Electrical synapse formation disrupts calcium-dependent exocytosis, but not vesicle mobilization

Electrical coupling exists prior to the onset of chemical connectivity at many developing and regenerating synapses. At cholinergic synapses in vitro, trophic factors facilitated the formation of electrical synapses and interfered with functional neurotransmitter release in response to photolytic elevations of intracellular calcium. In contrast, neurons lacking trophic factor induction and electrical coupling possessed flash-evoked transmitter release. Changes in cytosolic calcium and postsynaptic responsiveness to acetylcholine were not affected by electrical coupling. These data indicate that transient electrical synapse formation delayed chemical synaptic transmission by imposing a functional block between the accumulation of presynaptic calcium and synchronized, vesicular release. Despite the inability to release neurotransmitter, neurons that had possessed strong electrical coupling recruited secretory vesicles to sites of synaptic contact. These results suggest that the mechanism by which neurotransmission is disrupted during electrical synapse formation is downstream of both calcium influx and synaptic vesicle mobilization. Therefore, electrical synaptogenesis may inhibit synaptic vesicles from acquiring a readily releasable state. We hypothesize that gap junctions might negatively interact with exocytotic processes, thereby diminishing chemical neurotransmission.     

Retinal waves are governed by collective network properties.

Propagating neural activity in the developing mammalian retina is required for the normal patterning of retinothalamic connections. This activity exhibits a complex spatiotemporal pattern of initiation, propagation, and termination. Here, we discuss the behavior of a model of the developing retina using a combination of simulation and analytic calculation. Our model produces spatially and temporally restricted waves without requiring inhibition, consistent with the early depolarizing action of neurotransmitters in the retina. We find that highly correlated, temporally regular, and spatially restricted activity occurs over a range of network parameters; this ensures that such spatiotemporal patterns can be produced robustly by immature neural networks in which synaptic transmission by individual neurons may be unreliable. Wider variation of these parameters, however, results in several different regimes of wave behavior. We also present evidence that wave properties are locally determined by a single variable, the fraction of recruitable (i.e., nonrefractory) cells within the dendritic field of a retinal neuron. From this perspective, a given local area's ability to support waves with a wide range of propagation velocities-as observed in experiment-reflects the variability in the local state of excitability of that area. This prediction is supported by whole-cell voltage-clamp recordings, which measure significant wave-to-wave variability in the amount of synaptic input a cell receives when it participates in a wave. This approach to describing the developing retina provides unique insight into how the organization of a neural circuit can lead to the generation of complex correlated activity patterns.     

Ca(2+) signalling and gap junction coupling within and between pigment epithelium and neural retina in the developing chick

Development of the neural retina is controlled in part by the adjacent retinal pigment epithelium (RPE). To understand better the mechanisms involved, we investigated calcium signalling and gap junctional coupling within and between the RPE and the neural retina in embryonic day (E) 5 chick. We show that the RPE and the ventricular zone (VZ) of the neural retina display spontaneous Ca(2+) transients. In the RPE, these often spread as waves between neighbouring cells. In the VZ, the frequency of both Ca(2+) transients and waves was lower than in RPE, but increased two-fold in its presence. Ca(2+) signals occasionally crossed the boundary between the RPE and VZ in either direction. In both tissues, the frequency of propagating Ca(2+) waves, but not of individual cell transients, was reduced by gap junction blockers. Use of the gap junction permeant tracer Neurobiotin showed that neural retina cells are coupled into clusters that span the thickness of the retina, and that RPE cells are both coupled together and to clusters of cells in the neural retina. Immunolabelling for Cx43 showed this gap junction protein is present at the junction between the RPE and VZ and thus could potentially mediate the coupling of the two tissues. Immunolabelling for beta-tubulin and vimentin showed that clusters of coupled cells in the neural retina comprised mainly progenitor cells. We conclude that gap junctions between progenitor cells, and between these cells and the RPE, may orchestrate retinal proliferation/differentiation, via the propagation of Ca(2+) or other signalling molecules.     

