Emerging evidence supports an increased role for NG2/CSPG4-expressing cells in the process of neuroregeneration and synaptic plasticity, due to the increased production of multifunctional chondroitin sulfate proteoglycan (NG2/CSPG4). However, the response of NG2/CSPG4-expressing cells in spinal cord injury (SCI) remains to be elcudiated. Expression and distribution of NG2/CSPG4-expressing cells were studied by immunoelectron microscopy in the ventral horns (VH) of an intact and injured rat spinal cord. In the intact spinal cord, NG2/CSPG4 expression was detected on the cell membrane and in the cytoplasm of NG2 glia and was absent in neurons. Large amounts of NG2/CSPG4 were found on myelin membranes. The ability of intact astrocytes to produce NG2/CSPG4 was shown, although to a lesser extent than oligodendrocytes and NG2 glia. At 7 days after SCI at the Th8 level in the reactive glial zone of VH, the expression of NG2/CSPG4 sharply increased in NG2 glia at a distance of 3-5 mm and in reactive astrocytes were observed at all investigated distances caudally from the epicenter of injury. The obtained results indicate the presence of NG2/CSPG4-positive astrocytes in the intact spinal cord, and in the case of damage, an increase in the ability of reactive astrocytes to produce NG2/CSPG4. SCI leads to increased expression of NG2/CSPG4 by NG2 glia in the early stages after injury, which decreases with distance from the epicenter of the injury, as well as at later stages.

Organic cation transporters (OCTs) are expressed mainly in the kidney and liver. OCTs transport intrinsic organic cations, including monoamine, dopamine, serotonine and choline, across the plasma membrane. Here, we demonstrate that OCT2 (SLC22A2) is expressed in cholinergic neurons, motoneurons in the anterior horn of the spinal cord, and is implicated in acetylcholine (Ach) recycling in presynaptic terminals. Application of rabbit anti-peptide antibody revealed that OCT2 was expressed in the anterior horn of the spinal cord. Double immunostaining of muscle sections with anti-OCT2 and alpha-bungarotoxin (BTX) revealed that OCT2 was localized in the neuromuscular junctions (NMJs). Immunoelectron microscopy revealed that OCT2 was localized both in synaptic vesicles (SVs) in presynaptic terminals around the motoneurons (C-terminals) and in SVs in nerve terminals in NMJs. The similarity in the distribution of OCT2 in cholinergic neurons and that of vesicular acetyl choline transporter (VAchT), and the fact that OCT2 can transport choline suggest that OCT2 could work as a low-affinity and high-capacity choline transporter at presynaptic terminals in cholinergic neurons in a firing-dependent manner.

From our previous studies, myotonic dystrophy protein kinase: gene product of myotonic dystrophy is localized at the terminal cisternae of sarcoplasmic reticulum of human adult muscle. Now we have studied the developmental expression of myotonic dystrophy protein kinase in aneurally cultured human muscles and contracting cross-striated muscles innervated with fetal rat spinal cord using a semi-quantitative reverse transcription-polymerase chain reaction method for myotonic dystrophy protein kinase messenger RNA expression, Western blot analysis, immunohistochemical examinations by laser scanning confocal microscopy and immunoelectron microscopy. About 65,000 mol. wt myotonic dystrophy protein kinase was detected in aneurally cultured muscles. Myotonic dystrophy protein kinase messenger RNA was expressed in both aneurally and innervated cultured muscles, but in early innervated cultured muscles the message was transiently lower than in aneurally cultured muscles and innervated cultured muscles in long-term co-culture. In aneurally cultured muscles, immature aneurally cultured muscles show a diffuse and irregular distribution of myotonic dystrophy protein kinase in the deeper cytoplasm near the nuclei. Ultrastructurally the immuno-products against myotonic dystrophy protein kinase were observed as dense deposits in parts of the membranes near the mitochondria. In innervated cultured muscles, immunofluorescent microscopy showed myotonic dystrophy protein kinase to be localized regularly in the I bands and A-I junctions. Ultrastructurally myotonic dystrophy protein kinase was localized in branched duct-like membranes in the early stage of innervated cultured muscles and then in small sacs at the I bands and A-I junctions of the sarcolemma in the mature stage. Our present studies strongly suggest that innervation plays an important role in the localization of myotonic dystrophy protein kinase in human skeletal muscle during development. We conclude that the expression of myotonic dystrophy protein kinase during development is under neuronal influence.

