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PubMed1999

Syntaxin 1A interacts with multiple exocytic proteins to regulate neurotransmitter release in vivo.

Biochemical studies suggest that syntaxin 1A participates in multiple protein-protein interactions in the synaptic terminal, but the in vivo significance of these interactions is poorly understood. We used a targeted mutagenesis approach to eliminate specific syntaxin binding interactions and demonstrate that Drosophila syntaxin 1A plays multiple regulatory roles in neurotransmission in vivo. Syntaxin mutations that eliminate ROP/Munc-18 binding display increased neurotransmitter release, suggesting that ROP inhibits neurosecretion through its interaction with syntaxin. Syntaxin mutations that block Ca2+ channel binding also cause an increase in neurotransmitter release, suggesting that syntaxin normally functions in inhibiting Ca2+ channel opening. Additionally, we identify and characterize a syntaxin Ca2+ effector domain, which may spatially organize the Ca2+ channel, cysteine string protein, and synaptotagmin for effective excitation-secretion coupling in the presynaptic terminal.

Animals↗
PubMed2002

Localization of the Slack potassium channel in the rat central nervous system.

The Slack gene encodes a voltage-dependent K(+) channel that has a unitary conductance of approximately 60 pS. Evidence from heterologous expression studies suggests that Slack channel subunits can also combine with the Slo subunit to generate Ca(2+)-activated K(+) channels of larger conductances. Nonetheless, the function of Slack in the brain remains to be identified. We have now generated an affinity-purified antibody against the N-terminal of rat Slack, for biochemical and immunohistochemical studies. The antibody recognized Slack in transiently transfected CHO cells both by immunocytochemistry and by Western blot analysis. The antibody also detected a single band in rat brain membranes. The localization of Slack in rat brain slices was then determined using the antibody. Most prominent Slack immunoreactivity occurs in the brainstem, in particular the trigeminal system and reticular formation, where very intense staining was found in both cell bodies and axonal fibers of associated nuclei. Labeling was also very strong in the vestibular and oculomotor nuclei. Within the auditory system, the medial nucleus of the trapezoid had a robust signal consistent with staining of the giant presynaptic terminals. Strong Slack immunoreactivity was present in the olfactory bulb, red nucleus, and deep cerebellar nuclei. There was labeling also in the thalamus, substantia nigra, and amygdala. The only cortical region in which Slack immunoreactivity was detected was the frontal cortex. The subcellular and regional distribution of Slack differs from that previously reported for the Slo channel subunit and suggests that Slack may also have an autonomous role in regulating the firing properties of neurons.

Amino Acid Sequence↗
PubMed1993

Rapid recovery of structure and function of the cholinergic synapses in the cat superior cervical ganglion in vivo following stimulation-induced exhaustion.

Cat superior cervical ganglia (SCG) were tetanically stimulated in vivo at 30-100 Hz until neural transmission was exhausted, and then were allowed to rest and recover. Changes in their cholinergic synapses were examined electrophysiologically and morphologically during the time of tetanic stimulation and during recovery. For morphometric analysis the presynaptic terminal was subdivided into two areas: an area directly over the active zone, termed zone-I, (bounded by a hemicircle with a diameter equivalent to the active zone length), and the remaining preterminal area, termed zone-II. In control ganglia before stimulation synaptic vesicle density in zone-I (SVD-I) averaged 90 microns-2 and the number of vesicles actually attached to the active zone (SVA) averaged about 2.5 per single profile of nerve terminal. Upon stimulation, the postganglionic potential immediately began to decline in amplitude and disappeared after 1 min of stimulation. Simultaneously, SVD-I declined to less than 35 microns-2 and SVA declined to less than 1 per section. Thereafter, stimulation was terminated and the ganglion was allowed to rest. Recovery of the postganglionic potential was monitored by stimulation at 1 Hz. The postganglionic potential reached control levels after only 1 min of rest. Likewise, the structural parameters, SVD-I and SVA, also rapidly recovered, reaching control levels after only 30 sec of rest, slightly faster than the postganglionic potential. This illustrates that stimulation-induced fatigue of transmitter output and depletion of synaptic vesicles recover to the control level at a high rate in synapses of the cat SCG with a normal supply of blood. In fact, morphological recovery may be slightly faster than electrophysiological recovery. Mechanisms of vesicle formation and migration to the presynaptic area are discussed in light of these observations.