Emergence of realistic retinal networks in culture promoted by the superior colliculus

The developing retina is characterized by 'retinal waves', spontaneous depolarizations that propagate through a developing network of interneurons and retinal ganglion cells. Although the circuitry underlying retinal waves is well characterized, the secreted factors that are critical for its normal development are not defined. Dissociated cell culture provides an ideal system for defining these factors; however, it is difficult to recapitulate retinal circuitry in culture. Here we demonstrate that by culturing dissociated retinal neurons in the presence of cells from the superior colliculus (SC), retinal neurons form networks that are similar to those described in the intact retina. Whole-cell voltage clamp recordings reveal the presence of a spontaneously active network of interneurons. In addition, we observed spontaneous, propagating activity reminiscent of that observed in the intact retina. We propose that the presence of factors secreted from the SC results in the development of networks that reproduce critical features of the intact retina.     

Retinal TrkB receptors regulate neural development in the inner, but not outer, retina

BDNF signaling through its TrkB receptor plays a pivotal role in activity-dependent refinement of synaptic connectivity of retinal ganglion cells. Additionally, studies using TrkB knockout mice have suggested that BDNF/TrkB signaling is essential for the development of photoreceptors and for synaptic communication between photoreceptors and second order retinal neurons. Thus the action of BDNF on refinement of synaptic connectivity of retinal ganglion cells could be a direct effect in the inner retina, or it could be secondary to its proposed role in rod maturation and in the formation of rod to bipolar cell synaptic transmission. To address this matter we have conditionally eliminated TrkB within the retina. We find that rod function and synaptic transmission to bipolar cells is not compromised in these conditional knockout mice. Consistent with previous work, we find that inner retina neural development is regulated by retinal BDNF/TrkB signaling. Specifically we show here also that the complexity of neuronal processes of dopaminergic cells is reduced in conditional TrkB knockout mice. We conclude that retinal BDNF/TrkB signaling has its primary role in the development of inner retinal neuronal circuits, and that this action is not a secondary effect due to the loss of visual signaling in the outer retina.     

Glial modulation of synaptic transmission in the retina

Glial modulation of synaptic transmission and neuronal excitability in the mammalian retina is mediated by several mechanisms. Stimulation of glial cells evokes Ca(2+) waves, which propagate through the network of retinal astrocytes and Müller cells and result in the modulation of the activity of neighboring ganglion cells. Light-evoked spiking is enhanced in some ganglion cells and depressed in others. A facilitation or depression of light-evoked excitatory postsynaptic currents is also seen in ganglion cells following glial stimulation. In addition, stimulation of glial cells evokes a sustained hyperpolarizing current in ganglion cells which is mediated by ATP release from Müller cells and activation of neuronal A(1) adenosine receptors. Recent studies reveal that light-evoked activity in retinal neurons results in an increase in the frequency of Ca(2+) transients in Müller cells. Thus, there is two-way communication between neurons and glial cells, suggesting that glia contribute to information processing in the retina.     

Expression and function of the neuronal gap junction protein connexin 36 in developing mammalian retina

With the advent of transgenic mice, much has been learned about the expression and function of gap junctions. Previously, we reported that retinal ganglion cells in mice lacking the neuronal gap junction protein connexin 36 (Cx36) have nearly normal firing patterns at postnatal day 4 (P4) but many more asynchronous action potentials than wild-type mice at P10 (Torborg et al. [2005] Nat. Neurosci. 8:72-78). With the goal of understanding the origin of this increased activity in Cx36-/- mice, we used a transgenic mouse (Deans et al. [2001] Neuron 31:477-485) to characterize the developmental expression of a Cx36 reporter in the retina. We found that Cx36 was first detected weakly at P2 and gradually increased in expression until it reached an adult pattern at P14. Although the onset of expression varied by cell type, we identified Cx36 in the glycinergic AII amacrine cell, glutamatergic cone bipolar cell, and retinal ganglion cells (RGCs). In addition, we used calcium imaging and multielectrode array recording to characterize further the firing patterns in Cx36-/- mice. Both correlated and asynchronous action potentials in P10 Cx36-/- RGCs were significantly inhibited by bath application of an ionotropic glutamate receptor antagonist, indicating that the increase in activity was synaptically mediated. Hence, both the expression patterns and the physiology suggest an increasing role for Cx36-containing gap junctions in suppressing RGC firing between waves during postnatal retinal development.     

