Vesicular Glutamate Transporter Type Research Articles

C1 neurons excite locus coeruleus and A5 noradrenergic neurons along with sympathetic outflow in rats

C1 neurons activate sympathetic tone and stimulate the hypothalamic–pituitary–adrenal axis in circumstances such as pain, hypoxia or hypotension. They also innervate pontine noradrenergic cell groups, including the locus coeruleus (LC) and A5. Activation of C1 neurons reportedly inhibits LC neurons; however, because these neurons are glutamatergic and have excitatory effects elsewhere, we re-examined the effect of C1 activation on pontine noradrenergic neurons (LC and A5) using a more selective method. Using a lentivirus that expresses channelrhodopsin2 (ChR2) under the control of the artificial promoter PRSx8, we restricted ChR2 expression to C1 neurons (67%), retrotrapezoid nucleus neurons (20%) and cholinergic neurons (13%). The LC contained ChR2-positive terminals that formed asymmetric synapses and were immunoreactive for vesicular glutamate transporter type 2. Low-frequency photostimulation of ChR2-expressing neurons activated LC (38 of 65; 58%) and A5 neurons (11 of 16; 69%) and sympathetic nerve discharge. Locus coeruleus and A5 inhibition was not seen unless preceded by excitation. Locus coeruleus activation was eliminated by intracerebroventricular kynurenic acid. Stimulation of ChR2-expressing neurons at 20 Hz produced modest increases in LC and A5 neuronal discharge. In additional rats, the retrotrapezoid nucleus region was destroyed with substance P–saporin prior to lentivirus injection into the rostral ventrolateral medulla, increasing the proportion of C1 ChR2-expressing neurons (83%). Photostimulation in these rats activated the same proportion of LC and A5 neurons as in control rats but produced no effect on sympathetic nerve discharge owing to the destruction of bulbospinal C1 neurons. In conclusion, low-frequency stimulation of C1 neurons activates pontine noradrenergic neurons and sympathetic nerve discharge, possibly via the release of glutamate from monosynaptic C1 inputs.

Expression of vesicular glutamate transporters type 1 and 2 in sensory and autonomic neurons innervating the mouse colorectum

Vesicular glutamate transporters (VGLUTs) have been extensively studied in various neuronal systems, but their expression in visceral sensory and autonomic neurons remains to be analyzed in detail. Here we studied VGLUTs type 1 and 2 (VGLUT(1) and VGLUT(2) , respectively) in neurons innervating the mouse colorectum. Lumbosacral and thoracolumbar dorsal root ganglion (DRG), lumbar sympathetic chain (LSC), and major pelvic ganglion (MPG) neurons innervating the colorectum of BALB/C mice were retrogradely traced with Fast Blue, dissected, and processed for immunohistochemistry. Tissue from additional naïve mice was included. Previously characterized antibodies against VGLUT(1) , VGLUT(2) , and calcitonin gene-related peptide (CGRP) were used. Riboprobe in situ hybridization, using probes against VGLUT(1) and VGLUT(2) , was also performed. Most colorectal DRG neurons expressed VGLUT(2) and often colocalized with CGRP. A smaller percentage of neurons expressed VGLUT(1) . VGLUT(2) -immunoreactive (IR) neurons in the MPG were rare. Abundant VGLUT(2) -IR nerves were detected in all layers of the colorectum; VGLUT(1) -IR nerves were sparse. A subpopulation of myenteric plexus neurons expressed VGLUT2 protein and mRNA, but VGLUT1 mRNA was undetectable. In conclusion, we show 1) that most colorectal DRG neurons express VGLUT(2) , and to a lesser extent, VGLUT(1) ; 2) abundance of VGLUT2-IR fibers innervating colorectum; and 3) a subpopulation of myenteric plexus neurons expressing VGLUT(2). Altogether, our data suggests a role for VGLUT(2) in colorectal glutamatergic neurotransmission, potentially influencing colorectal sensitivity and motility.

