control of phase duration. The demonstration that spinal cord neurones growing in monolayer culture display typical bursting pacemaker activity raises the possibility that bursting pacemaker neurones in the mammalian spinal cord may be involved in a phasic pattern generator that could control such activities as walking and the respiratory rhythm.     

Summation of excitatory postsynaptic potentials in electrically-coupled neurones

Dendritic electrical coupling increases the number of effective synaptic inputs onto neurones by allowing the direct spread of synaptic potentials from one neurone to another. Here we studied the summation of excitatory postsynaptic potentials (EPSPs) produced locally and arriving from the coupled neurone (transjunctional) in pairs of electrically-coupled Retzius neurones of the leech. We combined paired recordings of EPSPs, the production of artificial excitatory postsynaptic potentials (APSPs) in neurone pairs with different coupling coefficients and simulations of EPSPs produced in the coupled dendrites. Summation of the EPSPs produced in the dendrites was always linear, suggesting that synchronous EPSPs are produced at two or more different pairs of coupled dendrites and not in both sides of any one gap junction. The different spatio-temporal relationships explored between pairs of EPSPs or APSPs produced three main effects. (1) Synchronous pairs of EPSPs or APSPs exhibited an elongation of their decay phase compared to singe EPSPs. (2) Asymmetries in the amplitudes between the pair of EPSPs added a “hump” to the smallest EPSP. (3) Modelling the inputs near the electrical synapse or anticipating the production of the transjunctional APSP increased the amplitude of the compound EPSP. The magnitude of all these changes depended on the coupling coefficient of the neurones. We also show that the hump improves the passive conduction of EPSPs by adding low frequency components. The diverse effects of summation of local and alien EPSPs shown here endow electrically-coupled neurones with a wider repertoire of adjustable integrative possibilities.     

Properties and origin of spikelets in thalamocortical neurones in vitro

Spikelets, or fast prepotentials as they are frequently referred to, are a common feature of the electrophysiology of central neurones and are invariably correlated with the presence of electrotonic coupling via gap junctions. Here we report that in the presence of the metabotropic glutamate receptor agonists, trans-ACPD or DHPG, thalamocortical neurones of the cat dorsal lateral geniculate nucleus maintained in vitro exhibit stereotypical spikelets that possess similar properties to those described in other brain areas. These spikelets were routinely observed in the presence of antagonists of fast chemical synaptic transmission, were resistant to the application of a variety of voltage-dependent Ca(2+) channel blockers but were abolished by tetrodotoxin. In addition, spikelets were reversibly blocked by the putative gap junction blocker carbenoxolone and were nearly always accompanied by dye-coupling. These results indicate that thalamocortical neurones may be electrotonically coupled via gap junctions with spikelets representing attenuated action potentials from adjoining cells. We suggest that the presence of electrotonic communication between thalamocortical neurones would have major implications for the understanding of both physiological (Steriade et al., 1993; Sillito et al., 1994; Alonso et al., 1996; Neuenschwander and Singer, 1996; Weliky and Katz, 1999) and pathological (Steriade and Contreras, 1995; Pinault et al., 1998) synchronised electrical activity in the thalamus.     

Brain stem afferents to the rat medial septum.

1. Activity of neurones in the medial septal nucleus and the diagonal band was recorded from urethane anaesthesized rats. Responses of the cells to electrical stimulation of the raphe nuclei and nucleus locus coeruleus (LC) were measured. 2. LC stimulation caused a long latency, 30-100 msec, and long duration, 100-300 msec cessation of spontaneous activity of most recorded neurones. When bursting-type neurones were recorded, the stimulation occasionally caused a synchronized repetitive bursting firing pattern. 3. Pre-treatment with drugs which interfere with catecholamine neurotransmission, i.e. reserpine and 6OHDA, prevented the appearance of cellular responses to LC stimulation. 4. Stimulation of the dorsal or the median raphe nuclei generated more complex and less clear-cut responses. These included several types of long (20-50 msec) and short (2-5 msec) latency responses. These responses were also accompanied in some cells by synchronized repetitive bursting. 5. Interference with serotonin neurotransmission with PPCA or reserpine reduced the detection of long latency responses. 6. Short latency responses accompanied by evoked field potentials were recorded also after stimulation of dorsal tegmental nucleus. 7. Rates of spontaneous firing cells were augmented after monoamine neurotransmission interruption whereas after fornix lesion, when there is supposedly an increased monoamine innervation of the septum, cells fire at lower rates than normal. 8. It is suggested that noradrenaline and serotonin may serve as neurotransmitters in the medial-septum-diagonal band areas.     