Photoreceptive Ganglion Cells Drive Circuits for Local Inhibition in the Mouse Retina

Intrinsically photosensitive retinal ganglion cells (ipRGCs) exhibit melanopsin-dependent light responses that persist in the absence of rod and cone photoreceptor-mediated input. In addition to signaling anterogradely to the brain, ipRGCs signal retrogradely to intraretinal circuitry via gap junction-mediated electrical synapses with amacrine cells (ACs). However, the targets and functions of these intraretinal signals remain largely unknown. Here, in mice of both sexes, we identify circuitry that enables M5 ipRGCs to locally inhibit retinal neurons via electrical synapses with a nonspiking GABAergic AC. During pharmacological blockade of rod- and cone-mediated input, whole-cell recordings of corticotropin-releasing hormone-expressing (CRH⁺) ACs reveal persistent visual responses that require both melanopsin expression and gap junctions. In the developing retina, ipRGC-mediated input to CRH⁺ ACs is weak or absent before eye opening, indicating a primary role for this input in the mature retina (i.e., in parallel with rod- and cone-mediated input). Among several ipRGC types, only M5 ipRGCs exhibit consistent anatomical and physiological coupling to CRH⁺ ACs. Optogenetic stimulation of local CRH⁺ ACs directly drives IPSCs in M4 and M5, but not M1-M3, ipRGCs. CRH⁺ ACs also inhibit M2 ipRGC-coupled spiking ACs, demonstrating direct interaction between discrete networks of ipRGC-coupled interneurons. Together, these results demonstrate a functional role for electrical synapses in translating ipRGC activity into feedforward and feedback inhibition of local retinal circuits.SIGNIFICANCE STATEMENT Melanopsin directly generates light responses in intrinsically photosensitive retinal ganglion cells (ipRGCs). Through gap junction-mediated electrical synapses with retinal interneurons, these uniquely photoreceptive RGCs may also influence the activity and output of neuronal circuits within the retina. Here, we identified and studied an electrical synaptic circuit that, in principle, could couple ipRGC activity to the chemical output of an identified retinal interneuron. Specifically, we found that M5 ipRGCs form electrical synapses with corticotropin-releasing hormone-expressing amacrine cells, which locally release GABA to inhibit specific RGC types. Thus, ipRGCs are poised to influence the output of diverse retinal circuits via electrical synapses with interneurons.     

Light-evoked synaptic activity of retinal ganglion and amacrine cells is regulated in developing mouse retina

Recent studies have shown a continued maturation of visual responsiveness and synaptic activity of retina after eye opening, including the size of receptive fields of retinal ganglion cells (RGCs), light-evoked synaptic output of RGCs, bipolar cell spontaneous synaptic inputs to RGCs, and the synaptic connections between RGCs and ON and OFF bipolar cells. Light deprivation retarded some of these age-dependent changes. However, many other functional and morphological features of RGCs are not sensitive to visual experience. To determine whether light-evoked synaptic responses of RGCs undergo developmental change, we directly examined the light-evoked synaptic inputs from ON and OFF synaptic pathways to RGCs in developing retinas, and found that both light-evoked excitatory and inhibitory synaptic currents decreased, but not increased, with age. We also examined the light-evoked synaptic inputs from ON and OFF synaptic pathways to amacrine cells in developing retinas and found that the light-evoked synaptic input of amacrine cells is also downregulated in developing mouse retina. Different from the developmental changes of RGC spontaneous synaptic activity, dark rearing has little effect on the developmental changes of light-evoked synaptic activity of both RGCs and amacrine cells. Therefore, we concluded that the synaptic mechanisms mediating spontaneous and light-evoked synaptic activity of RGCs and amacrine cells are likely to be different.     