Retinal horizontal cells (HCs) are inhibitory neurons, which modulate the transmission of light-elicited signals from photoreceptors to bipolar cells in the outer retina. HCs of the same physiological type are extensively coupled via gap junctions. In the zebrafish retina, the population of HCs comprises up to four morphologically distinct subtypes. Four different connexins (Cx52.6, Cx52.7, Cx52.9 and Cx55.5) were detected in these cells with overlapping expression patterns. In this study, we show that Cx52.6 is alternatively spliced in the retina, resulting in an additional isoform, designated as Cx53.4, which differs from the originally described Cx52.6 only by the final C-terminal peptide (12 vs. 4 aa). Further protein sequence alignments revealed that Cx53.4 represents the counterpart of alternatively spliced mouse Cx57 and human Cx62. RT-PCR analyses of mRNA expression in different adult zebrafish tissues showed that Cx53.4 is expressed exclusively in the retina. The localization of Cx53.4 protein within the retina was analyzed using a specific antibody. Immunofluorescence analyses demonstrated that the expression of Cx53.4 is restricted to HCs of all four subtypes. Further, immunoelectron microscopy confirmed the presence of Cx53.4 in gap junctions between HC dendrites and between their axon terminals.

Methionine enkephalin, an endogenous opioid peptide, participates in the regulation of growth in the developing brain. In the present study, enkephalin-like immunoreactivity was localized in the cerebellum of developing and adult rats by immunoelectron microscopy. In 10-day-old animals, enkephalin-like immunoreactivity was found in the somata of proliferating, migrating and differentiating neural cells, and was associated with the plasma membrane, microtubules, filaments, mitochondria, endoplasmic reticulum and nuclear envelope. Both neurons and glia in the cerebellum of the preweaning rat displayed a similar profile of immunoreactivity. Reaction product was also detected in the dendrites and dendritic spines of Purkinje cells where it was concentrated in postsynaptic densities. The majority of internal granule neurons in 10-day-old animals were not immunoreactive, nor were axons, glial processes and postsynaptic elements (with the exception of mossy fiber terminals). At weaning (Day 21), enkephalin-like immunoreactivity was confined primarily to the somata of Purkinje, basket and stellate neurons, and to Purkinje cell dendrites and synaptic spines. Adult rats (day 75) exhibited no enkephalin-like immunoreactivity. These results establish that enkephalin or an enkephalin-like substance can be detected during the ontogeny of both neurons and glia in the cerebellar cortex, and appears to be associated with certain structural elements.

Immunoelectron microscopy was used to investigate the ultrastructural features and synaptic relationships of serotonin-like immunoreactive amacrine cells in the larval tiger salamander retina. Serotonin-positive somas exhibited an evenly distributed peroxidase reaction product throughout their cytoplasm. Their nuclei were unstained and possessed indented nuclear membranes. Serotonin-immunoreactive processes were generally stained throughout with the exception of their mitochondria, whose morphology was often disrupted by the staining reaction. They were further characterized by an occasional dense-cored vesicle/s in addition to a generally homogeneous population of small, round, clear synaptic vesicles. Serotonin-immunoreactive amacrine cell processes formed conventional synapses that were characterized by symmetrical synaptic membrane densities. A total of 222 synaptic arrangements were observed that involved the immunostained processes of serotonin-amacrine cells. As presynaptic elements, they primarily contacted amacrine cells processes (37.8%). They also provided substantial synaptic input to processes that lacked synaptic vesicles (16.2%) and whose origin was unidentified. Serotonin-processes provided a far fewer number of synaptic contacts onto the processes of bipolar cells (1.4%) and the somas of cells in the amacrine cell layer (0.5%). As postsynaptic elements, they received synaptic inputs from amacrine cells (27.9%) and bipolar cells (16.2%). With the exception of their synapses onto bipolar cells and the somas of cells in the amacrine cell layer, each of the synaptic relationships of serotonin-amacrine cells was observed in each of sublayers 1-5 of the inner plexiform layer.

The electrogenic Na+/HCO3- cotransporter (NBCe1) has been identified as a key player for regulation of intracellular pH in several cell types. The present study was undertaken to determine expression and subcellular localization of the NH2-terminal solute carrier (SLC) 4A4 variants NBCe1-A and NBCe1-B in mouse brain using variant-specific antibodies by immunohistochemistry and immunoelectron microscopy. In addition, distribution of NBCe1 variants and activity-dependent regulation was investigated in mouse embryonic day 17.5 (E17.5) hippocampal primary cultures in vitro. The results showed NBCe1-A and NBCe1-B transcript expression in the mouse olfactory bulb, cerebral cortex, hippocampus and cerebellum. NBCe1-A was predominantly expressed in Purkinje cells, granule cells of the dentate gyrus, non-pyramidal cell bodies in cerebral cortex, and in periglomerular and mitral cells in the olfactory bulb. Pyramidal neurons in cerebral cortex and apical cell dendrites in the hippocampus were stained for both NBCe1-A and NBCe1-B. Moreover, NBCe1-B was present in Bergmann glia. At the ultrastructural level, NBCe1-B was preferentially expressed in perivascular astroglial lamellae, whereas both NBCe1 NH2-terminal variants were localized in pre- and postsynaptic compartments. Except for the olfactory bulb, NBCe1-A was always colocalized with calbindin. Treatment of E17.5 primary hippocampal cultures with KCl, showed dramatic downregulation of NBCe1-B mRNA and protein after 60 min, whereas NBCe1-A expression remained unchanged. These data demonstrate for the first time distinct cellular distribution of NBCe1 NH2-terminal variants in mouse brain. NBCe1 may be involved in neuronal modulation, and pH regulation during neuronal activity.