Synchronization of epileptiform bursts induced by 4-aminopyridine in the in vitro hippocampal slice preparation

Neuronal activity in hippocampal slices can be synchronized by drugs which either block synaptic inhibition (e.g. bicuculline methiodide) or do not (e.g. 4-aminopyridine). Here we compare these two drugs to assess the role of inhibition on the recruitment of neurones into synchronous epileptiform bursts. With 4-aminopyridine we recorded an acceleration of neuronal activity, and in most cells a slow depolarization (mean 6.8 mV), during the ca. 100 ms preceding the population burst. With bicuculline these changes occurred ca. 10 ms before the population bursts, and depolarizations reached a mean of 2.5 mV. We propose that bicuculline-sensitive synaptic inhibition retards the recruitment of neurones into epileptiform synchronous bursts.     

An ascending spinal pathway transmitting a central rhythmic pattern to the magnocellular red nucleus in the cat

The activity of cells in the magnocellular red nucleus (RNm) was recorded extra and intracellularly in the curarized thalamic cat performing fictive locomotion. The locomoter episodes were detected from the rhythmic activity recorded in the motor nerves of the contralateral hindlimb. It was confirmed that, during fictive locomotion, a large proportion of the rubrospinal cells (56% in our sample) exhibit a rhythmic pattern of activity which is synchronized with the efferent spinal motor nerve activity. On the basis of the intracellular recordings it was established that phases of intense synaptic activity with mixed excitatory postsynaptic potentials (EPSPs) and inhibitory postsynaptic potentials (IPSPs) are involved in this rhythmicity. After eliminating the cerebellar input to the RNm, it was observed that the cells still received intense excitatory and inhibitory inputs, resulting in a continuous modulation of their membrane potential, due to the occurrence of EPSPs and IPSPs. During fictive locomotor like activity and after elimination of the cerebellar afferents to the RNm, it was observed that the spontaneous PSPs in RNm cells (in the case of 45% of the cells) were organized in repetitive subthreshold bursts occurring in phase relationships with the activity recorded in the motor nerves. Some extracellularly recorded cells (12%) showed a rhythmic firing pattern. It is generally recognized that, in the thalamic cat preparation, the locomotor pattern observed in efferent nerves originates from the central pattern generator (CPG) of the spinal cord. It therefore seems likely that the rhythmicity observed here in the RNm may originate from the spinal CPG and be transmitted through the spino rubral pathway ascending in the ventral part of the cord. It is concluded that the spino rubral pathway may transmit both somatosensory information and corollary discharges relating to the activity of the spinal CPG for locomotion.     

Gap junctions between accessory medulla neurons appear to synchronize circadian clock cells of the cockroach Leucophaea maderae

The temporal organization of physiological and behavioral states is controlled by circadian clocks in apparently all eukaryotic organisms. In the cockroach Leucophaea maderae lesion and transplantation studies located the circadian pacemaker in the accessory medulla (AMe). The AMe is densely innervated by gamma-aminobutyric acid (GABA)-immunoreactive and peptidergic neurons, among them the pigment-dispersing factor immunoreactive circadian pacemaker candidates. The large majority of cells of the cockroach AMe spike regularly and synchronously in the gamma frequency range of 25-70 Hz as a result of synaptic and nonsynaptic coupling. Although GABAergic coupling forms assemblies of phase-locked cells, in the absence of synaptic release the cells remain synchronized but fire now at a stable phase difference. To determine whether these coupling mechanisms of AMe neurons, which are independent of synaptic release, are based on electrical synapses between the circadian pacemaker cells the gap-junction blockers halothane, octanol, and carbenoxolone were used in the presence and absence of synaptic transmission. Here, we show that different populations of AMe neurons appear to be coupled by gap junctions to maintain synchrony at a stable phase difference. This synchronization by gap junctions is a prerequisite to phase-locked assembly formation by synaptic interactions and to synchronous gamma-type action potential oscillations within the circadian clock.