Glial Plasma Membranes Research Articles

Nerve Structure-Function: Unusual Structural Details and Unmasking of Sulfhydryl Groups by Electrical Stimulation or Asphyxia in Axon Membranes and Gap Junctions.

This review describes and discusses unusual axonal structural details and evidence for unmasking sulfhydryl groups (-SH) in axoplasmic membranes resulting from electrical stimulation or asphyxia. Crayfish axons contain fenestrated septa (FS) that, in phase contrast, micrographs appear as repeated striations. In the electron microscope, each septum is made of two cross-sectioned membranes containing ~55 nm pores, each occupied by a microtubule. Thin filaments, which we believe are made of kinesin, bridge the microtubule to the edge of the pore. FS are believed to play a role in axoplasmic flow. The axons also display areas in which axon and sheath glial cell plasma membranes are sharply curved and project into the axoplasm. In freeze-fractures, the protoplasmic leaflet (P-face) of the projections appears as elongated indentations containing parallel chains of particles. The sheath glial cell plasma membrane also contains particles, but they are irregularly aggregated. The axons also display areas where axonal and glial plasma membranes fuse, creating intercellular pores. In axons fixed during electrical stimulation, the plasma membrane, the outer membrane of mitochondria, membranes of other cytoplasmic organelles, and gap junctions increase in electron opacity and thickness, resulting from unmasking of sulfhydryl groups (-SH). Similar changes occur in asphyxiated nerve cords.

Glycine Transporters in Glia Cells: Structural Studies.

Glycine, besides exerting essential metabolic functions, is an important inhibitory neurotransmitter in caudal areas of the central nervous system and also a positive neuromodulator at excitatory glutamate-mediated synapses. Glial cells provide metabolic support to neurons and modulate synaptic activity. Six transporters belonging to three solute carrier families (SLC6, SLC38, and SLC7) are capable of transporting glycine across the glial plasma membrane. The unique glial glycine-selective transporter GlyT1 (SLC6) is the main regulator of synaptic glycine concentrations, assisted by the neuronal GlyT2. The five additional glycine transporters ATB0,+, SNAT1, SNAT2, SNAT5, and LAT2 display broad amino acid specificity and have differential contributions to glial glycine transport. Glial glycine transporters are divergent in sequence but share a similar architecture displaying the 5 + 5 inverted fold originally characterized in the leucine transporter LeuT. The availability of protein crystals solved at high resolution for prokaryotic and, more recently, eukaryotic homologues of this superfamily has advanced significantly our understanding of the mechanism of glycine transport.

Organic cation transporter 3 (OCT3) is localized to intracellular and surface membranes in select glial and neuronal cells within the basolateral amygdaloid complex of both rats and mice

Organic cation transporter 3 (OCT3) is a high-capacity, low-affinity transporter that mediates corticosterone-sensitive uptake of monoamines including norepinephrine, epinephrine, dopamine, histamine and serotonin. OCT3 is expressed widely throughout the amygdaloid complex and other brain regions where monoamines are key regulators of emotional behaviors affected by stress. However, assessing the contribution of OCT3 to the regulation of monoaminergic neurotransmission and monoamine-dependent regulation of behavior requires fundamental information about the subcellular distribution of OCT3 expression. We used immunofluorescence and immuno-electron microscopy to examine the cellular and subcellular distribution of the transporter in the basolateral amygdaloid complex of the rat and mouse brain. OCT3-immunoreactivity was observed in both glial and neuronal perikarya in both rat and mouse amygdala. Electron microscopic immunolabeling revealed plasma membrane-associated OCT3 immunoreactivity on axonal, dendritic, and astrocytic processes adjacent to a variety of synapses, as well as on neuronal somata. In addition to plasma membrane sites, OCT3 immunolabeling was also observed associated with neuronal and glial endomembranes, including Golgi, mitochondrial and nuclear membranes. Particularly prominent labeling of the outer nuclear membrane was observed in neuronal, astrocytic, microglial and endothelial perikarya. The localization of OCT3 to neuronal and glial plasma membranes adjacent to synaptic sites is consistent with an important role for this transporter in regulating the amplitude, duration, and physical spread of released monoamines, while its localization to mitochondrial and outer nuclear membranes suggests previously undescribed roles for the transporter in the intracellular disposition of monoamines.

