Retinoic acid reduces the formation of, and acutely modulates, invertebrate electrical synapses.

Communication between cells in the nervous system is dependent on both chemical and electrical synapses. Factors that can affect chemical synapses have been well studied, but less is known about factors that influence electrical synapses. Retinoic acid, the vitamin A metabolite, is a known regulator of chemical synapses, but few studies have examined its capacity to regulate electrical synapses. In this study, we determine that retinoic acid is capable of rapidly altering the strength of electrical synapses in an isomer- and cell-dependent manner. Furthermore, we provide evidence that this acute effect might be independent of either the retinoid receptors or the activation of a protein kinase. In addition to the rapid modulatory effects of retinoic acid, we provide data to suggest that retinoic acid is also capable of regulating the formation of electrical synapses. Long-term exposure to both all-trans-retinoic acid or 9-cis-retinoic acid reduced the proportion of cell pairs forming electrical synapses, as well as reduced the strength of electrical synapses that did form. In summary, this study provides insights into the role that retinoids might play in both the formation and modulation of electrical synapses in the central nervous system.NEW & NOTEWORTHY Retinoids are known modulators of chemical synapses and mediate synaptic plasticity in the nervous system, but little is known of their effects on electrical synapses. Here, we show that retinoids selectively reduce electrical synapses in a cell- and isomer-dependent manner. This modulatory action on existing electrical synapses was rapid and nongenomic in nature. We also showed for the first time that longer retinoid exposures inhibit the formation of electrical synapses.

INX-18 and INX-19 play distinct roles in electrical synapses that modulate aversive behavior in Caenorhabditis elegans.

In order to respond to changing environments and fluctuations in internal states, animals adjust their behavior through diverse neuromodulatory mechanisms. In this study we show that electrical synapses between the ASH primary quinine-detecting sensory neurons and the neighboring ASK neurons are required for modulating the aversive response to the bitter tastant quinine in C. elegans. Mutant worms that lack the electrical synapse proteins INX-18 and INX-19 become hypersensitive to dilute quinine. Cell-specific rescue experiments indicate that inx-18 operates in ASK while inx-19 is required in both ASK and ASH for proper quinine sensitivity. Imaging analyses find that INX-19 in ASK and ASH localizes to the same regions in the nerve ring, suggesting that both sides of ASK-ASH electrical synapses contain INX-19. While inx-18 and inx-19 mutant animals have a similar behavioral phenotype, several lines of evidence suggest the proteins encoded by these genes play different roles in modulating the aversive quinine response. First, INX-18 and INX-19 localize to different regions of the nerve ring, indicating that they are not present in the same synapses. Second, removing inx-18 disrupts the distribution of INX-19, while removing inx-19 does not alter INX-18 localization. Finally, by using a fluorescent cGMP reporter, we find that INX-18 and INX-19 have distinct roles in establishing cGMP levels in ASK and ASH. Together, these results demonstrate that electrical synapses containing INX-18 and INX-19 facilitate modulation of ASH nociceptive signaling. Our findings support the idea that a network of electrical synapses mediates cGMP exchange between neurons, enabling modulation of sensory responses and behavior.

Neprilysin Controls the Synaptic Activity of Neuropeptides in the Intercalated Cells of the Amygdala.