Abnormalities in the Climbing Fiber-Purkinje Cell Circuitry Contribute to Neuronal Dysfunction inATXN1[82Q] Mice

One fundamental unanswered question in the field of polyglutamine diseases concerns the pathophysiology of neuronal dysfunction. Is there dysfunction in a specific neuronal population or circuit initially that contributes the onset of behavioral abnormalities? This study used a systems-level approach to investigate the functional integrity of the excitatory cerebellar cortical circuitry in vivo from several transgenic ATXN1 mouse lines. We tested the hypotheses that there are functional climbing fiber (CF)-Purkinje cell (PC) and parallel fiber (PF)-PC circuit abnormalities using flavoprotein autofluorescence optical imaging and extracellular field potential recordings. In early-symptomatic and symptomatic animals expressing ATXN1[82Q], there is a marked reduction in PC responsiveness to CF activation. Immunostaining of vesicular glutamate transporter type 2 demonstrated a decrement in CF extension on PC dendrites in symptomatic ATXN1[82Q] mice. In contrast, responses to PF stimulation were relatively normal. Importantly, the deficits in CF-PC synaptic transmission required expression of pathogenic ataxin-1 (ATXN1[82Q]) and for its entrance into the nucleus of PCs. Loss of endogenous mouse Atxn1 had no discernible effects. Furthermore, the abnormalities in CF-PC synaptic transmission were ameliorated when mutant transgene expression was prevented during postnatal cerebellar development. The results demonstrate the preferential susceptibility of the CF-PC circuit to the effects of ATXN1[82Q]. Further, this deficit likely contributes to the abnormal motor phenotype of ATXN1[82Q] mice. For polyglutamine diseases generally, the findings support a model whereby specific neuronal circuits suffer insults that alter function before cell death.

The morphological characteristics of corticostriatal and thalamostriatal neurons and their intrastriatal terminals in rats

The glutamatergic projection from the cerebral cortex and the thalamus extensively innervates the neostriatal neurons. However, some conflicts in the published literatures about cortical and thalamic intrastriatal synaptic terminals still need to be resolved. The present study intends to further elucidate the morphological characteristics of these two types of the terminals and their neurons. The corticostriatal and thalamostriatal terminals were immunolabeled for vesicular glutamate transporter type 1 (VGluT1) and 2 (VGluT2), respectively, and their neurons were retrograde labeled by biotinylated dextran amine 3,000 molecular weight (BDA3k) injection into the dorsolateral striatum of rats. The characteristics of the corticostriatal and thalamostriatal terminals were observed at the LM and EM levels, and the data were statistically analyzed with SPSS10.0 software. We observed that 63.53% of VGluT1+ terminals synapsed on dendritic spines, which was different from VGluT2+ terminals with the equal percentage of synapses on spines and dendrites (14.88 and 17.86%, respectively). Notably, VGluT1+ axospinous synaptic terminals were remarkably larger than VGluT2+ axospinous synaptic terminals. Terminal size-frequency distribution analysis showed that VGluT1+ terminals were within the size ranges of 0.4-0.5 and 0.8-0.9 μm, and VGluT2+ terminals were in the ranges of 0.4-0.5 and 0.6-0.7 μm. Perforated-postsynaptic densities (-PSDs) were more frequently found in VGluT1+ axospinous synaptic terminals than in VGluT2+ axospinous terminals. Furthermore, BDA3k-labeled corticostrital neurons were larger in perikaryal diameter than the thalamostriatal neurons, and they were also categorized as the two main populations based on their size-frequency distribution. The morphological characteristics of corticostriatal and thalamostriatal terminals and neurons have implications for understanding the roles of synaptic plasticity in adaptive motor control by the basal ganglia, and they have facilitations for understanding the complexities of basal ganglia function.

Glutamatergic and cholinergic pedunculopontine neurons innervate the thalamic parafascicular nucleus in rats: changes following experimental parkinsonism

The tegmental pedunculopontine nucleus (PPN) is a basal ganglia-related structure that has recently gained renewed interest as a potential surgical target for the treatment of several aspects of Parkinson's disease. However, the underlying anatomical substrates sustaining the choice of the PPN nucleus as a surgical candidate remain poorly understood. Here, we characterized the chemical phenotypes of different subtypes of PPN efferent neurons innervating the rat parafascicular (PF) nucleus. Emphasis was placed on elucidating the impact of unilateral nigrostriatal denervation on the expression patterns of the mRNA coding the vesicular glutamate transporter type 2 (vGlut2 mRNA). We found a bilateral projection from the PPN nucleus to the PF nucleus arising from cholinergic and glutamatergic efferent neurons, with a small fraction of projection neurons co-expressing both cholinergic and glutamatergic markers. Furthermore, the unilateral nigrostriatal depletion induced a bilateral twofold increase in the expression levels of vGlut2 mRNA within the PPN nucleus. Our results support the view that heterogeneous chemical profiles account for PPN efferent neurons innervating thalamic targets. Moreover, a bilateral enhancement of glutamatergic transmission arising from the PPN nucleus occurs following unilateral dopaminergic denervation, therefore sustaining the well-known hyperactivity of the PF nucleus in parkinsonian-like conditions. In conclusion, our data suggest that the ascending projections from the PPN that reach basal ganglia-related targets could play an important role in the pathophysiology of Parkinson's disease.