Closing the Gap between Glia and Neuroblast Proliferation

Reporting in this issue of Developmental Cell, Spéder and Brand (2014) show that gap junctions are required in blood-brain barrier glial cells to reactivate proliferation of quiescent neuroblasts. Gap junctions allow synchronous Ca(2+) waves and control insulin-like protein Dipl6 expression and secretion to trigger neuroblast division.

Tonically balancing intracortical excitation and inhibition by GABAergic gliotransmission.

For sensory cortices to respond reliably to feature stimuli, the balancing of neuronal excitation and inhibition is crucial. A typical example might be the balancing of phasic excitation within cell assemblies and phasic inhibition between cell assemblies. The former controls the gain of and the latter the tuning of neuronal responses. A change in ambient GABA concentration might affect the dynamic behavior of neurons in a tonic manner. For instance, an increase in ambient GABA concentration enhances the activation of extrasynaptic receptors, augments an inhibitory current, and thus inhibits neurons. When a decrease in ambient GABA concentration occurs, the tonic inhibitory current is reduced, and thus the neurons are relatively excited. We simulated a neural network model in order to examine whether and how such a tonic excitatory-inhibitory mechanism could work for sensory information processing. The network consists of cell assemblies. Each cell assembly, comprising principal cells (P), GABAergic interneurons (Ia, Ib), and glial cells (glia), responds to one particular feature stimulus. GABA transporters, embedded in glial plasma membranes, regulate ambient GABA levels. Hypothetical neuron-glia signaling via inhibitory (Ia-to-glia) and excitatory (P-to-glia) synaptic contacts was assumed. The former let transporters import (remove) GABA from the extracellular space and excited stimulus-relevant P cells. The latter let them export GABA into the extracellular space and inhibited stimulus-irrelevant P cells. The main finding was that the glial membrane transporter gave a combinatorial excitatory-inhibitory effect on P cells in a tonic manner, thereby improving the gain and tuning of neuronal responses. Interestingly, it worked cooperatively with the conventional, phasic excitatory-inhibitory mechanism. We suggest that the GABAergic gliotransmission mechanism may provide balanced intracortical excitation and inhibition so that the best perceptual performance of the cortex can be achieved.

Balanced Crossmodal Excitation and Inhibition Essential for Maximizing Multisensory Gain

We examined whether and how the balancing of crossmodal excitation and inhibition affects intersensory facilitation. A neural network model, comprising lower-order unimodal networks (X, Y) and a higher-order multimodal network (M), was simulated. Crossmodal excitation was made by direct activation of principal cells of the X network by the Y network. Crossmodal inhibition was made in an indirect manner: the Y network activated glial cells of the X network. This let glial plasma membrane transporters export GABA molecules into the extracellular space and increased the level of ambient GABA. The ambient GABA molecules were accepted by extrasynaptic GABAa receptors and tonically inhibited principal cells of the X network. Namely, crossmodal inhibition was made through GABAergic gliotransmission. Intersensory facilitation was assessed in terms of multisensory gain: the difference between the numbers of spikes evoked by multisensory (XY) stimulation and unisensory (X-alone) stimulation. The maximal multisensory gain (XY-X) could be achieved at an intermediate noise level by balancing crossmodal excitation and inhibition. This result supports an experimentally derived conclusion: intersensory facilitation under noisy environmental conditions is not necessarily in accord with the principle of inverse effectiveness; rather, multisensory gain is maximal at intermediate signal-to-noise ratio (SNR) levels. The maximal multisensory gain was available at the weakest signal if noise was not present, indicating that the principle of inverse effectiveness is a special case of the intersensory facilitation model proposed here. We suggest that the balancing of crossmodal excitation and inhibition may be crucial for intersensory facilitation. The GABAergic glio-transmission-mediated crossmodal inhibitory mechanism effectively works for intersensory facilitation and on determining the maximal multisensory gain in the entire SNR range between the two extremes: low and high SNRs.

