The mormyrid brainstem--III. Ultrastructure and synaptic organization of the medullary "pacemaker" nucleus

The ultrastructure and synaptic organization of the presumed medullary pacemaker nucleus, nucleus c of the weakly electric mormyrid fish, Gnathonemus petersii has been investigated. Nucleus c consists of about 12-15 small (20-25 micron) neurones (P-cells), which form a group situated ventrally to the medullary relay nucleus and embedded in a neuropil of myelinated fibres and dendritic processes. The P-cells often exhibit an enhanced electron density of their cytoplasm and dendroplasm. They possess several dendrites of different diameter, a short, thin axon initial segment and a thickly myelinated axon running in dorsal direction. The pacemaker neurons are interconnected by complex electronic coupling, established by somatosomatic, dendrosomatic and dendrodendritic gap junctions. Perikarya and dendrites are frequently interconnected serially by gap junctions; dendrites showed sometimes triadic gap-junction arrangement. It is suggested that this high degree of electrotonic coupling amongst the pacemaker cells represents the first level of the highly ordered synchronization processes which characterize the electric discharge command system of Gnathonemus. Pacemaker cells receive synaptic input from club endings with mixed synapses and from bouton-like terminals with chemical synapses, both of them originating from medium-sized myelinated fibres and contacting mainly neuronal perikarya and dendritic processes. The axon initial segment receives only few synaptic inputs. Bouton-like terminals were found to be of two types according to their vesicle content, namely, boutons with ovoid, clear synaptic vesicles forming Gray type-1 synapses and boutons with pleomorphic clear synaptic vesicles forming Gray type-2 synapses. Different functional roles for the two types of boutons in modulating pacemaker cell activity are suggested.     

Distal gap junctions and active dendrites can tune network dynamics

Gap junctions allow direct electrical communication between CNS neurons. From theoretical and modeling studies, it is well known that although gap junctions can act to synchronize network output, they can also give rise to many other dynamic patterns including antiphase and other phase-locked states. The particular network pattern that arises depends on cellular, intrinsic properties that affect firing frequencies as well as the strength and location of the gap junctions. Interneurons or GABAergic neurons in hippocampus are diverse in their cellular characteristics and have been shown to have active dendrites. Furthermore, parvalbumin-positive GABAergic neurons, also known as basket cells, can contact one another via gap junctions on their distal dendrites. Using two-cell network models, we explore how distal electrical connections affect network output. We build multi-compartment models of hippocampal basket cells using NEURON and endow them with varying amounts of active dendrites. Two-cell networks of these model cells as well as reduced versions are explored. The relationship between intrinsic frequency and the level of active dendrites allows us to define three regions based on what sort of network dynamics occur with distal gap junction coupling. Weak coupling theory is used to predict the delineation of these regions as well as examination of phase response curves and distal dendritic polarization levels. We find that a nonmonotonic dependence of network dynamic characteristics (phase lags) on gap junction conductance occurs. This suggests that distal electrical coupling and active dendrite levels can control how sensitive network dynamics are to gap junction modulation. With the extended geometry, gap junctions located at more distal locations must have larger conductances for pure synchrony to occur. Furthermore, based on simulations with heterogeneous networks, it may be that one requires active dendrites if phase-locking is to occur in networks formed with distal gap junctions.     

Effect of electrical coupling on ionic current and synaptic potential measurements

Recent studies have found electrical coupling to be more ubiquitous than previously thought, and coupling through gap junctions is known to play a crucial role in neuronal function and network output. In particular, current spread through gap junctions may affect the activation of voltage-dependent conductances as well as chemical synaptic release. Using voltage-clamp recordings of two strongly electrically coupled neurons of the lobster stomatogastric ganglion and conductance-based models of these neurons, we identified effects of electrical coupling on the measurement of leak and voltage-gated outward currents, as well as synaptic potentials. Experimental measurements showed that both leak and voltage-gated outward currents are recruited by gap junctions from neurons coupled to the clamped cell. Nevertheless, in spite of the strong coupling between these neurons, the errors made in estimating voltage-gated conductance parameters were relatively minor (<10%). Thus in many cases isolation of coupled neurons may not be required if a small degree of measurement error of the voltage-gated currents or the synaptic potentials is acceptable. Modeling results show, however, that such errors may be as high as 20% if the gap-junction position is near the recording site or as high as 90% when measuring smaller voltage-gated ionic currents. Paradoxically, improved space clamp increases the errors arising from electrical coupling because voltage control across gap junctions is poor for even the highest realistic coupling conductances. Furthermore, the common procedure of leak subtraction can add an extra error to the conductance measurement, the sign of which depends on the maximal conductance.     