Acetylcholine↗
PubMed1994

Spatiotemporal pattern of quantal release of ATP and noradrenaline from sympathetic nerves: consequences for neuromuscular transmission.

The recent explosive development in research concerning the fundamental mechanisms of synaptic transmission helps put the present paper in context. It is now evident that not all transmitter vesicles in a nerve terminal, not even all those docked at its active zones, are immediately available for release (36). We watch, fascinated, the unraveling of the amazingly complex cellular mechanisms and molecular machinery that determine whether or not a vesicle is "exocytosis-competent" (77,78,39,79). Studies on quantal release in different systems show that neurons are fundamentally similar in one respect: that transmitter release from individual active zones is monoquantal (2). But they also show that active zones in different neurons differ drastically in the probability of monoquantal release and in the number of quanta immediately available for release (3). This implies that one should not extrapolate directly from transmitter release in one set of presynaptic terminals (e.g., in neuromuscular endplate or squid giant synapse) to that in other nerve terminals, especially if they have a very different morphology. As shown here, one should not even extrapolate from transmitter release in sympathetic nerves in one tissue (e.g., rat tail artery) to that in other tissues or species (e.g., mouse vas deferens). It is noteworthy that most studies of quantal release are based on electrophysiological analysis and therefore deal with release of fast, ionotropic transmitters from small synaptic vesicles at the active zones, especially in neurons in which these events may be examined with high resolution (49,48,46,33,32). Such data are useful as general models of the release of both fast and slow transmitters from small synaptic vesicles at active zones in other systems, provided that these transmitters are released in parallel, as are apparently ATP and NA in sympathetic nerves. They tell us little or nothing, however, about the release of transmitters (e.g., neuropeptides) from the large vesicles, nor about the spatiotemporal pattern of monoquantal release from small synaptic vesicles in the many neurons that have boutons-en-passent terminals. They show that the time course of effector responses to fast, rapidly inactivated transmitters such as ACh or ATP is necessarily release related. But they do not even address the possibility that the effector responses to slow transmitters such as NA, co-released from the same terminals, may obey completely different rules and perhaps rather be clearance related (7).(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1980

Antibody-induced modulation of rhabdovirus infection of neurons in vitro.

Dissociated neuron cultures of mice were inoculated with vesicular stomatitis virus (VSV) and subsequently fed with medium containing sufficient antiviral antibody (AB) to neutralize all free virus. In contrast to control acute neuronal infection, which lasts one to two days, AB-treated neuron cultures were maintained as long as two weeks. This protection was not obtained in infected monkey kidney cells treated with AB. Protected neuron cultures were examined with immunolabeling and EM techniques. During the first days of AB treatment, viral buds and surface antigens were often grouped in clusters instead of being diffuse on the neuronal surface. In addition, phagocytic cells often in contact with viruses released by neurons progressively engulfed aggregates of these viruses in their lysosomal systems. Dendritic processes in AB-treated cultures contained more viral antigen and ribonucleoproteins, but less assembly sites than in acutely-infected neurons. However, budding sites were frequent on the side of the post-synaptic density and some viruses seemed to enter directly into the lateral side of the presynaptic terminal to which the productive dendrite was connected. Removal of AB at this stage resulted in reactivation of viral infection in more neurons than those originally infected, but complete viral maturation and release occurred only 2 to 3 days after removal of AB. Chance of reactivation decreased with increasing length of AB treatment, and no viral antigens or budding sites were detected during the second week of AB treatment. Continuous treatment of VSV-infected neuron cultures with antiviral AB first induces (1) activation of non-neuronal cells to phagocytize clusters of viruses forming at the neuron surface, and (2) restriction of virus maturation sites mostly to the postsynaptic area where virus spreading to presynaptic endings seems favored. Prolonged treatment with AB later results in the apparent curing of the infection.