Neurons Regulate Glial Cell Surface Proteome (P07.089)

Objective: To identify glial cell surface receptors that transduce neuronal signals across glial plasma membrane. Background Bidirectional neuro-glia interactions are known to play important roles in maintaining normal functioning and architecture of CNS in health and disease. For example, neuronal signals reportedly influence overall gene expression in glial cells, but how these signals are transduced at the glial cell surface into glial specific functional responses remains undefined. Design/Methods: To identify receptors important in transducing neuro-glial communication, we performed a proteome screen on glial surface-enriched molecules obtained from primary glia, exposed to soluble neuronal factors or left un-treated as control. Results: While levels of proteins in the total glial cell lysates remained unchanged, cell surface biotinylation, followed by unbiased proteomics, identified significant changes in key signaling and adhesion molecules. Surprisingly, we observed that exposure to the neuronal factors caused contrasting regulation of several key molecules on the surface of astrocytes compared to oligodendrocytes, including adhesion molecules like N-cadherin and β-catenin along with neurotrophin receptors, known to positively regulate myelination. These molecules were strongly down-regulated on astrocytic surface whereas they were significantly up-regulated on oligodendrocytes. Consistent with the changes in expression of receptors, we found presence of secreted neurotrophins and processed N-cadherin ecto-domain in the neuronal media. We are now confirming that shed ecto-domain of N-cadherin and neurotrophins released by neurons can modulate cell surface expression of their receptors in a cell type specific manner, leading to different downstream activation events in distinct CNS glia. Conclusions: The distinct regulation of above mentioned cell surface receptors will be important in understanding the neuron-glial interactions in both normal development and neurological disorders. We hypothesise that normal neuro-glial cross talk is disrupted under inflamed CNS conditions; and understanding the mechanism of such a perturbed cellular communication is the subject of our ongoing studies. Disclosure: Dr. Awasthi has nothing to disclose. Dr. Morisse has nothing to disclose. Dr. Colman has nothing to disclose. Dr. Dhaunchak has nothing to disclose. Dr. Bar-Or has received personal compensation for activities with Aventis Pharmaceuticals, Bayhill Therapeutics, Biogen Idec, Berlex Laboratories, Eli Lilly & Company, Genentech, Inc., GlaxoSmithKline, Ono Pharmaceutical, Diogenix, Roche Diagnostics Corporation, Merck Serono, Novartis, Teva Neuroscience.

Phosphatidylinositol 4,5-Bisphosphate-Dependent Interaction of Myelin Basic Protein with the Plasma Membrane in Oligodendroglial Cells and Its Rapid Perturbation by Elevated Calcium

Myelin basic protein (MBP) is an essential structural component of CNS myelin. The electrostatic association of this positively charged protein with myelin-forming membranes is a crucial step in myelination, but the mechanism that regulates myelin membrane targeting is not known. Here, we demonstrate that phosphatidylinositol 4,5-bisphosphate (PIP2) is important for the stable association of MBP with cellular membranes. In oligodendrocytes, overexpression of synaptojanin 1-derived phosphoinositide 5-phosphatase, which selectively hydrolyzes membrane PIP2, causes the detachment of MBP from the plasma membrane. In addition, constitutively active Arf6/Q67L induces the formation of PIP2-enriched endosomal vacuoles, leading to the redistribution of MBP to intracellular vesicles. Fluorescence resonance energy transfer imaging revealed an interaction of the PIP2 sensing probe PH-PLCdelta1 with wild-type MBP, but not with a mutant MBP isoform that fails to associate with the plasma membrane. Moreover, increasing intracellular Ca(2+), followed by phospholipase C-mediated PIP2 hydrolysis, as well as reduction of the membrane charge by ATP depletion, resulted in the dissociation of MBP from the glial plasma membrane. When the corpus callosum of mice was analyzed in acute brain slices by electron microscopy, the reduction of membrane surface charge led to the loss of myelin compaction and rapid vesiculation. Together, these results establish that PIP2 is an essential determinant for stable membrane binding of MBP and provide a novel link between glial phosphoinositol metabolism and MBP function in development and disease.