Connexin36 vs. connexin32, "miniature" neuronal gap junctions, and limited electrotonic coupling in rodent suprachiasmatic nucleus

Suprachiasmatic nucleus (SCN) neurons generate circadian rhythms, and these neurons normally exhibit loosely-synchronized action potentials. Although electrotonic coupling has long been proposed to mediate this neuronal synchrony, ultrastructural studies have failed to detect gap junctions between SCN neurons. Nevertheless, it has been proposed that neuronal gap junctions exist in the SCN; that they consist of connexin32 or, alternatively, connexin36; and that connexin36 knockout eliminates neuronal coupling between SCN neurons and disrupts circadian rhythms. We used confocal immunofluorescence microscopy and freeze-fracture replica immunogold labeling to examine the distributions of connexin30, connexin32, connexin36, and connexin43 in rat and mouse SCN and used whole-cell recordings to re-assess electrotonic and tracer coupling. Connexin32-immunofluorescent puncta were essentially absent in SCN but connexin36 was relatively abundant. Fifteen neuronal gap junctions were identified ultrastructurally, all of which contained connexin36 but not connexin32, whereas nearby oligodendrocyte gap junctions contained connexin32. In adult SCN, one neuronal gap junction was >600 connexons, whereas 75% were smaller than 50 connexons, which may be below the limit of detectability by fluorescence microscopy and thin-section electron microscopy. Whole-cell recordings in hypothalamic slices revealed tracer coupling with neurobiotin in <5% of SCN neurons, and paired recordings (>40 pairs) did not reveal obvious electrotonic coupling or synchronized action potentials, consistent with few neurons possessing large gap junctions. However, most neurons had partial spikes or spikelets (often <1 mV), which remained after QX-314 [N-(2,6-dimethylphenylcarbamoylmethyl)triethylammonium bromide] had blocked sodium-mediated action potentials within the recorded neuron, consistent with spikelet transmission via small gap junctions. Thus, a few "miniature" gap junctions on most SCN neurons appear to mediate weak electrotonic coupling between limited numbers of neuron pairs, thus accounting for frequent detection of partial spikes and hypothetically providing the basis for "loose" electrical or metabolic synchronization of electrical activity commonly observed in SCN neuronal populations during circadian rhythms.     

Gap junction effects on precision and frequency of a model pacemaker network

We investigated the precision of spike timing in a model of gap junction-coupled oscillatory neurons. The model incorporated the known physiology, morphology, and connectivity of the weakly electric fish's high-frequency and extremely precise pacemaker nucleus (Pn). Two neuron classes, pacemaker and relay cells, were each modeled with two compartments containing Hodgkin-Huxley sodium and potassium currents. Isolated pacemaker cells fired periodically, due to a constant current injection; relay cells were silent but slightly depolarized at rest. When coupled by gap junctions to other neurons, a model neuron, like its biological correlate, spiked at frequencies and amplitudes that were largely independent of current injections. The phase distribution in the network was labile to intracellular current injections and to gap junction conductance changes. The model predicts a biologically plausible gap junction conductance of 4-5 nS (200-250 MOmega). This results in a coupling coefficient of approximately 0.02, as observed in vitro. Network parameters were varied to test which could improve the temporal precision of oscillations. Increased gap junction conductances and larger numbers of cells (holding total junctional conductance per cell constant) both substantially reduced the coefficient of variation (CV = standard deviation/mean) of relay cell spike times by 74-85% and more, and did so with lower gap junction conductance when cells were contacted axonically compared with somatically. Pacemaker cell CV was only reduced when the probability of contact was increased, and then only moderately: a fivefold increase in the probability of contact reduced CV by 35%. We conclude that gap junctions facilitate synchronization, can reduce CV, are most effective between axons, and that pacemaker cells must have low intrinsic CV to account for the low CV of cells in the biological network.     