Endogenous opioid peptides in the amygdala regulate many of our behaviors and emotional responses. In particular, the endogenous opioid enkephalin plays a significant role in regulating amygdala activity, but its action is strongly limited by peptidases, which degrade enkephalin into inactive fragments. Inhibiting peptidases may be an attractive method to enhance endogenous opioid signaling; however, we do not know which specific peptidase(s) to target. Using inhibition of glutamate release onto the intercalated cells of the amygdala as an assay for enkephalin activity, we applied specific peptidase inhibitors to determine which peptidase(s) regulate enkephalin signaling in this region. Thiorphan (10 µM), captopril (1 µM), or bestatin (10 µM) were used to inhibit the activity of neprilysin, angiotensin-converting enzyme, or aminopeptidase N, respectively. In rat brain slices containing the intercalated cells, we found that inhibition of glutamate release by a submaximal concentration of enkephalin was doubled by application of all three peptidase inhibitors combined. Then, we tested inhibitors individually and found that inhibition of neprilysin alone could enhance enkephalin responses to the same extent as inhibitors of all three peptidases combined. This indicates neprilysin is the predominant peptidase responsible for degrading enkephalins in the intercalated cells of the amygdala. This differs from the striatum, locus coeruleus, and spinal cord, where multiple peptidases metabolize enkephalin. These data highlight the importance of knowing which specific peptidase(s) control opioid actions in the relevant neural circuit and how they change in disease states to allow rational choices of drugs targeting the specific peptidase of interest. SIGNIFICANCE STATEMENT: Endogenous opioids modulate many of our emotional and behavioral responses. In the amygdala, they modulate our pain, fear, and addictive behaviors. Their actions are terminated when they are catabolized into inactive fragments by at least three different peptidases. In this study, we found that neprilysin selectively controls endogenous opioid concentrations at synapses in the intercalated cells of the amygdala. This peptidase may be a target for regulation of endogenous opioid modulation of amygdala-mediated emotional and behavioral responses.

Gap junctional coupling between retinal amacrine and ganglion cells underlies coherent activity integral to global object perception.

Coherent spike activity occurs between widely separated retinal ganglion cells (RGCs) in response to a large, contiguous object, but not to disjointed objects. Since the large spatial separation between the RGCs precludes common excitatory inputs from bipolar cells, the mechanism underlying this long-range coherence remains unclear. Here, we show that electrical coupling between RGCs and polyaxonal amacrine cells in mouse retina forms the synaptic mechanism responsible for long-range coherent activity in the retina. Pharmacological blockade of gap junctions or genetic ablation of connexin 36 (Cx36) subunits eliminates the long-range correlated spiking between RGCs. Moreover, we find that blockade of gap junctions or ablation of Cx36 significantly reduces the ability of mice to discriminate large, global objects from small, disjointed stimuli. Our results indicate that synchronous activity of RGCs, derived from electrical coupling with amacrine cells, encodes information critical to global object perception.

Electrical synapses mediate synergism between pheromone and food odors in Drosophila melanogaster.

In Drosophila melanogaster, the sex pheromone produced by males, cis-vaccenyl acetate (cVA), evokes a stereotypic gender-specific behavior in both males and females. As Drosophila adults feed, mate, and oviposit on food, they perceive the pheromone as a blend against a background of food odors. Previous studies have reported that food odors enhance flies' behavioral response to cVA, specifically in virgin females. However, how and where the different olfactory inputs interact has so far remained unknown. In this study, we elucidated the neuronal mechanism underlying the response at an anatomical, functional, and behavioral level. Our data show that in virgin females cVA and the complex food odor vinegar evoke a synergistic response in the cVA-responsive glomerulus DA1. This synergism, however, does not appear at the input level of the glomerulus, but is restricted to the projection neuron level only. Notably, it is abolished by a mutation in gap junctions in projection neurons and is found to be mediated by electrical synapses between excitatory local interneurons and projection neurons. As a behavioral consequence, we demonstrate that virgin females in the presence of vinegar become receptive more rapidly to courting males, while male courtship is not affected. Altogether, our results suggest that lateral excitation via gap junctions modulates odor tuning in the antennal lobe and drives synergistic interactions between two ecologically relevant odors, representing food and sex.

Protein Kinase C Enhances Electrical Synaptic Transmission by Acting on Junctional and Postsynaptic Ca2+ Currents.