A rapid Percoll gradient procedure for preparation of synaptosomes

Homogenization of fresh brain tissue in isotonic medium shears plasma membranes causing nerve terminals to become separated from their axons and postsynaptic connections. The nerve terminal membranes then reseal to form synaptosomes. The discontinuous Percoll gradient procedure described here is designed to isolate synaptosomes from brain homogenates in the minimum time to allow functional experiments to be performed. Synaptosomes are isolated using a medium-speed centrifuge, while maintaining isotonic conditions and minimizing mechanically damaging resuspension steps. This protocol has advantages over other procedures in terms of speed and by producing relatively homogeneous synaptosomes, minimizing the presence of synaptic and glial plasma membranes and extrasynaptosomal mitochondria. The purified synaptosomes are viable and take up and release neurotransmitters very efficiently. A typical yield of synaptosomes is between 2.5 and 4 mg of synaptosomal protein per gram rat brain. The procedure takes approximately 1 h from homogenization of the brain until collection of the synaptosomal suspension from the Percoll gradient.

Developmental expression of glycine receptor subunits in rat cerebellum

The distribution of the glycine receptor subunits α1–3 and β in the developing rat cerebellum was studied from postnatal day 1 to adulthood by means of quantitative RT-PCR and immunohistochemistry. qRT-PCR of postnatal cerebella indicated the presence of mRNA for each subunit, with a relative expression of α2>α3>α1>β. The immunohistochemistry indicated a strong α2 signal in the Purkinje cells, internal and external granular layers. The α1–3 subunits had weak signals in the Purkinje cells and molecular layer. The α1 subunit was expressed at a low level and was also found in the white matter. The function of these receptors in neuronal and glial plasma membranes in early postnatal development remains to be determined.

Involvement of adenylate cyclase and tyrosine kinase signaling pathways in response of crayfish stretch receptor neuron and satellite glia cell to photodynamic treatment

Neuroglial interactions are most profound during development or damage of nerve tissue. We studied the responses of crayfish stretch receptor neurons (SRN) and satellite glial cells to photosensitization with sulfonated aluminum phthalocyanine Photosens. Although Photosens was localized mainly in the glial envelope, neurons were very sensitive to photodynamic treatment. Photosensitization gradually inhibited and then abolished neuron activity. Neuronal and glial nuclei shrank. Some neurons and glial cells lost the integrity of the plasma membrane and died through necrosis after the treatment. The nuclei of other glial cells but not neurons become fragmented, indicating apoptosis. The number of glial nuclei around neuron soma increased, probably indicating proliferation for enhanced neuron protection. Adenylate cyclase (AC) inhibition by MDL-12330A, or tyrosine kinase (TK) inhibition by genistein, shortened neuron lifetime, whereas AC activation by forskolin or protein tyrosine phosphatases (PTP) inhibition by sodium orthovanadate prolonged neuronal activity. Therefore, cAMP and phosphotyrosines produced by AC and TK, respectively, protected SRN against photoinactivation. AC inhibition reduced photodamage of the plasma membrane and subsequent necrosis in neuronal and glial cells. AC activation prevented apoptosis in photosensitized glial cells and stimulated glial proliferation. TK inhibition protected neurons but not glia against photoinduced membrane permeabilization and subsequent necrosis whereas PTP inhibition more strongly protected glial cells. Therefore, both signaling pathways involving cAMP and phosphotyrosines might contribute to the maintenance of neuronal activity and the integrity of the neuronal and glial plasma membranes. Adenylate cyclase but not phosphotyrosine signaling pathways modulated glial apoptosis and proliferation under photooxidative stress.