Animals↗
PubMed1995

Plasticity of complex terminals in lamina II in partially deafferented spinal cord: the cat spared root preparation.

Projections to the dorsal horn change in adult mammals in response to complete or partial deafferentation. The number of synaptic terminals remains constant after complete lumbosacral deafferentation, indicating replacement of lost dorsal root terminals by newly formed terminals from spared intrinsic systems. The density of a spared central projection of a dorsal root is increased in dorsal horn after partial deafferentation, consistent with sprouting by the axons in the spared root. In this study, we have used electron microscopy to study morphological changes in a specific class of terminals in the dorsal horn induced by partial deafferentation. Complex terminals (CTs) in the dorsal horn originate exclusively from dorsal roots and are readily distinguished morphologically. The CTs and the postsynaptic densities (PSDs) associated with CTs were measured in lamina II at L5 and L6 in cats subjected to unilateral spared root (L6) dorsal rhizotomies and compared to CTs in the control side. Acutely following partial deafferentation, the number of CT profiles decreased. At more chronic survivals, the number of CT profiles were restored to normal levels, and both the number and the length of PSDs were increased. The changes in CTs and PSDs suggest sprouting and synaptogenesis by the spared dorsal root fibers that produce changes in the postsynaptic neuron. Spared root deafferentation thus elicits compensatory changes in presynaptic terminals of the spared root and also in their postsynaptic target neurons.

Animals↗
PubMed1988

Angiotensin receptors and the vagal system.

Angiotensin II (Ang II) is known to attenuate the vagal component of the baroreflex at both central and peripheral cardiac sites. Ang II receptor binding sites occur in both the nucleus of the solitary tract (NTS), where they are associated with vagal afferent terminals, and in the dorsal motor nucleus of vagus. In this study we have examined the distribution of Ang II binding sites in the cell bodies of vagal afferents in the nodose ganglion, and investigated whether these receptors are transported in the vagus nerve. Dense Ang II receptor binding was observed over neuronal cell bodies in the nodose ganglion and, in streaks, in the vagus nerve. Vagal ligation distal to the nodose ganglion resulted in a marked accumulation of receptor binding sites, proximal to the ligature, with a moderate increase on the distal side. These results demonstrate that Ang II receptor binding sites occur in the nodose ganglion and are transported centrally in the vagus to be located on presynaptic terminals in the NTS and also peripherally where they may occur on terminals of the vagus.

Animals↗
PubMed1988

Giant Aplysia neuron R2 has distal dendrites: evidence for protein sorting and a second spike initiation site.

Because of its anatomy, the neuron R2 of Aplysia has been used to study how proteins are distributed to their appropriate destinations within the cell. The R2 cell body resides in the abdominal ganglion, while its axons terminate on glands in the skin. Using intracellular injection of HRP and intraxonal recordings, we found that R2 has a dendritic (receptive) arborization in the pleural ganglion. The structure of these dendrites was examined after injecting the soma with 3H-L-fucose, thereby labeling glycoproteins that are transported to all regions of the cell. Light- and electron-microscope autoradiography show that the openings to the dendrites are not on the periphery, but are suspended inside the axon by glial cell infoldings. All of the organelles seen in the axon are found in the dendrites, including 2 types of vesicles. Neither the axon nor the dendrites contain ribosomes. Thus, R2 has 3 functionally distinct regions--cell body, dendrites, presynaptic terminals--that are separated from each other by at least 4 cm. This implies that pre- and postsynaptic proteins made in the cell body are transported along the axon to the pleural ganglion, where they are sorted. To investigate this idea, we exposed the abdominal ganglion to 35S-methionine to label R2's proteins. Analyses by SDS-PAGE of the rapidly transported labeled proteins from R2 consistently showed a 78 kDa band that accumulated in the pleural ganglion and did not move into the peripheral nerves. This then is a putative dendritic constituent.