By synchronizing neuronal activity, electrical transmission influences the coordination, pattern, and/or frequency of firing. In the hemaphroditic marine-snail, Aplysia calfornica, the neuroendocrine bag cell neurons use electrical synapses to synchronize a 30 min afterdischarge of action potentials for the release of reproductive hormone. During the afterdischarge, protein kinase C (PKC) is activated, although its impact on bag cell neuron electrical transmission is unknown. This was investigated here by monitoring electrical synapses between paired cultured bag cell neurons using dual whole-cell recording. Voltage clamp revealed a largely voltage-independent junctional current, which was enhanced by treating with a PKC activator, PMA, before recording. We also examined the transfer of presynaptic action potential-like waveforms (generated in voltage clamp) to the postsynaptic cell (measured in current clamp). For control pairs, the presynaptic spike-like waveforms mainly evoked electrotonic potentials; however, when PKC was triggered, these stimuli consistently produced postsynaptic action potentials. To assess whether this involved changes to postsynaptic responsiveness, single bag cell neurons were injected with junctional-like current mimicking that evoked by a presynaptic action potential. Unlike control neurons, which were less likely to spike, cells in PMA always fired action potentials to the junctional-like current. Furthermore, PKC activation increased a postsynaptic voltage-gated Ca2+ current, which was recruited even by modest depolarization associated with an electrotonic potential. Whereas PKC inhibits gap junctions in most systems, bag cell neurons are rather unique, as the kinase potentiates the electrical synapse; in turn, this synergizes with augmented postsynaptic Ca2+ current to promote synchronous firing.SIGNIFICANCE STATEMENT Electrical coupling is a fundamental form of communication. For the bag cell neurons of Aplysia, electrical synapses coordinate a prolonged burst of action potentials known as the afterdischarge. We looked at how protein kinase C, which is upregulated with the afterdischarge, influences information transfer across the synapse. The kinase activation increased junctional current, a remarkable finding given that this enzyme is largely considered inhibitory for gap junctions. There was also an augmentation in the ability of a presynaptic neuron to provoke postsynaptic action potentials. This increased excitability was, in part, due to enhanced postsynaptic voltage-dependent Ca2+ current. Thus, protein kinase C improves the fidelity of electrotonic transmission and promotes synchronous firing by modulating both junctional and membrane conductances.

The role of retinoic acid in the formation and modulation of invertebrate central synapses.

Trophic factors can influence many aspects of nervous system function, such as neurite outgrowth, synapse formation, and synapse modulation. The vitamin A metabolite, retinoic acid, can exert trophic effects to promote neuronal survival and outgrowth in many species and is also known to modulate vertebrate hippocampal synapses. However, its role in synaptogenesis has not been well studied, and whether it can modulate existing invertebrate synapses is also not known. In this study, we first examined a potential trophic effect of retinoic acid on the formation of excitatory synapses, independently of its role in neurite outgrowth, using cultured neurons of the mollusc Lymnaea stagnalis We also investigated its role in modulating both chemical and electrical synapses between various Lymnaea neurons in cell culture. Although we found no evidence to suggest retinoic acid affected short-term synaptic plasticity in the form of post-tetanic potentiation, we did find a significant cell type-specific modulation of electrical synapses. Given the prevalence of electrical synapses in invertebrate nervous systems, these findings highlight the potential for retinoic acid to modulate network function in the central nervous system of at least some invertebrates. NEW & NOTEWORTHY: This study performed the first electrophysiological analysis of the ability of the vitamin A metabolite, retinoic acid, to exert trophic influences during synaptogenesis independently of its effects in supporting neurite outgrowth. It was also the first study to examine the ability of retinoic acid to modify both chemical and electrical synapses in any invertebrate, nonchordate species. We provide evidence that all-trans retinoic acid can modify invertebrate electrical synapses of central neurons in a cell-specific manner.

Activation of Group I and Group II Metabotropic Glutamate Receptors Causes LTD and LTP of Electrical Synapses in the Rat Thalamic Reticular Nucleus.