Gap junction-mediated bidirectional signaling between human fetal hippocampal neurons and astrocytes

Gap junctions are clusters of intercellular channels that connect the interiors of coupled cells. In the brain, gap junctions function as electrotonic synapses between neurons and as pathways for the exchange of metabolites and second-messenger molecules between glial cells. Astrocytes, the most abundant glial cell type coupled by gap junctions, are intimately involved in the active control of neuronal activity including synaptic transmission and plasticity. Previous studies have suggested that astrocytic-neuronal signaling may involve gap junction-mediated intercellular connections; this issue remains unresolved. In this study, we demonstrate that second-trimester human fetal hippocampal neurons and astrocytes in culture are coupled by gap junctions bidirectionally; we show that human fetal neurons and astrocytes express both the same and different connexin subtypes. The formation of functional homotypic and heterotypic gap junction channels between neurons and astrocytes may add versatility to the signaling between these cell types during human hippocampal ontogeny; disruption of such signaling may contribute to CNS dysfunction during pregnancy.     

Rod pathways in the mammalian retina use connexin 36.

Many neurons in the mammalian retina are coupled by means of gap junctions. Here, we show that, in rabbit retina, an antibody to connexin 36 heavily labels processes of AII amacrine cells, a critical interneuron in the rod pathway. Image analysis indicates that Cx36 is primarily located at dendritic crossings between overlapping AII amacrine cells. This finding suggests that Cx36 participates in homotypic gap junctions between pairs of AII amacrine cells. Cx36 was also found at AII/cone bipolar contacts, previously shown to be gap junction sites. This finding suggests that Cx36 participates at gap junctions that may be heterotypic. These results place an identified neuronal connexin in the context of a well-defined retinal circuit. The absence of Cx36 in many other neurons known to be coupled suggests the presence of additional unidentified connexins in mammalian neurons. Conversely, Cx36 labeling in other regions of the retina is not associated with AII amacrine cells, indicating some other cell types use Cx36.     

Visual afferents from an eye in the terrestrial slug Limax valentianus

Terrestrial gastropods have a lens-bearing eye on the tip of their tentacles. There are two morphologically distinct photoreceptors, called Type-I and Type-II photoreceptors, in the retina. Type-I photoreceptors are equipped with highly developed photoreceptive microvilli in their outer rhabdomeric segment, whereas Type-II photoreceptors have short and fewer microvilli. Although both types of photoreceptors send afferent projections directly to the brain, their destinations in the brain, called optic neuropiles, have not been sufficiently investigated. Our recent studies revealed that there are commissural fibers in the cerebral ganglia that transmit photic information acquired by bilateral eyes. Moreover, some of the retinal photoreceptors are connected by gap junctions to the photosensitive brain neurons, suggesting the functional interaction of the photic information between the eye and brain photoreceptors, as well as between bilateral eyes. However, it has not been clarified which type of retinal photoreceptors send commissural projections to the contralateral hemiganglion nor interact with the brain photoreceptors. In the present study, we demonstrated by molecular histological analyses and tracer injections that (1) Type-I and Type-II photoreceptors send glutamatergic afferent projections to the medial and lateral lobes of the ipsilateral optic neuropile, respectively, (2) direct synaptic interaction between bilateral optic nerves occurs in the medial lobe of the optic neuropile, and (3) brain photosensory neurons form gap junctions with the medial lobe of the contralateral optic neuropile. These results reveal an ordered pattern of afferent projections from the retina and provide insight into the different functional roles of retinal photoreceptors.     

A High-Density Narrow-Field Inhibitory Retinal Interneuron with Direct Coupling to Müller Glia