Bergmann Glia GABAAReceptors Concentrate on the Glial Processes That Wrap Inhibitory Synapses

We studied the cellular and subcellular distribution of GABA(A) receptors in the Bergmann glia and Purkinje cells in the molecular layer of the cerebellum by using electron microscopy postembedding immunogold techniques. Gold particles corresponding to alpha2 and gamma1 immunoreactivity were localized in Bergmann glia processes that wrapped Purkinje cell somata, dendritic shafts, and some dendritic spines. The gold particles were mainly located on the glial plasma membrane or intracellularly but near the plasma membrane. The density of gold particles corresponding to alpha2 and gamma1 GABA(A) receptor subunits was 4.3-fold higher in the glial processes wrapping Purkinje cell somata than in the glial processes wrapping Purkinje cell dendritic spines. Moreover, the Bergmann glia GABA(A) receptors were often located in close proximity to the type II GABAergic synapses made by the basket cell axons on Purkinje cell somata. These GABAergic synapses were enriched in neuronal GABA(A) receptors containing alpha1 and beta2/3 subunits. Unexpectedly, 2.8% of the Purkinje cell dendritic spines also showed immunoreactivity for the neuronal alpha1 or beta2/3 subunits, which were located on the spine in type I synapses or extrasynaptically. Double-labeling immunogold experiments showed that approximately 50% of the dendritic spines that were immunolabeled with the neuronal GABA(A) receptors were wrapped by Bergmann glia processes containing glial GABA(A) receptors. These results are consistent with a role of the Bergmann glial GABA(A) receptors in sensing GABAergic synaptic function.

High-affinity neurotensin receptors in the rat nucleus accumbens: subcellular targeting and relation to endogenous ligand.

Neurotensin is present in selective mesolimbic dopaminergic projections to the nucleus accumbens (NAc) shell but also is synthesized locally in this region and in the motor-associated NAc core. We examined the electron microscopic immunolabeling of the high-affinity neurotensin receptor (NTR) and neurotensin in these subdivisions of rat NAc to determine the sites for receptor activation and potential regional differences in distribution. Throughout the NAc, NTR immunoreactivity was localized discretely within both neurons and glia. NTR-labeled neuronal profiles were mainly axons and axon terminals with diverse synaptic structures, which resembled dopaminergic and glutamatergic afferents, as well as collaterals of inhibitory projection neurons. These terminals had a significantly higher numerical density in the NAc core than in the shell but were prevalent in both regions, suggesting involvement in both motor and limbic functions. In each region, neurotensin was detected in a few NTR-immunoreactive axon terminals and in terminals that formed symmetric, inhibitory type synapses with NTR-labeled somata and dendrites. The NTR labeling, however, was not seen within these synapses and, instead, was localized to segments of dendritic and glial plasma membranes often near excitatory type synapses. Neuronal NTR immunoreactivity also was associated with cytoplasmic tubulovesicles and nuclear membranes. Our results suggests that, in the NAc shell and core, NTR is targeted mainly to presynaptic sites, playing a role in the regulated secretion and/or retrograde signaling in diverse, neurotransmitter-specific neurons. The findings also support a volume mode of neurotensin actions, specifically affecting excitatory transmission through activation of not only axonal but also dendritic and glial NTR.

Direct immunogold labeling of aquaporin-4 in square arrays of astrocyte and ependymocyte plasma membranes in rat brain and spinal cord.

Aquaporin (AQP) water channels are abundant in the brain and spinal cord, where AQP1 and AQP4 are believed to play major roles in water metabolism and osmoregulation. Immunocytochemical analysis of the brain recently revealed that AQP4 has a highly polarized distribution, with marked expression in astrocyte end-feet that surround capillaries and form the glia limitans; however, the structural organization of AQP4 has remained unknown. In freeze-fracture replicas, astrocyte end-feet contain abundant square arrays of intramembrane particles that parallel the distribution of AQP4. To determine whether astrocyte and ependymocyte square arrays contain AQP4, we employed immunogold labeling of SDS-washed freeze-fracture replicas and stereoscopic confirmation of tissue binding. Antibodies to AQP4 directly labeled approximately 33% of square arrays in astrocyte and ependymocyte plasma membranes in rat brain and spinal cord. Overall, 84% of labels were present beneath square arrays; 11% were beneath particle clusters that resembled square arrays that had been altered during fixation or cleaving; and 5% were beneath the much larger areas of glial plasma membrane that were devoid of square arrays. Based on this evidence that AQP4 is concentrated in glial square arrays, freeze-fracture methods may now provide biophysical insights regarding neuropathological states in which abnormal fluid shifts are accompanied by alterations in the aggregation state or the molecular architecture of square arrays.