Compared with the extensive characterization of chemical synaptic plasticity, electrical synaptic plasticity remains poorly understood. Electrical synapses are strong and prevalent among the GABAergic neurons of the rodent thalamic reticular nucleus. Using paired whole-cell recordings, we show that activation of Group I metabotropic glutamate receptors (mGluRs) induces long-term depression of electrical synapses. Conversely, activation of the Group II mGluR, mGluR3, induces long-term potentiation of electrical synapses. By testing downstream targets, we show that modifications induced by both mGluR groups converge on the same signaling cascade--adenylyl cyclase to cAMP to protein kinase A--but with opposing effects. Furthermore, the magnitude of modification is inversely correlated to baseline coupling strength. Thus, electrical synapses, like their chemical counterparts, undergo both strengthening and weakening forms of plasticity, which should play a significant role in thalamocortical function.

Auditory Golgi cells are interconnected predominantly by electrical synapses.

The mossy fiber-granule cell-parallel fiber system conveys proprioceptive and corollary discharge information to principal cells in cerebellum-like systems. In the dorsal cochlear nucleus (DCN), Golgi cells inhibit granule cells and thus regulate information transfer along the mossy fiber-granule cell-parallel fiber pathway. Whereas excitatory synaptic inputs to Golgi cells are well understood, inhibitory and electrical synaptic inputs to Golgi cells have not been examined. Using paired recordings in a mouse brain slice preparation, we find that Golgi cells of the cochlear nucleus reliably form electrical synapses onto one another. Golgi cells were only rarely electrically coupled to superficial stellate cells, which form a separate network of electrically coupled interneurons in the DCN. Spikelets had a biphasic effect on the excitability of postjunctional Golgi cells, with a brief excitatory phase and a prolonged inhibitory phase due to the propagation of the prejunctional afterhyperpolarization through gap junctions. Golgi cells and stellate cells made weak inhibitory chemical synapses onto Golgi cells with low probability. Electrical synapses are therefore the predominant form of synaptic communication between auditory Golgi cells. We propose that electrical synapses between Golgi cells may function to regulate the synchrony of Golgi cell firing when electrically coupled Golgi cells receive temporally correlated excitatory synaptic input.

Attenuation of Neuropathic Pain by Inhibiting Electrical Synapses in the Anterior Cingulate Cortex.

BACKGROUND: Synaptic mechanisms and neuronal oscillations have been proposed to be responsible for neuropathic pain formation. Many studies have also highlighted the important role of electrical synapses in synaptic plasticity and in neuronal oscillations. Thus, electrical synapses may contribute to neuropathic pain generation. However, previous studies have primarily focused on the role of chemical synapses, while ignoring the role of electrical synapses, in neuropathic pain generation. METHODS: The authors adopted microinjection, RNA interference techniques, and behavioral tests to verify the link between connexin 36 (Cx36) and neuropathic pain. They also studied the selective Cx36 blocker mefloquine in rat chronic constriction injury and spared nerve injury model of neuropathic pain. Electrophysiologic recordings were used to further confirm the behavioral data. RESULTS: The authors found that Cx36, which constitutes the neuron-neuron electrical synapses, was up-regulated in the anterior cingulate cortex after nerve injury (n = 5). Meanwhile, Cx36-mediated neuronal oscillations in the gamma frequency range (30 to 80 Hz) (n = 7 to 8) and the neuronal synaptic transmission (n = 13 to 19) were also enhanced. Neuropathic pain was relieved by disrupting Cx36 function or expression in the anterior cingulate cortex. They also found that mefloquine, which are clinically used for treating malaria, affected gamma oscillations and synaptic plasticity, leading to a sustained pain relief in chronic constriction injury and spared nerve injury models (n = 7 to 12). CONCLUSION: The electrical synapses blocker mefloquine could affect gamma oscillations and synaptic plasticity in the anterior cingulate cortex and relieve neuropathic pain. Cx36 may be a new therapeutic target for treating chronic pain.

Conflicting effects by antibodies against connexin36 during the action of intracellular Cyclic-AMP onto electrical synapses of retinal ganglion cells.