Potential pathways for intercellular communication within the calbindin subnucleus of the hamster suprachiasmatic nucleus

In mammals, the suprachiasmatic nucleus (SCN) is the master circadian pacemaker. Within the caudal hamster SCN, a cluster of neurons containing the calcium binding protein, calbindin-D28K (CB), has been implicated in circadian locomotion. However, calbindin-immunoreactive (CB+) neurons in the calbindin subnucleus (CBsn) do not display a circadian rhythm in spontaneous firing [Eur J Neurosci 16 (2002) 2469]. Previously, we proposed that intercellular communication might be essential in integrating outputs from rhythmic (CB-) neurons and nonrhythmic (CB+) neurons to produce a circadian output in the intact animal. The primary aim of this study is to provide a neuroanatomical framework to better understand intercellular communication within the CBsn. Using reconstructions of previously recorded neurons, we demonstrate that CB+ neurons have significantly more dendrites than CB- neurons. In addition, CBsn neurons have dorsally oriented dendritic arbors. Using double-label confocal microscopy, we show that GABA colocalizes with CB+ neurons and GABA(A) receptor subunits make intimate contacts with neurons in the CBsn. Transforming growth factor alpha (TGFalpha), a substance shown to inhibit locomotion [Science 294 (2001) 2511], is present within the CBsn. In addition, neurons in this region express the epidermal growth factor receptor, the only receptor for TGFalpha. Lastly, we show that CB+ neurons are coupled to CB+ and CB- neurons by gap junctions. The current study provides a structural framework for synaptic communication, electrical coupling, and signaling via a growth factor within the CBsn of the hamster SCN. Our results reveal connections that have the potential for integrating cellular communication within a subregion of the SCN that is critically involved in circadian locomotion.     

Morphological correlates of electrotonic coupling in the magnocellular mesencephalic nucleus of the weakly electric fish Gymnotus carapo.

The magnocellular mesencephalic nucleus (MMN) of Gymnotus carapo was studied by electron microscopy. This particular nucleus, characteristic of weakly electric fish, contains two principal classes of neuron. (1) Large neurons (25-35 mum): these are rounded unipolar cells, with the perikaryon partially covered by a sheath of compact myelin. The axon leaves the neuron as a short thick unmyelinated process not resembling the initial segment of multipolar neurons. The axon branches profusely and becomes myelinated very close to its origin. The perikaryal surface not covered by the myelin sheath receives abundant club endings. The synaptic interface between club endings and large neurons is characterized by alternating gap junctions and attachment plaques. In addition, at the periphery of the club endings, "active" zones are generally present, and this synapse is therefore a "mixed" synapse. (2) Small neurons (5-12 mum): these are uni- or bipolar cells, scattered throughout the nucleus, and occasionally, grouped in small clusters. Gap junctions were not observed between neuronal perikarya in such clusters. The synaptic investment of small neurons is formed by long cup endings which almost completely encircle the perikarya. The synaptic interface between cup endings and the perikarya of small neurons is characterized by large areas of gap junctions. A single cup ending establishing gap junctions with two small neurons within the plane of the section was frequently observed and this arrangement provides a morphological basis for electrotonic coupling between small neurons by way of presynaptic fibres. In the neuropil of the MMN, there are abundant synaptic islands constituted by a large axon terminal in synaptic contact with small unidentified profiles; both synaptic elements are surrounded by numerous thin glial lamellae. At the synaptic interface, in the islands, both gap junctions and "active" zones are present. The synaptic islands must also be considered as "mixed" synapses. The morphological results presented here correlate with electrophysiological data (Szabo et al., 1975). The very short delay (0.8-1.3 ms) of the MMS response to the fish's own electric organ discharge can only be explained by the existence of electrotonic transmission along the neuronal chain of the electrosensory pathway. The presence of gap junctions between club endings and large neurons provides a morphological basis for electrotonic transmission at the mesencephalic level of the electrosensory pathway.     