Amacrine cells are interneurons composing the most diverse cell class in the mammalian retina. They help encode visual features, such as edges or directed motion, by mediating excitatory and inhibitory interactions between input (i.e., bipolar) and output (i.e., ganglion) neurons in the inner plexiform layer (IPL). Like other brain regions, the retina also contains glial cells that contribute to neurotransmitter uptake, metabolic regulation, and neurovascular control. Here, we report that, in mouse retina (of either sex), an abundant, though previously unstudied inhibitory amacrine cell is coupled directly to Müller glia. Electron microscopic reconstructions of this amacrine type revealed chemical synapses with known retinal cell types and extensive associations with Müller glia, the processes of which often completely ensheathe the neurites of this amacrine cell. Microinjecting small tracer molecules into the somas of these amacrine cells led to selective labeling of nearby Müller glia, leading us to suggest the name “Müller glia-coupled amacrine cell,” or MAC. Our data also indicate that MACs release glycine at conventional chemical synapses, and viral retrograde transsynaptic tracing from the dorsal lateral geniculate nucleus showed selective connections between MACs and a subpopulation of retinal ganglion cell types. Visually evoked responses revealed a strong preference for light increments; these “ON” responses were primarily mediated by excitatory chemical synaptic input and direct electrical coupling with other cells. This initial characterization of the MAC provides the first evidence for neuron-glia coupling in the mammalian retina and identifies the MAC as a potential link between inhibitory processing and glial function.SIGNIFICANCE STATEMENT Gap junctions between pairs of neurons or glial cells are commonly found throughout the nervous system and play multiple roles, including electrical coupling and metabolic exchange. In contrast, gap junctions between neurons and glia cells have rarely been reported and are poorly understood. Here we report the first evidence for neuron-glia coupling in the mammalian retina, specifically between an abundant (but previously unstudied) inhibitory interneuron and Müller glia. Moreover, viral tracing, optogenetics, and serial electron microscopy provide new information about the neuron''s synaptic partners and physiological responses.     

Limited intravascular coupling in the rodent brainstem and retina supports a role for glia in regional blood flow

Regional synaptic activity induces local increases in perfusion that are coupled to upstream vasodilation and improved blood flow. In the cerebral circulation, it has been proposed that astrocytes mediate the link between the initiating stimulus and local vasodilation through propagated intracellular calcium waves. In the systemic circulation the mechanism by which local vasodilation triggers upstream alterations in blood flow involves electrotonic propagation of hyperpolarization via endothelial gap junctions, although less is known concerning the cerebral circulation. The present study aimed to investigate the extent of coupling in microvessels of the rodent brainstem and retina and the subtypes of intracellular calcium stores that might mediate astrocytic signaling. Within the brainstem, connexins (Cxs) 37 and 40 were restricted to the endothelium of pial vessels and larger penetrating arterioles, whereas astrocytic Cxs30 and 43 were found closely associated with pre- and postsynaptic neurons and nearby microvessels. Within the rat retina, Cxs37 and 40 were expressed in large radiating arterioles, but were not found in smaller vessels on the retinal surface or in the deeper retinal layers. These Cxs were absent from all retinal vessels in mice. Astrocytes, expressing Cxs30 and 43 in the rat, but only Cx43 in the mouse, were found closely associated with superficial, but not deeper blood vessels. Inositol-trisphosphate receptors (IP(3)R) 1 and 2 were expressed within brainstem astrocytes, whereas IP(3)R1 and 3 were expressed within retinal astrocytes. Limited intravascular coupling and the proximity of astrocytic networks to blood vessels supports a role for glia in activity-dependent alterations in central blood flow.     

Horizontal cell electrical coupling in the giant danio: synaptic modulation by dopamine and synaptic maintenance by calcium

Electrical synapses, and their structural manifestation, gap junctions, are critical elements of retinal circuitry. These synapses are subject to both rapid modulation and slower structural changes by physiological signals which mediate changes in the adaptational state of the retina. The electrical synapses of fish retinal horizontal cells are an excellent preparation for in vitro studies of electrical synapses. We have examined the rapid modulation of electrical coupling by dopamine and effects on the expression and maintenance of electrical synapses by cell calcium in pairs of horizontal cells isolated from retinas of the giant danio (Danio aquipinnatus). We report that rapid modulation by dopamine reduces junctional conductance by modifying gap junction channel gating, while maintaining cells in reduced calcium medium, and lowering; intracellular calcium concentration, results in the loss of electrical coupling. The effects of calcium on synaptic maintenance may be related to structural changes observed in horizontal cell electrical synapses during light adaptation.     