Alpha-type retinal ganglion cells (alpha cells) of the same class in mammalian retina are connected by gap junctions. Electrical synapses between alpha cells were examined using combined techniques of dual patch-clamp recordings, intracellular labeling and electron microscopy in the albino rat retina. In simultaneous dual whole-cell recordings from pairs of neighboring alpha cells, bidirectional electrical synapses with symmetrical junction conductance were observed in pairs with cells of the same morphological type. Regulatory domains of gap junction protein subunit connexins in electrical synapses between alpha cells by extracellular and intracellular ligands investigated by dual whole-patch clamp recordings. I examined how passage currents through electrical synapses between alpha cells are modulated by specific antibodies against connexin36 proteins, and extracellular or intracellular application of ligands. Control conditions led us to observe large passage currents between connected cells and adequate transjunctional conductance (Gj) (1.35[Formula: see text][Formula: see text][Formula: see text]0.51[Formula: see text]nS). Experimental results show that high level of intracellular cyclic AMP within examined cells suppress electrical synapses between the neighboring cells. Gj between examined cells reduced to 0.15[Formula: see text][Formula: see text][Formula: see text]0.04[Formula: see text]nS. Under application of dopamine (1.25[Formula: see text][Formula: see text][Formula: see text]0.06[Formula: see text]nS) or intracellular cyclic GMP (0.98[Formula: see text][Formula: see text][Formula: see text]0.23[Formula: see text]nS), however, Gj also remains as in the control level. Intracellular application of an antibody against the cytoplasmic loop of connexin36 reduced Gj (0.98[Formula: see text][Formula: see text][Formula: see text]0.23[Formula: see text]nS). Cocktail of the antibody against cytoplasmic connexin36 and intracellular cyclic AMP leaves Gj as in the level by single involvement of the cytoplasmic antibody. The elimination of Gj by the cytoplasmic antibody was in a dose-dependent manner. These results suggest that binding domains against cyclic AMP may be present in the cytoplasmic sites of connexin proteins to regulate channel opening of gap junctions between mammalian retinal alpha ganglion cells.

Opioids potentiate electrical transmission at mixed synapses on the Mauthner cell.

Opioid receptors were shown to modulate a variety of cellular processes in the vertebrate central nervous system, including synaptic transmission. While the effects of opioid receptors on chemically mediated transmission have been extensively investigated, little is known of their actions on gap junction-mediated electrical synapses. Here we report that pharmacological activation of mu-opioid receptors led to a long-term enhancement of electrical (and glutamatergic) transmission at identifiable mixed synapses on the goldfish Mauthner cells. The effect also required activation of both dopamine D1/5 receptors and postsynaptic cAMP-dependent protein kinase A, suggesting that opioid-evoked actions are mediated indirectly via the release of dopamine from varicosities known to be located in the vicinity of the synaptic contacts. Moreover, inhibitory inputs situated in the immediate vicinity of these excitatory synapses on the lateral dendrite of the Mauthner cell were not affected by activation of mu-opioid receptors, indicating that their actions are restricted to electrical and glutamatergic transmissions co-existing at mixed contacts. Thus, as their chemical counterparts, electrical synapses can be a target for the modulatory actions of the opioid system. Because gap junctions at these mixed synapses are formed by fish homologs of the neuronal connexin 36, which is widespread in mammalian brain, it is likely that this regulatory property applies to electrical synapses elsewhere as well.

Trafficking of gap junction channels at a vertebrate electrical synapse in vivo.