Cortical and Thalamic Innervation of Direct and Indirect Pathway Medium-Sized Spiny Neurons in Mouse Striatum

The striatum receives major excitatory inputs from the cortex and thalamus that predominantly target the spines of medium-sized spiny neurons (MSNs). We aimed to determine whether there is any selectivity of these two excitatory afferents in their innervation of direct and indirect pathway MSNs. To address this, we used bacterial artificial chromosome transgenic mice, in which enhanced green fluorescent protein (EGFP) reports the presence of D(1) or D(2) dopamine receptor subtypes, markers of direct and indirect pathway MSNs, respectively. Excitatory afferents were identified by the selective expression of vesicular glutamate transporter type 1 (VGluT1) by corticostriatal afferents and vesicular glutamate transporter type 2 (VGluT2) by thalamostriatal afferents. A quantitative electron microscopic analysis was performed on striatal tissue from D(1) and D(2) mice that was double immunolabeled to reveal the EGFP and VGluT1 or VGluT2. We found that the proportion of synapses formed by terminals derived from the cortex and thalamus was similar for both direct and indirect pathway MSNs. Furthermore, qualitative analysis revealed that individual cortical or thalamic terminals form synapses with both direct and indirect pathway MSNs. Similarly, we observed a convergence of cortical and thalamic inputs onto individual MSNs of both direct and indirect pathway: individual EGFP-positive structures received input from both VGluT2-positive and VGluT2-negative terminals. These findings demonstrate that direct and indirect pathway MSNs are similarly innervated by cortical and thalamic afferents; both projections are thus likely to be critical in the control of MSNs and hence play fundamental roles in the expression of basal ganglia function.

VGLUT2-Dependent Glutamate Release from Nociceptors Is Required to Sense Pain and Suppress Itch

Itch can be suppressed by painful stimuli, but the underlying neural basis is unknown. We generated conditional null mice in which vesicular glutamate transporter type 2 (VGLUT2)-dependent synaptic glutamate release from mainly Nav1.8-expressing nociceptors was abolished. These mice showed deficits in pain behaviors, including mechanical pain, heat pain, capsaicin-evoked pain, inflammatory pain, and neuropathic pain. The pain deficits were accompanied by greatly enhanced itching, as suggested by (1) sensitization of both histamine-dependent and histamine-independent itch pathways and (2) development of spontaneous scratching and skin lesions. Strikingly, intradermal capsaicin injection promotes itch responses in these mutant mice,as opposed to pain responses in control littermates. Consequently, coinjection of capsaicin was no longer able to mask itch evoked by pruritogenic compounds. Our studies suggest that synaptic glutamate release from a group of peripheral nociceptors is required to sense pain and suppress itch. Elimination of VGLUT2 in these nociceptors creates a mouse model of chronic neurogenic itch.

Neuregulin‐1 at synapses on phrenic motoneurons

The neuregulin (NRG) family of trophic factors is present in the central and peripheral nervous systems and participates in the survival, proliferation, and differentiation of many different cell types, including motoneurons. NRG1 was first characterized by its role in the formation of the neuromuscular junction, and recently it was shown to play a crucial role in modulating glutamatergic and cholinergic transmission in the central nervous system of adult rats. However, little is known about NRG1's role in adult motor systems. Motoneurons receive dense glutamatergic and cholinergic input. We hypothesized that NRG1 is present at synapses on phrenic motoneurons. Confocal microscopy and 3D reconstruction techniques were used to determine the distribution of NRG1 and its colocalization with these different neurotransmitter systems. We found that NRG1 puncta are present around retrogradely labeled motoneurons and are distributed predominantly at motoneuron somata and primary dendrites. NRG1 is present exclusively at synaptic sites (identified using the presynaptic marker synaptophysin), making up ∼30% of all synapses at phrenic motoneurons. Overall, NRG1 immunoreactivity is found predominantly at cholinergic synapses (75% ± 14% colocalize with the vesicular acetylcholine transporter; VAChT). Nearly all (99% ± 1%) VAChT-immunoreactive synapses expressed NRG1. NRG1 also is present at a subset of glutamatergic synapses expressing the vesicular glutamate transporter (VGLUT) type 2 (∼6%) but not those expressing VGLUT type 1. Overall, 26% ± 6% of NRG1 synapses are VGLUT2 immunoreactive. These findings provide the first evidence suggesting that NRG1 may modulate synaptic activity in adult motor systems.