Ultrastructural correlates of electrical-chemical synaptic transmission in goldfish cranial motor nuclei

The output connections of the cranial relay neurons, part of the Mauthner cell network, were examined in goldfish with light and electron microscopic techniques. Either lucifer yellow or horseradish peroxidase (HRP) was injected into cranial relay neuron axons to demonstrate that they diverge to several motor nuclei and to many motoneurons within one nucleus. Retrograde transport of the enzyme from injections of mandibular muscles was used to label the trigeminal motoneurons. In the electron microscope, cranial relay neuron processes were distinguished by the granular appearance of the electron-opaque polymer formed enzymatically by HRP, while the retrogradely labeled motoneurons had the polymer enclosed in lysosomes. The cranial relay neuron terminals contained many presynaptic vesicles which concentrated the HRP reaction product. Active zones and synaptic clefts were evident. At some synapses, both gap junctions and presynaptic vesicles were found. The mechanism of synaptic transmission was investigated by simultaneous recording with two intracellular microelectrodes from cranial relay neuron-motoneuron pairs. Composite postsynaptic potentials in a trigeminal motoneuron were evoked by intracellular stimulation of a cranial relay neuron axon. The earliest excitatory postsynaptic potential (EPSP) component had a latency of 0.25 msec and had a peak amplitude that was not depressed by repetitive stimulation. A second component had larger peak amplitudes which were reduced easily by repetitive stimulation. Antidromic action potentials were not transmitted from motoneurons to the cranial relay neuron axons. Thus, both electrical and chemical transmission probably occur at the cranial relay neuron-motoneuron synapses. Since the cranial relay neurons fire synchronously and receive excitatory chemical synapses, the function of the gap junctions and electrical transmission is unclear. Perhaps the importance of these gap junctions is more for transport of small molecules than for impulse transmission.     

Expression of gap junction connexin36 in adult rat retinal ganglion cells

Electrophysiological and ultrastructural studies have demonstrated that gap junctions connect diverse types of neurons in the central nervous system, permitting direct electrical and metabolic coupling. A member of gap junction channel subunit connexin36 (Cx36), is probed for the location of cell-to-cell communication in the mammalian retina, where gap junction networks of major classes of neurons are present. We present an analysis of the expression and localization of Cx36 protein in adult Wistar rat retina, using a newly generated polyclonal antibody against a sequence in the predicted cytoplasmic loop of the Cx36 amino acid alignment, deduced from the cDNA sequence. The affinity-purified antibody, recognizing a single 36-kDa protein, consistently labeled discrete puncta of subcellular structures likely to be associated with gap junctions in the inner plexiform layer, and also cytoplasm within somata and dendrites of retinal amacrine and ganglion cells, following examination with various fixation protocols and double labeling immuno-fluorescence. These results provide that prominent cell-to-cell communication appears in mature excitatory neurons such as retinal ganglion cells, in addition to inhibitory amacrine cells, mediated by gap junctions in the adult retina.     

Retinal waves are likely to instruct the formation of eye-specific retinogeniculate projections

Prior to eye-opening and the development of visual responses, the retina exhibits highly correlated spontaneous firing pattens termed retinal waves. Disruption of the normal spontaneous firing pattern either genetically or pharmacologically prevents the eye-specific refinement of retinogeniculate afferents. Here I provide the evidence that retinal waves play an instructive role in this process. In addition, I argue that a full understanding requires an identification of the features of retinal activity that drive the refinement as well as an understanding of mechanisms that transform these signals into axonal rearrangements.     

Dopamine modulates evoked transmission at cholinergic synapses formed by rat retinal neurons with muscle cells

The purpose of this study was to investigate the effect of dopamine on synaptic transmission mediated by cholinergic neurons derived from the rat retina. We used a cell culture system that permitted the physiological monitoring of acetylcholine released at synapses formed between retinal neurons and striated muscle cells. Dopamine was found to facilitate evoked transmission by a mechanism mediated by D₁ dopamine receptors. The results support the hypothesis that dopamine may have a modulatory role in information processing by the mammalian retina.