Molecular biology of glycinergic neurotransmission.

Glycine is a major inhibitory neurotransmitter in the spinal cord and brainstem of vertebrates. Glycine is accumulated into synaptic vesicles by a proton-coupled transport system and released to the synaptic cleft after depolarization of the presynaptic terminal. The inhibitory action of glycine is mediated by pentameric glycine receptors (GlyR) that belong to the ligand-gated ion channel superfamily. The synaptic action of glycine is terminated by two sodium- and chloride-coupled transporters, GLYT1 and GLYT2, located in the glial plasma membrane and in the presynaptic terminals, respectively. Dysfunction of inhibitory glycinergic neurotransmission is associated with several forms of inherited mammalian myoclonus. In addition, glycine could participate in excitatory neurotransmission by modulating the activity of the NMDA subtype of glutamate receptor. In this article, we discuss recent progress in our understanding of the molecular mechanisms that underlie the physiology and pathology of glycinergic neurotransmission.

GAT-3, a High-Affinity GABA Plasma Membrane Transporter, Is Localized to Astrocytic Processes, and It Is Not Confined to the Vicinity of GABAergic Synapses in the Cerebral Cortex

The termination of GABA synaptic action by high-affinity, Na(+)-dependent, neuronal, and glial plasma membrane transporters plays an important role in regulating neuronal activity in physiological and pathological conditions. We have investigated the cellular localization and distribution in the cerebral cortex of adult rats of one GABA transporter (GAT), GAT-3, by immunocytochemistry with affinity-purified polyclonal antibodies directed to its predicted C terminus that react monospecifically with a protein of approximately 70 kDa. Light microscopic studies revealed specific GAT-3 immunoreactivity (ir) in small punctate structures, and it was never observed in fibers or cell bodies. No changes in immunostaining were observed in sections incubated with GAT-3 antibodies preadsorbed with the related rat GAT-1 or mouse GAT-2/ BGT-1 C-terminal peptides, whereas in sections incubated with GAT-3 antibodies preadsorbed with rat GAT-3 C-terminal peptide, ir was not present. The highest number of GAT-3-positive puncta was in layer IV and in a narrow band corresponding to layer Vb, followed by layers II and III. Many GAT-3-positive puncta were in close association with pyramidal and nonpyramidal neuron cell bodies. Ultrastructural studies showed that GAT-3 ir was localized exclusively to astrocytic processes, which were found in the neuropil and adjacent to axon terminals having either symmetric or asymmetric specializations. In sections processed by both preembedding labeling for GAT-3 and postembedding immunogold labeling for GABA, only some of the GAT-3-positive astrocytic processes were found close to GABAergic profiles. These findings on the localization of GAT-3 in the cerebral cortex indicate that this transporter mediates GABA uptake into glial cells, and suggest that glial GABA uptake may function to limit the spread of GABA from the synapse, as well as to regulate overall GABA levels in the neuropil.

Qualitative enzyme histochemistry and microanalysis reveals changes in ultrastructural distribution of calcium and calcium-activated ATPases after microwave irradiation of the medial habenula.