VGLUT3 (Vesicular Glutamate Transporter Type 3) Contribution to the Regulation of Serotonergic Transmission and Anxiety

Three different subtypes of H(+)-dependent carriers (named VGLUT1-3) concentrate glutamate into synaptic vesicles before its exocytotic release. Neurons using other neurotransmitter than glutamate (such as cholinergic striatal interneurons and 5-HT neurons) express VGLUT3. It was recently reported that VGLUT3 increases acetylcholine vesicular filling, thereby, stimulating cholinergic transmission. This new regulatory mechanism is herein designated as vesicular-filling synergy (or vesicular synergy). In the present report, we found that deletion of VGLUT3 increased several anxiety-related behaviors in adult and in newborn mice as early as 8 d after birth. This precocious involvement of a vesicular glutamate transporter in anxiety led us to examine the underlying functional implications of VGLUT3 in 5-HT neurons. On one hand, VGLUT3 deletion caused a significant decrease of 5-HT(1A)-mediated neurotransmission in raphe nuclei. On the other hand, VGLUT3 positively modulated 5-HT transmission of a specific subset of 5-HT terminals from the hippocampus and the cerebral cortex. VGLUT3- and VMAT2-positive serotonergic fibers show little or no 5-HT reuptake transporter. These results unravel the existence of a novel subset of 5-HT terminals in limbic areas that might play a crucial role in anxiety-like behaviors. In summary, VGLUT3 accelerates 5-HT transmission at the level of specific 5-HT terminals and can exert an inhibitory control at the raphe level. Furthermore, our results suggest that the loss of VGLUT3 expression leads to anxiety-associated behaviors and should be considered as a potential new target for the treatment of this disorder.

Caffeine and an adenosine A2A receptor antagonist prevent memory impairment and synaptotoxicity in adult rats triggered by a convulsive episode in early life

Seizures early in life cause long-term behavioral modifications, namely long-term memory deficits in experimental animals. Since caffeine and adenosine A(2A) receptor (A(2A)R) antagonists prevent memory deficits in adult animals, we now investigated if they also prevented the long-term memory deficits caused by a convulsive period early in life. Administration of kainate (KA, 2 mg/kg) to 7-days-old (P7) rats caused a single period of self-extinguishable convulsions which lead to a poorer memory performance in the Y-maze only when rats were older than 90 days, without modification of locomotion or anxiety-like behavior in the elevated-plus maze. In accordance with the relationship between synaptotoxicity and memory dysfunction, the hippocampus of these adult rats treated with kainate at P7 displayed a lower density of synaptic proteins such as SNAP-25 and syntaxin (but not synaptophysin), as well as vesicular glutamate transporters type 1 (but not vesicular GABA transporters), with no changes in PSD-95, NMDA receptor subunits (NR1, NR2A, NR2B) or alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate receptor subunits (GluR1, GluR2) compared with controls. Caffeine (1 g/L) or the A(2A)R antagonist, KW6002 (3 mg/kg) applied in the drinking water from P21 onwards, prevented these memory deficits in P90 rats treated with KA at P7, as well as the accompanying synaptotoxicity. These results show that a single convulsive episode in early life causes a delayed memory deficit in adulthood accompanied by a glutamatergic synaptotoxicity that was prevented by caffeine or adenosine A(2A)R antagonists.