Precision of the pacemaker nucleus in a weakly electric fish: network versus cellular influences

We investigated the relative influence of cellular and network properties on the extreme spike timing precision observed in the medullary pacemaker nucleus (Pn) of the weakly electric fish Apteronotus leptorhynchus. Of all known biological rhythms, the electric organ discharge of this and related species is the most temporally precise, with a coefficient of variation (CV = standard deviation/mean period) of 2 x 10(-4) and standard deviation (SD) of 0.12-1.0 micros. The timing of the electric organ discharge is commanded by neurons of the Pn, individual cells of which we show in an in vitro preparation to have only a slightly lesser degree of precision. Among the 100-150 Pn neurons, dye injection into a pacemaker cell resulted in dye coupling in one to five other pacemaker cells and one to three relay cells, consistent with previous results. Relay cell fills, however, showed profuse dendrites and contacts never seen before: relay cell dendrites dye-coupled to one to seven pacemaker and one to seven relay cells. Moderate (0.1-10 nA) intracellular current injection had no effect on a neuron's spiking period, and only slightly modulated its spike amplitude, but could reset the spike phase. In contrast, massive hyperpolarizing current injections (15-25 nA) could force the cell to skip spikes. The relative timing of subthreshold and full spikes suggested that at least some pacemaker cells are likely to be intrinsic oscillators. The relative amplitudes of the subthreshold and full spikes gave a lower bound to the gap junctional coupling coefficient of 0.01-0.08. Three drugs, called gap junction blockers for their mode of action in other preparations, caused immediate and substantial reduction in frequency, altered the phase lag between pairs of neurons, and later caused the spike amplitude to drop, without altering the spike timing precision. Thus we conclude that the high precision of the normal Pn rhythm does not require maximal gap junction conductances between neurons that have ordinary cellular precision. Rather, the spiking precision can be explained as an intrinsic cellular property while the gap junctions act to frequency- and phase-lock the network oscillations.     

Short duty cycle destabilizes a half-center oscillator, but gap junctions can restabilize the anti-phase pattern

Mutually inhibitory pacemaker neurons with duty cycle close to 50% operate as a half-center oscillator (anti-phase coordination, i.e., 180 degrees out of phase), even in the presence of weak to modest gap junctional coupling. For electrical coupling strength above a critical value synchronization occurs. But, as shown here with modeling studies, the effects of electrical coupling depend critically on a cell's duty cycle. Instead of oscillating either in-phase or anti-phase, model cells with short duty cycle express additional rhythmic patterns, and different transitions between them, depending on electrical coupling strength. For weak or no electrical coupling, cells do not oscillate in anti-phase but instead exhibit almost in-phase activity. Strengthening this weak coupling leads to stable anti-phase activity. With yet stronger electrical coupling stable inphase (synchrony) emerges but it coexists with the anti-phase pattern. Thus the network shows bistability for an intermediate range of coupling strength. For sufficiently strong electrical coupling synchrony is the network's only attracting rhythmic state. Our results, numerical and analytical (phase plane analysis), are based on a minimal but biophysically motivated pacemaker model for the slowly oscillating envelope of bursting neurons. However, illustrations for an Hodgkin-Huxley model suggest that some of our results for short duty cycle may extend to patterning of repetitive spikes. In particular, electrical coupling of intermediate strength may promote anti-phase activity and provide bistability of anti-phase and in-phase spiking.     

Synaptology of the command (pacemaker) nucleus in the brain of the weakly electric fish, Sternarchus (Apteronotus) albifrons

The high frequency electric emission of the weakly electric fish Sternarchus (Apteronotus) albifrons depends on the pacemaker activity of a specific brainstem nucleus located in the ventral part of the rhombencephalic reticular formation. The general morphology and fine structure of this nucleus has been investigated, with particular reference to its synaptic connections.Three neuronal components could be distinguished in the nucleus; namely large cells of 80–100 μm diameter, small cells of 30–50 μm diameter and bundles of thin, myelinated fibres. These elements are embedded in a network of thick myelinated fibres. The large cells have a few small and short dendrites whereas the small neurons have long branching dendrites. Large and small neurons possess thick myelinated axons, but only those of the latter show branching patterns and send collaterals which have intranuclear courses only. Two types of synaptic terminals have been found on both neurons: large club endings exclusively with gap junctions and small bouton-like terminals with polarized chemical synapses. Serial semi-thin and ultra-thin sections revealed that the large club endings belong to the pacemaker cells, whereas the small terminals are found in the thin myelinated axons of extranuclear origin.The findings indicate that the small neurons are connected 1) to each other and 2) to the large neurons, by way of their large myelinated axons. Both, small (pacemaker) as well as large (relay), neurons receive chemical synapses from myelinated fine fibers probably originating from higher encephalic centers. Thus, electric organ discharge rhythm can be modulated at the level of pacemaker as well as of the relay cells. No somatosomatic, dendrodendritic or dendrosomatic connections were found between large, small or large and small cells.     