The localization of calcium and calcium-activated ATPases was investigated electron microscopically in the medial habenula of mice after whole body irradiation with modulated microwaves. In non-irradiated animals calcium-containing precipitates were seen in different subcellular compartments and were often localized on the luminal side of membranes of synaptic vesicles in nerve terminals. At 1 h after 16-Hz modulated microwave irradiation, the number of synaptic vesicles containing calcium precipitates decreased, and reaction products appeared at new locations: in the synaptic clefts and on non-synaptic surfaces of the neuronal plasma membrane. This modified calcium distribution remained unchanged for 24 h following irradiation. Calcium-activated "ecto"-localized ATPase was detected as a punctuated-linear distribution of the reaction product outlining whole areas of glial and neuronal plasma membrane in the habenula of control animals. This pattern did not change on microwave irradiation. However, a quercetin-sensitive "endo"-localized Ca(2+)-ATPase activity appeared in some nerve terminals 24 h after irradiation. Thus, microwave irradiation can influence neuronal calcium homeostasis by inducing Ca2+ redistribution across the plasma membrane and by modifying Ca(2+)-ATPase activity. However, no direct correlation between these effects could be demonstrated by the present study.

Ultrastructural Immunocytochemical Localization of μ-Opioid Receptors in Rat Nucleus Accumbens: Extrasynaptic Plasmalemmal Distribution and Association with Leu5-Enkephalin

mu-Opioid receptors and their endogenous ligands, including Leu5-enkephalin (LE), are distributed abundantly in the nucleus accumbens (NAC), a region implicated in mechanisms of opiate reinforcement. We used immunoperoxidase and/or immunogold-silver methods to define ultrastructural sites for functions ascribed to mu-opioid receptors and potential sites for activation by LE in the NAC. An antipeptide antibody raised against an 18 amino acid sequence of the cloned mu-opioid receptor (MOR) C terminus showed that MOR-like immunoreactivity (MOR-LI) was localized predominantly to extrasynaptic sites along neuronal plasma membranes. The majority of neuronal profiles containing MOR-LI were dendrites and dendritic spines. The dendritic plasma membranes immunolabeled for MOR were near sites of synaptic input from LE-labeled terminals and other unlabeled terminals forming either inhibitory or excitatory type synapses. Unmyelinated axons and axon terminals were also intensely but less frequently immunoreactive for MOR. Observed sites for potential axonal associations with LE included coexistence of MOR and LE within the same terminal, as well as close appositions between differentially labeled axons. Astrocytic processes rarely contained detectable MOR-LI, but also were sometimes observed in apposition to LE-labeled terminals. We conclude that in the rat NAC, MOR is localized prominently to extrasynaptic neuronal and more rarely to glial plasma membranes that are readily accessible to released LE and possibly other opioid peptides and opiate drugs. The close affiliation of MOR with spines receiving excitatory synapses and dendrites receiving inhibitory synapses provides the first direct morphological evidence that MOR selectively modulates postsynaptic responses to cortical and other afferents.

Ultrastructural association of hyaluronan with rat unmyelinated nerve fibres.

A potential association between hyaluronan and unmyelinated nerve fibres in the PNS (rat skin and iris) and CNS (rat retinal inner plexiform and nerve fibre layers) was investigated at the ultrastructural level using two different hyaluronan-binding probes (link protein-gold and aggrecan-gold). Neuronal and glial cell plasma membranes, as well as the periaxonal space in between, were specifically labelled, suggesting that hyaluronan is secreted by these cells and utilized locally. Intracelluarly, weak labelling of mitochondrial profiles was observed.

Glutamate transporters in glial plasma membranes: Highly differentiated localizations revealed by quantitative ultrastructural immunocytochemistry

The glutamate transporters GLT-1 and GLAST were studied by immunogold labeling on ultrathin sections of rat brain tissue embedded in acrylic resins at low temperature after freeze substitution. Both proteins were selective markers of astrocytic plasma membranes. GLT-1 was much higher in hippocampal astrocytes than in cerebellar astrocytes. Astroglial membrane GLAST densities ranked as follows: Bergmann > cerebellar granular layer ≈ hippocampus > cerebellar white matter. No astrocyte appeared unlabeled. Astrocytic membranes facing capillaries, pia, or stem dendrites were lower in glutamate transporters than those facing nerveterminals, axons, and spines. Parallel fiber boutons (glutamatergic) synapsing on interneuron dendritic shafts were surrounded by lower transporter densities than those synapsing on Purkinje cell spines. Our findings suggest the localizations of glutamate transporters are carefully regulated.