In the vertebrate retina, horizontal cells (HCs) reveal homologous coupling by gap junctions (gj), which are thought to consist of different connexins (Cx). However, recent studies in mouse, rabbit and zebrafish retina indicate that individual HCs express more than one connexin. To provide further insights into the composition of gj connecting HCs and to determine whether HCs express multiple connexins, we examined the molecular identity and distribution of gj between HCs of the carp retina. We have cloned four carp connexins designated Cx49.5, Cx55.5, Cx52.6 and Cx53.8 with a close relationship to connexins previously reported in HCs of mouse, rabbit and zebrafish, respectively. Using in situ hybridization, Cx49.5 expression was detected in different subpopulations of retinal neurons including HCs, whereas the Cx52.6 transcript was localized exclusively in HCs. Using specific antibodies, Cx55.5 and Cx53.8 were detected on dendrites of all four HC subtypes and axon terminals. Immunoelectron microscopy confirmed the presence of Cx55.5 and Cx53.8 in gap junctions between these processes and Cx55.5 was additionally observed in HC dendrites invaginating cone pedicles, suggesting its participation in the modulation of photoreceptor output in the carp retina. Furthermore, using single-cell RT-PCR, all four connexins were detected in different subtypes of HCs, suggesting overlapping expression patterns. Thus, the composition of gj mediating homologous coupling between subtypes of carp HCs appears to be more complex than expected. Moreover, BLAST searches of the preliminary carp genome, using novel sequences as query, suggest that most of the analyzed connexin genes are duplicated in carp.

The basolateral amygdala receives a very dense cholinergic innervation from the basal forebrain that is important for memory consolidation. Although behavioral studies have shown that both M1 and M2 muscarinic receptors are critical for these mnemonic functions, there have been very few neuroanatomical and electrophysiological investigations of the localization and function of different types of muscarinic receptors in the amygdala. In the present study we investigated the subcellular localization of M2 muscarinic receptors (M2Rs) in the anterior basolateral nucleus (BLa) of the mouse, including the localization of M2Rs in parvalbumin (PV) immunoreactive interneurons, using double-labeling immunoelectron microscopy. Little if any M2R-immunoreactivity (M2R-ir) was observed in neuronal somata, but the neuropil was densely labeled. Ultrastructural analysis using a pre-embedding immunogold-silver technique (IGS) demonstrated M2R-ir in dendritic shafts, spines, and axon terminals forming asymmetrical (excitatory) or symmetrical (mostly inhibitory) synapses. In addition, about one-quarter of PV+ axon terminals and half of PV+ dendrites, localized using immunoperoxidase, were M2R+ when observed in single thin sections. In all M2R+ neuropilar structures, including those that were PV+, about one-quarter to two-thirds of M2R+ immunoparticles were plasma-membrane-associated, depending on the structure. The expression of M2Rs in PV+ and PV-negative terminals forming symmetrical synapses indicates M2R modulation of inhibitory transmission. Electrophysiological studies in mouse and rat brain slices, including paired recordings from interneurons and pyramidal projection neurons, demonstrated M2R-mediated suppression of GABA release. These findings suggest cell-type-specific functions of M2Rs and shed light on organizing principles of cholinergic modulation in the BLa.