Trafficking and turnover of transmitter receptors required to maintain and modify the strength of chemical synapses have been characterized extensively. In contrast, little is known regarding trafficking of gap junction components at electrical synapses. By combining ultrastructural and in vivo physiological analysis at identified mixed (electrical and chemical) synapses on the goldfish Mauthner cell, we show here that gap junction hemichannels are added at the edges of GJ plaques where they dock with hemichannels in the apposed membrane to form cell-cell channels and, simultaneously, that intact junctional regions are removed from centers of these plaques into either presynaptic axon or postsynaptic dendrite. Moreover, electrical coupling is readily modified by intradendritic application of peptides that interfere with endocytosis or exocytosis, suggesting that the strength of electrical synapses at these terminals is sustained, at least in part, by fast (in minutes) turnover of gap junction channels. A peptide corresponding to a region of the carboxy terminus that is conserved in Cx36 and its two teleost homologs appears to interfere with formation of new gap junction channels, presumably by reducing insertion of hemichannels on the dendritic side. Thus, our data indicate that electrical synapses are dynamic structures and that their channels are turned over actively, suggesting that regulated trafficking of connexons may contribute to the modification of gap junctional conductance.

Electrical coupling between Aplysia bag cell neurons: characterization and role in synchronous firing.

In neuroendocrine cells, hormone release often requires a collective burst of action potentials synchronized by gap junctions. This is the case for the electrically coupled bag cell neurons in the reproductive system of the marine snail, Aplysia californica. These neuroendocrine cells are found in two clusters, and fire a synchronous burst, called the afterdischarge, resulting in neuropeptide secretion and the triggering of ovulation. However, the physiology and pharmacology of the bag cell neuron electrical synapse are not completely understood. As such, we made dual whole cell recordings from pairs of electrically coupled cultured bag cell neurons. The junctional current was nonrectifying and not influenced by postsynaptic voltage. Furthermore, junctional conductance was voltage independent and, not surprisingly, strongly correlated with coupling coefficient magnitude. The electrical synapse also acted as a low-pass filter, although under certain conditions, electrotonic potentials evoked by presynaptic action potentials could drive postsynaptic spikes. If coupled neurons were stimulated to spike simultaneously, they presented a high degree of action potential synchrony compared with not-coupled neurons. The electrical synapse failed to pass various intracellular dyes, but was permeable to Cs(+), and could be inhibited by niflumic acid, meclofenamic acid, or 5-nitro-2-(3-phenylpropylamino)benzoic acid. Finally, extracellular and sharp-electrode recording from the intact bag cell neuron cluster showed that these pharmacological uncouplers disrupted both electrical coupling and afterdischarge generation in situ. Thus electrical synapses promote bag cell neuron firing synchrony and may allow for electrotonic spread of the burst through the network, ultimately contributing to propagation of the species.

Is the amyloid hypothesis of Alzheimer's disease therapeutically relevant?

The conventional view of AD (Alzheimer's disease) is that much of the pathology is driven by an increased load of ß-amyloid in the brain of AD patients (the 'Amyloid Hypothesis'). Yet, many therapeutic strategies based on lowering ß-amyloid have so far failed in clinical trials. This failure of ß-amyloid-lowering agents has caused many to question the Amyloid Hypothesis itself. However, AD is likely to be a complex disease driven by multiple factors. In addition, it is increasingly clear that ß-amyloid processing involves many enzymes and signalling pathways that play a role in a diverse array of cellular processes. Thus the clinical failure of ß-amyloid-lowering agents does not mean that the hypothesis itself is incorrect; it may simply mean that manipulating ß-amyloid directly is an unrealistic strategy for therapeutic intervention, given the complex role of ß-amyloid in neuronal physiology. Another possible problem may be that toxic ß-amyloid levels have already caused irreversible damage to downstream cellular pathways by the time dementia sets in. We argue in the present review that a more direct (and possibly simpler) approach to AD therapeutics is to rescue synaptic dysfunction directly, by focusing on the mechanisms by which elevated levels of ß-amyloid disrupt synaptic physiology.

Synchronization dynamics induced on pairs of neurons under applied weak alternating magnetic fields.