Animals↗
PubMed1997

Intercellular communication that mediates formation of the neuromuscular junction.

Reciprocal signals between the motor axon and myofiber induce structural and functional differentiation in the developing neuromuscular junction (NMJ). Elevation of presynaptic acetylcholine (ACh) release on nerve-muscle contact and the correlated increase in axonal-free calcium are triggered by unidentified membrane molecules. Restriction of axon growth to the developing NMJ and formation of active zones for ACh release in the presynaptic terminal may be induced by molecules in the synaptic basal lamina, such as S-laminin, heparin binding growth factors, and agrin. Acetylcholine receptor (AChR) synthesis by muscle cells may be increased by calcitonin gene-related peptide (CGRP), ascorbic acid, and AChR-inducing activity (ARIA)/heregulin, which is the best-established regulator. Heparin binding growth factors, proteases, adhesion molecules, and agrin all may be involved in the induction of AChR redistribution to form postsynaptic-like aggregates. However, the strongest case has been made for agrin's involvement. "Knockout" experiments have implicated agrin as a primary anterograde signal for postsynaptic differentiation and muscle-specific kinase (MuSK), as a putative agrin receptor. It is likely that both presynaptic and postsynaptic differentiation are induced by multiple molecular signals. Future research should reveal the physiological roles of different molecules, their interactions, and the identity of other molecular participants.

Animals↗
PubMed1988

Characteristics of local excitatory circuits studied with glutamate microapplication in the CA3 area of rat hippocampal slices.

1. Local neuronal circuits in CA3 of hippocampal slices were studied by recording excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs) intracellularly during glutamate microapplication in CA3. Control experiments validated this approach by providing evidence that glutamate microdrops stimulated neurons but not axons-of-passage or axon terminals in CA3. 2. Glutamate microdrops (10-20 mM, 10-20 microns diam) increased the firing frequency of extracellularly recorded dentate granule cells for 5-10 s when applied to their somata but not when applied to their mossy fiber axons and terminals in the hilus and in CA3. 3. Glutamate microapplications to granule cell somata, but not to mossy fiber axons, also increased the frequency of intracellularly recorded EPSPs in CA3 pyramidal cells for 5-10 s. This provided a second line of evidence that glutamate did not cause firing in mossy fiber axons synapsing in CA3. 4. In slices where the CA3 region was surgically separated from the dentate gyrus and CA2, glutamate microdrops placed in the CA3 stratum pyramidale within 400 microns of intracellularly recorded pyramidal cells increased the frequency of EPSPs and IPSPs. Tetrodotoxin (1 microgram/ml) blocked these increases in PSP frequency, indicating that they did not result from glutamate-induced depolarization and associated transmitter release from presynaptic terminals. Increases in PSP frequency were interpreted to reflect glutamate activations of CA3 neurons with local synaptic connections to recorded cells. 5. Low concentrations of picrotoxin (PTX, 5-10 microM) blocked glutamate-induced increases in IPSP frequency and often revealed increases in EPSP frequency where they were not previously observed. This suggests that recurrent inhibitory circuits normally mask or block transmission through recurrent excitatory pathways in CA3. 6. In five experiments following PTX treatment (7.5-10 microM), large and prolonged (up to 2 min) increases in EPSP frequency were observed in CA3 pyramidal cells to glutamate microapplications in CA3. Rhythmic epileptiform bursts eventually occurred in two of these cases, suggesting that the protracted increases in EPSP frequency represent a form of reverberating excitation during a transition from normal to epileptic states. 7. Sixteen CA3 pyramidal cells were recorded in PTX (5-10 microM) during glutamate microapplications at 200 and 400 microns on each side of the recording site. The most consistent glutamate-induced increases in EPSP frequency occurred to microapplications 200 microns from recording sites on the hilar side.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2005

Zinc accumulation after target loss: an early event in retrograde degeneration of thalamic neurons.