Although the importance of neuropeptide Y (NPY) in the regulation of gonadotropin releasing hormone (GnRH) and reproduction has been highlighted in recent years, the neuroanatomical substrate within which these substances might interact has not been fully elucidated. Present work was undertaken with a view to define the anatomical-physiological correlates underlying the role exercised by NPY in the regulation of GnRH in the forebrain of the teleost Clarias batrachus. Application of double immunocytochemistry revealed close associations as well as colocalizations of the two peptides in the olfactory receptor neurons (ORNs), olfactory nerve fibers and their terminals in the glomeruli, ganglion cells of nervus terminalis, medial olfactory tract, fibers in the area ventralis telencephali/pars supracommissuralis and cells as well as fibers in the pituitary. NPY containing axons were found to terminate in the vicinity of GnRH cells in the pituitary with light as well as electron microscopy. Double immunoelectron microscopy demonstrated gold particles for NPY and GnRH colocalized on the membrane and in dense core of the secretory granules in the cells distributed in all components of the pituitary gland. To assess the physiological implication of these observations, NPY was injected via the intracranial route and the response of GnRH immunoreactive system was evaluated by relative quantitative morphometry as well as high performance liquid chromatography (HPLC) analysis. Two hours following NPY (20 ng/g body weight) administration, a dramatic increase was observed in the GnRH immunoreactivity in the ORNs, in the fibers of the olfactory bulb (163%) and medial olfactory tract (351%). High performance liquid chromatography-electrospray ionization-mass spectrometric analysis confirmed the immunocytochemical data. Significant rise in the salmon GnRH (sGnRH)-like peptide content was observed in the olfactory organ (194.23%), olfactory bulb (146.64%), telencephalon+preoptic area (214.10%) and the pituitary (136.72%) of the NPY-treated fish. However, GnRH in the hypothalamus was below detection limit in the control as well as NPY-treated fish. Present results suggest the involvement of NPY in the up-regulation of sGnRH containing system at different level of neuraxis extending from the olfactory epithelium to the pituitary in the forebrain of C. batrachus.

The ultrastructural features and synaptic interactions of tyrosine hydroxylase-like-immuno-reactive amacrine cells in the larval tiger salamander retina were examined using routine immunoelectron microscopy. The somas of tyrosine hydroxylase-like-immunoreactive amacrine cells were immunostained evenly throughout their cytoplasm. Their nuclei were generally unstained and possessed indented nuclear membranes. The processes of tyrosine hydroxylase-like-immunoreactive amacrine cells were homogeneously stained with the exception of their mitochondria, whose morphology was often disrupted by the staining procedure. Tyrosine hydroxylase-like-immunoreactive amacrine cell processes were characterized by an occasional dense-cored vesicle(s), in addition to a generally homogeneous population of small, round, agranular synaptic vesicles. They formed conventional synaptic junctions that were characterized by symmetrical synaptic membrane densities. A total of 168 synapses were observed that involved tyrosine hydroxylase-like-immunoreactive amacrine cell processes. A large percentage (79.8%) of these synaptic arrangements were found in sublayer 1 of the inner plexiform layer, while substantially lower percentages were observed in sublayers 3 (9.5%) and 5 (10.7%). They served as pre- and postsynaptic elements 63.1 and 36.9% of the time, respectively. Tyrosine hydroxylase-like-immunoreactive amacrine cell processes were presynaptic to amacrine cell processes (36.9% of total synaptic involvement) and processes that lack synaptic vesicles and whose origin remains uncertain (26.2%). They received synaptic input primarily from amacrine cell processes (31.0%). Tyrosine hydroxylase-like-immunoreactive amacrine cell processes also received a few ribbon synapses from bipolar cells (5.9%). Each of these synaptic relationships were observed in each of sublayers 1, 3 and 5 of the inner plexiform layer, with the majority of each arrangement being found in sublayer 1.

Synuclein was initially named for its localization in both presynaptic nerve terminals and portions of nuclear envelope. However, subsequent studies only confirmed the presynaptic localization of this protein in the brain; its nuclear localization in the neurons remained elusive. Here, two new monoclonal antibodies against alpha-synuclein (alpha-SYN) were produced. Epitope mapping using phage peptide display showed that the epitopes of the two antibodies were localized in two distinct specific sequences of the C-terminal domain of alpha-SYN. One antibody named 3D5 recognized amino acids 115-121 of alpha-SYN and the other antibody named 2E3 identified the amino acids 134-138 of the protein. Western blot analysis demonstrated that both 2E3 and 3D5 detected a 19 kD protein from rat and human brain homogenates, which was identical to the molecular size of recombinant alpha-SYN. However, immunohistochemical staining on normal adult rat brain sections showed that the two antibodies revealed distinct patterns of subcellular localization of alpha-SYN immunoreactivity. Both 3D5 and 2E3 detected the presynaptic alpha-SYN but only 3D5 detected the nuclear alpha-SYN. The nuclear localization of alpha-SYN was further confirmed by Western blot analysis in isolated nuclear fraction where the same size of alpha-SYN was detected, and by immunoelectron microscopy using colloidal gold probes where gold particles were specifically localized in portions of peri- and intra-nucleus. The nuclear positive neurons were distributed extensively in almost all the brain regions. This is the first report well characterizing the extensive localization of alpha-SYN in the neuronal nuclei throughout the brain in normal conditions. This finding indicates an important physiological function of this molecule in the nuclei of brain neurons, which deserves further investigations.