Pairs of Helix aspersa neurons show an alternating magnetic field dependent frequency synchronization (AMFS) when exposed to a weak (amplitude B0 between 0.2 and 150 Gauss (G)) alternating magnetic field (AMF) of extremely low frequency (ELF, fM = 50 Hz). We have compared the AMFS patterns of discharge with: i) the synaptic activity promoted by glutamate and acetylcholine; ii) the activity induced by caffeine; iii) the bioelectric activity induced on neurons interconnected by electric synapses. AMFS activity reveals several specific features: i) a tight coincidence in time of the pattern and frequency, f, of discharge; ii) it is induced in the time interval of field application; iii) it is dependent on the intensity of the sinusoidal applied magnetic field; iv) elicited biphasic responses (excitation followed by inhibition) run in parallel for the pair of neurons; and v) some neuron pairs either spontaneously or AMF synchronized can be desynchronized under applied higher AMF. Our electron microscopy studies reveal gap-like junctions confirming our immunocytochemistry results about expression of connexin 26 (Cx26) in 4.7% of Helix neurons. AMF and carbenoxolone did not induce any significant effect on spontaneous synchronization through electric synapses.

[The fundaments of neuronal plasticity].

Plasticity of the nervous system is determined by the modification of efficacy ofsynaptic transmission: long-term potentiation and long-term depression. Different modern technical approaches such as: registration of ionic currents in single neuron, molecular-genetic analysis, neurovisualization, and others reveal the molecular mechanisms of synaptic plasticity. The understanding of these mechanisms, in its turn, stimulates the development of methods of pharmacological correction of different forms of brain pathology such as Alzheimer disease, parkinsonism, alcoholism, aging and others.

cAMP controls a trafficking mechanism that maintains the neuron specificity and subcellular placement of electrical synapses.

Electrical synapses are established between specific neurons and within distinct subcellular compartments, but the mechanisms that direct gap junction assembly in the nervous system are largely unknown. Here, we show that a developmental program tunes cAMP signaling to direct the neuron-specific assembly and placement of electrical synapses in the C. elegans motor circuit. We use live-cell imaging to visualize electrical synapses in vivo and an optogenetic assay to confirm that they are functional. In ventral A class (VA) motor neurons, the UNC-4 transcription factor blocks expression of cAMP antagonists that promote gap junction miswiring. In unc-4 mutants, VA electrical synapses are established with an alternative synaptic partner and are repositioned from the VA axon to soma. cAMP counters these effects by driving gap junction trafficking into the VA axon for electrical synapse assembly. Thus, our experiments establish that cAMP regulates gap junction trafficking for the biogenesis of functional electrical synapses.

Electrical coupling of astrocytes in rat hippocampal slices under physiological and simulated ischemic conditions.

Mammalian protoplasmic astrocytes are extensively coupled through gap junction channels but the biophysical properties of these channels under physiological and ischemic conditions in situ are not well defined. Using confocal morphometric analysis of biocytin-filled astrocytic syncytia in rat hippocampal CA1 stratum radiatum we found that each astrocyte directly couples, on average, to 11 other astrocytes with a mean interastrocytic distance of 45 microm. Voltage-independent and bidirectional transjunctional currents were always measured between directly coupled astrocyte pairs in dual voltage-clamp recordings, but never from astrocyte-NG2 glia or astrocyte-interneuron pairs. The electrical coupling ratio varied considerably among astrocytes in developing postnatal day 14 rats (P14, 0.5-12.4%, mean = 3.6%), but became more constant in young adult P21 rats (0.18-3.9%, mean = 1.6%), and the coupling ratio declined exponentially with increasing pair distance. Electrical coupling was not affected by short-term oxygen-glucose deprivation (OGD) treatment, but showed delayed inhibition in an acidic extracellular pH of 6.4. Combination of acidic pH (6.4) and OGD, a condition that better represents cerebral ischemia in vivo, accelerated the inhibition of electrical coupling. Our results show that, under physiological conditions, 20.7-24.2% of K(+) induced currents can travel from any astrocytic soma in CA1 stratum radiatum to the gap junctions of the nearest neighbor astrocytes, but this should be severely inhibited as a consequence of the OGD and acidosis seen in the ischemic brain.