Accumulation of cytoplasmic zinc is linked with a cascade of events leading to neuronal death. In many in vivo models of zinc-induced cell death, toxic concentrations of synaptically released zinc enter vulnerable neurons via neurotransmitter- or voltage-gated ion channels. In vitro studies demonstrate, in addition, that zinc can be liberated from intracellular stores following oxidative stress and contribute to cell death processes, including apoptosis. Here we describe accumulation of intracellular zinc in an in vivo model of cell death in the absence of presynaptic zinc release. We focused on the lateral geniculate nucleus (LGN) because LGN neurons undergo apoptosis when separated from their target, the primary visual cortex (V1), and the LGN is mostly devoid of zinc-containing presynaptic terminals. Infant and adult rats and adult mice received unilateral ablation of V1, either by aspiration or kainate injection. One to 14 days later, brain sections were stained with selenium autometallography or fluorescently labeled to localize zinc, or stained immunochemically for activated caspase-3. V1 lesions led to zinc accumulation in LGN neurons in infant and adult subjects. Zinc-containing neurons were evident 1-3 days after aspiration lesions, depending on age, but not until 14 days after kainate injection. Zinc accumulation was followed rapidly by immunostaining for activated caspase-3. Our data indicate that like neurotrauma and excitotoxicity, target deprivation leads to accumulation of zinc in apoptotic neurons. Moreover, zinc accumulation in vivo can occur in the absence of presynaptic zinc release. Together these findings suggest that accumulation of intracellular zinc is a ubiquitous component of the cell death cascade in neurons.

Animals↗
PubMed2005

Overexpression of drebrin A in immature neurons induces the accumulation of F-actin and PSD-95 into dendritic filopodia, and the formation of large abnormal protrusions.

Drebrin A is a neuron-specific F-actin binding protein, and plays a pivotal role in the spine formation. In this study, we expressed drebrin A tagged with green fluorescent protein (GFP-DA) in hippocampal neurons at 7-9 days in vitro when presynaptic terminals are not fully maturated. GFP-DA was accumulated in dendritic protrusions and formed large abnormal structures. Since these structures were similar to filopodia in terms of lack of MAP2 immunostaining, we named them "megapodia" meaning large dendritic filopodia. F-actin and PSD-95 were also accumulated in megapodia, and their amounts were significantly correlated with that of GFP-DA. However, the expression of GFP-DA did not result in the promotion of the morphological change from filopodia into spines. These results demonstrate that drebrin A accumulates spine-resident proteins via protein-protein interaction in filopodia, and suggest that the spine formation requires the concurrence of the increase of drebrin-A expression and the functional presynaptic contact.

Actins↗
PubMed1990

Immunocytochemical localization of endopeptidase-24.11 in the nucleus tractus solitarius of the rat brain.

Recently, it has been hypothesized that the N-terminal portion of substance P (SP), SP(1-7), which results from the action of endopeptidase 24.11 (EC3.4.24.11), could be involved in mediating the depressor effects of baroreceptor afferent activation via its action on cells in the nucleus tractus solitarius (NTS). In this study, the binding of a monoclonal antibody to endopeptidase 24.11 was examined immunohistochemically at the level of the caudal medulla of the rat brain. By light microscopy, intense immunoreactivity was seen in the NTS, in fibers bordering the area postrema, and in the area postrema itself. After electron microscopy, endopeptidase 24.11-like immunoreactivity was seen to be associated with the cytoskeleton and plasma membrane in axons, dendrites and glial processes. Antigen was also associated with synaptic vesicles and plasma membranes in presynaptic terminals forming mainly axo-dendritic synapses typical of vagal afferent terminals involved in the baroreceptor reflex. Thus, endopeptidase 24.11 appears to be localized at sites where it could effectively process SP prior to its binding to postsynaptic receptors.

Amino Acid Sequence↗
PubMed1978

Depressant action of lithium at the crayfish neuromuscular junction: pre- and postsynaptic effects.