The extent and strength of electrical coupling between inferior olivary neurons is heterogeneous

Gap junctions constitute the only form of synaptic communication between neurons in the inferior olive (IO), which gives rise to the climbing fibers innervating the cerebellar cortex. Although its exact functional role remains undetermined, electrical coupling was shown to be necessary for the transient formation of functional compartments of IO neurons and to underlie the precise timing of climbing fibers required for cerebellar learning. So far, most functional considerations assume the existence of a network of permanently and homogeneously coupled IO neurons. Contrasting this notion, our results indicate that coupling within the IO is highly variable. By combining tracer-coupling analysis and paired electrophysiological recordings, we found that individual IO neurons could be coupled to a highly variable number of neighboring neurons. Furthermore, a given neuron could be coupled at remarkably different strengths with each of its partners. Freeze-fracture analysis of IO glomeruli revealed the close proximity of glutamatergic postsynaptic densities to connexin 36-containing gap junctions, at distances comparable to separations between chemical transmitting domains and gap junctions in goldfish mixed contacts, where electrical coupling was shown to be modulated by the activity of glutamatergic synapses. On the basis of structural and molecular similarities with goldfish mixed synapses, we speculate that, rather than being hardwired, variations in coupling could result from glomerulus-specific long-term modulation of gap junctions. This striking heterogeneity of coupling might act to finely influence the synchronization of IO neurons, adding an unexpected degree of complexity to olivary networks.     

Proximally targeted GABAergic synapses and gap junctions synchronize cortical interneurons

Networks of GABAergic interneurons are implicated in synchronizing cortical activity at gamma frequencies (30-70 Hz). Here we demonstrate that the combined electrical and GABAergic synaptic coupling of basket cells instantaneously entrained gamma-frequency postsynaptic firing in layers 2/3 of rat somatosensory cortex. This entrainment was mediated by rapid curtailment of gap junctional coupling potentials by GABAA receptor-mediated IPSPs. Electron microscopy revealed spatial proximity of gap junctions and GABAergic synapses on somata and dendrites. Electrical coupling alone entrained postsynaptic firing with a phase lag, whereas unitary GABAergic connections were ineffective in gamma-frequency phasing. These observations demonstrate precise spatiotemporal mechanisms underlying action potential timing in oscillating interneuronal networks.     

Clock controls circadian period in isolated suprachiasmatic nucleus neurons

The suprachiasmatic nucleus (SCN) is the master circadian pacemaker in mammals, and one molecular regulator of circadian rhythms is the Clock gene. Here we studied the discharge patterns of SCN neurons isolated from Clock mutant mice. Long-term, multielectrode recordings showed that heterozygous Clock mutant neurons have lengthened periods and that homozygous Clock neurons are arrhythmic, paralleling the effects on locomotor activity in the animal. In addition, cells in dispersals expressed a wider range of periods and phase relationships than cells in explants. These results suggest that the Clock gene is required for circadian rhythmicity in individual SCN cells and that a mechanism within the SCN synchronizes neurons and restricts the range of expressed circadian periods.     

The lateral vestibular nucleus of the toadfish Opsanus tau: Ultrastructural and electrophysiological observations with special reference to electrotonic transmission