1. The effects of replacement of external sodium ions with lithium have been studied at the excitatory neuromuscular junction of the crayfish. 2. Intracellularly recorded excitatory junctional potentials fall 45% in amplitude in the first 10 min after lithium substitution, and fail irreversibly in 48--120 min. 3. The quantal content of extracellularly recorded excitatory junctional potentials declines 25--70% within the first 10 min of Li+ exposure. During the next 40--120 min the nerve terminal potentials and quantal release at individual synapses fail simultaneously and irreversibly. 4. The mean amplitude of the spontaneous miniature excitatory junctional potentials (m.e.j.p.s) is reduced 13% by Li+ substitution, but recovers upon restoration of sodium. The mean frequency of m.e.j.p.s rises steadily during Li+ exposure, and continues to increase after reintroduction of Na+. 5. The postsynaptic response to iontophoretically applied L-glutamate falls 35--40% in 10 min, but never falls below 45% of the control level. The effect on the glutamate response is completely reversible with sodium restoration. 6. The effective resistance of the postsynpatic cells is unaffected or only increased slightly by lithium substitution. 7. Thus, the primary mechanism of transmission block by lithium appears to be decreased transmitter release and inexcitability of presynaptic terminals, probably as a result of intracellular accumulation of lithium.

Animals↗
PubMed1983

The pineal organ of Raja clavata: opsin immunoreactivity and ultrastructure.

The pineal organ of Raja clavata was studied by light and electron microscopy, including the immunocytochemical antiopsin reaction. The pineal organ of the ray consists of three portions: (i) a large proximal pineal, (ii) a long tube-like connecting stalk, and (iii) a short distal terminal enlargement. This latter end-vesicle lies in the deep connective tissue layers of the braincase. All portions of the pineal are composed of pinealocytes, intrinsic neurons, ependymal/glial cells, and bundles of nerve fibers embedded in thin neuropil formations. The inner segments of the pinealocytes protrude into the lumen in all parts of the organ and usually contain basal bodies and numerous mitochondria. Often, two outer segments were found to arise from the basal bodies of a single inner segment. By means of light-microscopic immunocytochemistry the outer segments showed a strong antiopsin reaction. The axons of the pinealocytes form ribbon-containing synapses on dendrite-like profiles, which appear to belong to the intrinsic pineal neurons. There are other axo-dendritic synapses established by presynaptic terminals lacking ribbons and containing granular and synaptic vesicles. Pineal neurons may contain granular vesicles approximately 60-100 nm in diameter; their processes contribute to the bundles of unmyelinated axons. The fine structural organization of the pineal organ and the opsin immunoreactivity of the outer segments of the pinealocytes indicate a photoreceptive capacity of the organ. The double outer segments represent a peculiar multiplication of the photoreceptor structures.

Animals↗
PubMed1986

The transneuronal transport of horseradish peroxidase in the visual system of the frog, Rana pipiens.

During the course of experiments designed to study synaptic relationships between the terminals of retinal axons and the various cell populations in the optic tectum of the frog, Rana pipiens, we found that neurons in many of the retinorecipient nuclei, including the tectum, are labeled transneuronally following injections of horseradish peroxidase into the optic nerve. In the optic tectum, particular cell groups are labeled to the extent that their dendrites as well as their somas are filled with reaction product while other cell types, which, on the basis of the location of their somas or dendrites, seem equally likely to receive direct retinal projections, remain free of label. Electron microscopic investigation of the optic tectum reveals that the label is confined to pre- and postsynaptic processes. These results suggest that the transneuronal transport depends on a transfer of horseradish peroxidase from presynaptic terminals to postsynaptic cells rather than on a widespread diffusion of the enzyme through the neuropil followed by a selective uptake by particular cell groups. These results also suggest that only some of the tectal cell groups which receive direct retinal projections may be transneuronally labeled. The transneuronal transport of horseradish peroxidase is useful since it reveals the morphology as well as the location of at least some of the retinorecipient cells. Moreover, the robust nature of this phenomenon makes the frog a good choice for future studies of the mechanism of transneuronal transport.