The lateral vestibular nucleus of the toadfish Opsanus tau was localized by means of axonal iontophoresis of Procion Yellow. The ultrastructure of the lateral vestibular nucleus neurons was then correlated with their electrophysiological properties. The lateral vestibular nucleus consists of neurons of various sizes which are distributed in small clusters over a heavily myelinated neuropil. The perikarya and main dendrites of the large and the small neurons are surrounded by a synaptic bed, which is separated from the neighboring neuropil by a layer of thin astrocytic processes. The synaptic bed contains three main classes of axon terminals, club endings, large and small terminals, the first being quite infrequent. All the large terminals as well as the occasionally observed club endings contain a pure population of rounded synaptic vesicles. In some of the small axon terminals there are also rounded vesicles; however, the majority contain flattened vesicles or a pleomorphic population. These data indicate that the small terminals originate from different afferent sources. The synaptic interfaces of the large boutons and of the club endings bear three types of junctional complexes: attachment plates, gap junctions and active zones. Those showing both gap junctions and active zones were designated as morphologically ‘mixed synapses’. Gap junctions, although in large number, have only been observed at the synaptic interfaces between terminals with rounded vesicles and the perikarya or the dendrite of the lateral vestibular nucleus neurons. Therefore electrotonic coupling would only be possible by way of presynaptic fibers. Some axons observed in the neuropil were found to establish gap junctional complexes with two different dendritec profiles and this observation is in favour of electrotonic coupling by way of presynaptic terminals.Field and intracellular potentials were recorded in the lateral vestibular nucleus. The field potential evoked by stimulation of the vestibular nerve consisted of an early positive-negative wave followed by a slow negativity, and that evoked by spinal cord stimulation was composed of an antidromic potential followed by a slow negative wave. Vestibulo-spinal neurons were identified by their antidromic spikes. In these cells, stimulation of the ipsilateral vestibular nerve evoked an excitatory postsynaptic potential with two components. The short delay of the first component of this excitatory postsynaptic potential and its ability to follow paired stimulation at close intervals without reduction of the second response suggest that it is transmitted electrotonically from primary vestibular afferent fibers. By contrast the latency of the second peak of the vestibular evoked excitatory postsynaptic potential and its sensitivity to high stimulus frequencies are compatible with monosynaptic chemically mediated transmission from primary vestibular afferents. Spinal stimulation evoked graded antidromic depolarizations in vestibulo-spinal neurons. The latency of these potentials was too short to allow for chemical transmission through afferents or recurrent collaterals and suggests electrotonic spread of antidromic activity from neighboring neurons. An important finding is that the graded antidromic depolarizations can initiate spikes; thus coupling between neurons in the lateral vestibular nucleus is sufficiently close that a cell can be excited by activity spread from neighboring cells. Similar graded depolarizations were recorded in identified primary vestibular afferents; their latencies and time course indicate that they were brought about by electrotonic spread of postsynaptic potentials and spikes to the impaled presynaptic fibers; this confirms the morphological evidence that coupling between lateral vestibular nucleus neurons occurs, at least in part, by way of presynaptic vestibular axons. As the spinal stimulus strength was increased, these graded depolarizations became large enough to initiate spikes which presumably propagate to the vestibular receptors. Thus antidromic invasion of the presynaptic terminals may provide negative feedback by preventing their re-excitation at short intervals after a synchronous discharge of an adequate number of postsynaptic cells. Excitatory inputs to the neurons of the lateral vestibular nucleus were identified from the spinal cord and from the contralateral vestibular nerve. Long latency excitatory postsynaptic potentials large enough to excite the cells were recorded following spinal stimulation; the threshold intensity for evoking them was consistently higher than that adequate to generate the graded antidromic depolarizations. Field potentials recorded after stimulation of the contra lateral vestibular nerve consisted of an initial positive negative wave followed by a slow negative wave. the stimulus intensity for evoking these potentials was the same or slightly above the threshold for those evoked in the lateral vestibular nucleus on the stimulated side. Also lateral vestibular nucleus neurons exhibited excitatory postsynaptic potentials large enough to excite the cells following stimulation of the contralateral vestibular nerve. but no inhibitory postsynaptic potentials were detected. This lack of commissural inhibition indicates a qualitative difference between the central organization of these cells in the toadfish and in mammals.The presence of neurons in the lateral vestibular nucleus which send their axons to the labyrinth was confirmed by their heavy staining with Procion Yellow following axonal iontophoresis. In a number of vestibular neurons. abruptly rising spikes were evoked at short latencies after adequate stimulation of the ipsilateral vestibular nerve. Graded stimuli applied to the vestibular nerve evoked graded short latency depolarizations as well as long latency excitatory postsynaptic potentials in these presumed efferent neurons to the labyrinth; the former could indicate electrotonic coupling of the efferent cells or electrotonic transmission from primary afferents, resulting in a short latency feedback loop.From these studies, the synaptic organization of the lateral vestibular nucleus neurons is compared with that of the Mauthner cells of teleosts, and the possibility of a dual mode of transmission, electrical and chemical, by primary vestibular afferents is discussed.     

Aging is associated with an increase in dye coupling and in gap junction number in satellite glial cells of murine dorsal root ganglia

Glial cells in both central and peripheral nervous systems are connected by gap junctions, which allow electrical and metabolic coupling between them. In spite of the great current interest in aging of the nervous system, the effect of aging on glial cell coupling received little attention. We examined coupling between satellite glial cells in murine dorsal root ganglia using the dye coupling technique and electron microscopy. We studied mice at ages of postnatal 90-730 days. Dye coupling incidence between satellite glial cells associated with a single neuron increased from 24.2% at postnatal day 90 to 50.5% at postnatal day 730. Dye coupling between satellite glial cells that are in contact with two or more neurons increased from 2.7% at postnatal day 90 to 18.6% at postnatal day 730 (P<0.05). Examination of the ganglia with the electron microscope showed that the number of gap junctions per 100 microm2 of surface area of satellite glial cells increased from 0.22 at postnatal day 90 to 1.56 at postnatal day 730 (P<0.01). The mean length of individual gap junctions did not change with age. These results provide strong evidence for an increase of functional coupling between satellite glial cells during life. This increase is apparently due to an increase in the total area of the system of gap junctions connecting these cells.     

Distinct synaptic dynamics of heterogeneous pacemaker neurons in an oscillatory network

Many rhythmically active networks involve heterogeneous populations of pacemaker neurons with potentially distinct synaptic outputs that can be differentially targeted by extrinsic inputs or neuromodulators, thereby increasing possible network output patterns. To understand the roles of heterogeneous pacemaker neurons, we characterized differences in synaptic output from the anterior burster (AB) and pyloric dilator (PD) neurons in the lobster pyloric network. These intrinsically distinct neurons are strongly electrically coupled, coactive, and constitute the pyloric pacemaker ensemble. During pyloric oscillations, the pacemaker neurons produce compound inhibitory synaptic connections to the follower lateral pyloric (LP) and pyloric constrictor (PY) neurons, which fire out of phase with AB/PD and with different delay times. Using pharmacological blockers, we separated the synapses originating from the AB and PD neurons and investigated their temporal dynamics. These synapses exhibited distinct short-term dynamics, depending on the presynaptic neuron type, and had different relative contributions to the total synaptic output depending on waveform shape and cycle frequency. However, paired comparisons revealed that the amplitude or dynamics of synapses from either the AB or PD neuron did not depend on the postsynaptic neuron type, LP or PY. To address the functional implications of these findings, we examined the correlation between synaptic inputs from the pacemakers and the burst onset phase of the LP and PY neurons in the ongoing pyloric rhythm. These comparisons showed that the activity of the LP and PY neurons is influenced by the peak phase and amplitude of the synaptic inputs from the pacemaker neurons.     

Computational model of electrically coupled, intrinsically distinct pacemaker neurons

Electrical coupling between neurons with similar properties is often studied. Nonetheless, the role of electrical coupling between neurons with widely different intrinsic properties also occurs, but is less well understood. Inspired by the pacemaker group of the crustacean pyloric network, we developed a multicompartment, conductance-based model of a small network of intrinsically distinct, electrically coupled neurons. In the pyloric network, a small intrinsically bursting neuron, through gap junctions, drives 2 larger, tonically spiking neurons to reliably burst in-phase with it. Each model neuron has 2 compartments, one responsible for spike generation and the other for producing a slow, large-amplitude oscillation. We illustrate how these compartments interact and determine the dynamics of the model neurons. Our model captures the dynamic oscillation range measured from the isolated and coupled biological neurons. At the network level, we explore the range of coupling strengths for which synchronous bursting oscillations are possible. The spatial segregation of ionic currents significantly enhances the ability of the 2 neurons to burst synchronously, and the oscillation range of the model pacemaker network depends not only on the strength of the electrical synapse but also on the identity of the neuron receiving inputs. We also compare the activity of the electrically coupled, distinct neurons with that of a network of coupled identical bursting neurons. For small to moderate coupling strengths, the network of identical elements, when receiving asymmetrical inputs, can have a smaller dynamic range of oscillation than that of its constituent neurons in isolation.     