Animals↗
PubMed2000

The neuregulin receptor ErbB-4 interacts with PDZ-containing proteins at neuronal synapses.

Neuregulins regulate the expression of ligand- and voltage-gated channels in neurons and skeletal muscle by the activation of their cognate tyrosine kinase receptors, ErbB 1-4. The subcellular distribution and mechanisms that regulate the localization of ErbB receptors are unknown. We have found that ErbB receptors are present in brain subcellular fractions enriched for postsynaptic densities (PSD). The ErbB-4 receptor is unique among the ErbB proteins because its C-terminal tail (T-V-V) conforms to a sequence that binds to a protein motif known as the PDZ domain. Using the yeast two-hybrid system, we found that the C-terminal region of ErbB-4 interacts with the three related membrane-associated guanylate kinases (MAGUKs) PSD-95/SAP90, PSD-93/chapsyn-110, and SAP 102, which harbor three PDZ domains, as well as with beta(2)-syntrophin, which has a single PDZ domain. As with N-methyl-D-aspartate (NMDA) receptors, ErbB4 interacts with the first two PDZ domains of PSD-95. Using coimmunoprecipitation assays, we confirmed the direct interactions between ErbB-4 and PSD-95 in transfected heterologous cells, as well as in vivo, where both proteins are coimmunoprecipitated from brain lysates. Moreover, evidence for colocalization of these proteins was also observed by immunofluorescence in cultured hippocampal neurons. ErbB-4 colocalizes with PSD-95 and NMDA receptors at a subset of excitatory synapses apposed to synaptophysin-positive presynaptic terminals. The capacity of ErbB receptors to interact with PDZ-domain proteins at cell junctions is conserved from invertebrates to mammals. As discussed, the interactions found between receptor tyrosine kinases and MAGUKs at neuronal synapses may have important implications for activity-dependent plasticity.

Animals↗
PubMed2003

Immunolocalization of the acid-sensing ion channel 2a in the rat cerebellum.

The acid-sensing ion channels (ASICs) are members of the DEG/ENaC superfamily of Na+ channels. Acid-gated cation currents have been detected in neurons from multiple regions of the brain including the cerebellum, but little is known about their molecular identity and function. Recently, one of ASICs (ASIC1a) was implicated in synaptic plasticity. In this study we examined the subcellular distribution of ASIC2a in rat cerebellum by immunostaining and confocal microscopy. Monoclonal antibodies for labeling of defined brain structures, for example, astroglia, Purkinje cell dendrites, nuclei, and presynaptic terminals were used for colocalization analyses. In the gray matter, the anti-ASIC2a antibody intensively stained dendrite branches of Purkinje cells evenly distributed throughout the entire molecular layer (ML). In the granule cell layer (GL), anti-ASIC2a antibody stained synaptic glomeruli. Neuronal localization of ASIC2a was confirmed by lack of co-staining with glial fibrillary acidic protein. Anti-ASIC2a staining in the ML colocalized with metabotropic glutamate receptor 1alpha (mGluR1alpha) in Purkinje cell dendrites and dendritic spines. Both proteins, mGluR1alpha and ASIC2a, were enriched in a crude synaptic membrane fraction prepared from cerebellum, suggesting synaptic expression of these proteins. Dual staining with anti-syntaxin 1A and anti-ASIC2a antibodies demonstrates characteristic complementary distribution of two proteins in both ML and GL. Because syntaxin 1A localized in presynaptic membranes and synaptic vesicles, complementary distribution with ASIC2a suggests postsynaptic localization of ASIC2a in these structures. This study shows specific localization of ASIC2a in both Purkinje and granule cell dendrites of the cerebellum and enrichment of ASIC2a in a crude cerebellar synaptic membrane fraction. The study is the first report of synaptic localization of ASIC2a in the CNS. The synaptic localization of ASIC2a in the cerebellum makes this channel a candidate for a role in motor coordination and learning.

Acid Sensing Ion Channels↗