Electron microscopy of synapses in reptile spinal cord

The spinal cord of the reptile Anolis carolinensis was examined by electron microscopy. Motor neurons appear as multipolar cells 30-60 micrometer in diameter. Two types of synaptic endings are endings are present on motor neurons. The first type is characterized by distinct synaptic clefts measuring 15-20 nm between pre- and postsynaptic membranes, and by clear presynaptic vesicles. The second type of synapse, which is less common, is characterized by gap junctions between pre- and postsynaptic membranes. At these synapses, there are also clusters of clear vesicles close to the presynaptic membrane adjacent to the gap junction. These findings indicate that both chemical and electrical synaptic transmission are present in the spinal cord of Anolis.     

A mechanism for production of phase shifts in a pattern generator

During motor activity of the pyloric system of the lobster stomatogastric ganglion, there are rhythmic alternations between activity in the pyloric dilator (PD) and pyloric (PY) motor neurons. We studied the phase relations between PD motor neuron activity and PY motor neuron activity in preparations cycling at a wide range of frequencies and after altering the activity of the PD neurons. The PY neurons fall into two classes, early (PE) and late (PL) (21), distinguished by the different phases in the pyloric cycle at which they fire. The phase at which PE neurons fired and the phase at which PL neurons fired was independent of pyloric cycle frequency over a range of frequencies from 0.5 to 2.25 Hz. The anterior burster (AB) interneuron is electrically coupled to the PD motor neurons. Together the AB and PD neurons form the pacemaker for the pyloric system. Synchronous depolarization of the AB and PD neurons evokes a complex inhibitory post-synaptic potential (IPSP) in PY neurons. This IPSP has two components: an early, AB neuron-derived component and a late, PD neuron-derived component. Deletion of the PD neurons from the pyloric circuit by photoinactivation removed the PD-evoked component of the pacemaker-evoked IPSP. This resulted in a decrease in the duration of the IPSP evoked by pacemaker depolarization and a significant advance in the firing phase of PY neurons. Bath application of dopamine was used to hyperpolarize and inhibit the PD neurons (30), causing them to release less neurotransmitter. As a consequence, the duration of the IPSP evoked by pacemaker depolarization was decreased and the firing phase of the PY neurons was significantly advanced. Stimulation of the inferior ventricular nerve (IVN) produces a slow excitation of the PD neurons (30), causing them to release more neurotransmitter. Consequently, the duration of the IPSP evoked by pacemaker depolarization was increased and the firing phase of the PY neurons was significantly retarded for several cycles of pyloric activity following IVN stimulation. Thus, modulation of the strength of PD-evoked inhibition in PY neurons is responsible for altering the firing phase of the PY neurons with respect to the pyloric pacemaker. We suggest that frequency of the pyloric output and the phase relations of the elements within the pyloric cycle can be regulated independently. The potential implications of these data for modulation of synaptic efficacy in other preparations are discussed.     

Gap junctions are required for NMDA receptor dependent cell death in developing neurons

A number of studies have indicated an important role for N-methyl-D-aspartate (NMDA) receptors in cell survival versus cell death decisions during neuronal development, trauma, and ischemia. Coupling of neurons by electrical synapses (gap junctions) is high or increases in neuronal networks during all three of these conditions. However, whether neuronal gap junctions contribute to NMDA receptor-regulated cell death is not known. Here we address the role of neuronal gap junction coupling in NMDA receptor-regulated cell death in developing neurons. We report that inactivation or hyperactivation of NMDA receptors induces neuronal cell death in primary hypothalamic cultures, specifically during the peak of developmental gap junction coupling. In contrast, increasing or decreasing NMDA receptor function when gap junction coupling is low has no or greatly reduced impact on cell survival. Pharmacological inactivation of gap junctions or knockout of neuronal connexin 36 prevents the cell death caused by NMDA receptor hypofunction or hyperfunction. The results indicate the critical role of neuronal gap junctions in cell death caused by increased or decreased NMDA receptor function in developing neurons. Based on these data, we propose the novel hypothesis that NMDA receptors and gap junctions work in concert to regulate neuronal survival.