Lateral habenula projections to dopamine and GABA neurons in the rat ventral tegmental area

Ventral tegmental area (VTA) dopamine (DA) neurons and their forebrain projections are critically involved in reward processing and cognitive functions. Descending projections from the lateral habenula (LHb) play a central role in inhibiting DA cell activity in response to the absence of expected rewards. As LHb efferents are reportedly glutamatergic, their ability to inhibit DA cells would theoretically require a disynaptic connection involving VTA GABA neurons and their local collateral inputs to DA cells. We therefore used anterograde tract-tracing from the LHb to investigate the relative selectivity of LHb synapses onto GABA versus DA VTA neurons. LHb axons were visualized using immunoperoxidase, and DA and GABA cells were marked by immunogold-silver labeling for tyrosine hydroxylase (TH) or GABA, respectively. By ultrastructural analysis, 16% of LHb axons were observed to form synaptic contacts in the VTA, and most of these were of an intermediate morphological type that did not exhibit definitive asymmetric or symmetric character. LHb axons synaptically targeted TH- and GABA-labeled dendrites to a comparable extent (45 and 52% observed incidence, respectively). Pre-embedding immunogold labeling for the vesicular glutamate transporter type 2 and post-embedding immunogold staining for GABA confirmed that approximately 85% of LHb terminals were glutamatergic and not GABAergic. These results suggest that the robust inhibition of DA cells evoked by the LHb is unlikely to arise from a selective innervation of VTA GABA neurons. Moreover, the LHb may mediate a direct excitation of DA cells that is over-ridden by indirect inhibition originating from an extrinsic source.

Synaptic and Extrasynaptic Factors Governing Glutamatergic Retinal Waves

In the few days prior to eye-opening in mice, the excitatory drive underlying waves switches from cholinergic to glutamatergic. Here, we describe the unique synaptic and spatiotemporal properties of waves generated by the retina's glutamatergic circuits. First, knockout mice lacking vesicular glutamate transporter type 1 do not have glutamatergic waves, but continue to exhibit cholinergic waves, demonstrating that the two wave-generating circuits are linked. Second, simultaneous outside-out patch and whole-cell recordings reveal that retinal waves are accompanied by transient increases in extrasynaptic glutamate, directly demonstrating the existence of glutamate spillover during waves. Third, the initiation rate and propagation speed of retinal waves, as assayed by calcium imaging, are sensitive to pharmacological manipulations of spillover and inhibition, demonstrating a role for both signaling pathways in shaping the spatiotemporal properties of glutamatergic retinal waves.

Synaptic contacts of vesicular glutamate transporter 2 fibres on chemically identified neurons of the hypothalamic suprachiasmatic nucleus of the rat

The hypothalamic suprachiasmatic nucleus (SCN), which plays a pivotal role in the control of circadian rhythms, consists of several neuronal subpopulations characterized by different neuroactive substances. This prominent cell group has a fairly rich glutamatergic innervation, but the cell types that are targeted by this innervation are unknown. Therefore, the purpose of the present study was to examine the relationship between the afferent glutamatergic axon terminals and the vasoactive intestinal polypeptide (VIP)-, arginine-vasopressin (AVP)- and gamma-aminobutyric acid (GABA)-positive neurons of the SCN. Glutamatergic elements were revealed via immunocytochemical double-labelling for vesicular glutamate transporter type 1 (VGluT1) and type 2 (VGluT2), and brain sections were imaged via confocal laser-scanning microscopy and electron microscopy. Numerous VGluT2-immunoreactive axons were observed to be in synaptic contact with VIP- and GABA-positive neurons, and only a few synapses were detected between VGluT2 boutons and AVP neurons. VGluT1 axon terminals exhibiting very moderate distribution in this cell group were observed to be in synaptic contact with chemically unidentified neurons. The findings provide the first morphological data on the termination of presumed glutamatergic fibres on chemically identified neurons of the rat SCN, and indicate that all three prominent cell types of the cell group receive glutamatergic afferents.

Distinct immunohistochemically defined areas in the medial amygdala in the developing and adult mouse

The medial amygdala has been considered a subpallial structure, but various studies have shown that is a somewhat complex structure expressing both pallial and subpallial gene markers. In this regard, we analyzed the immunohistochemical expression of the neurotransmitter GABA, the vesicular glutamate transporter type 2 (VGLUT2), the neuronal nitric oxide synthase (nNOS), and the calcium-binding proteins calbindin-D28k (CB) and calretinin (CR) in the developing and adult mouse medial amygdala. From intermediate embryonic stages on, neurochemical data show a distinctive superficial region forming a band all along the medial amygdalar surface. This superficial band displays a strong VGLUT2-immunoreactive neuropil and numerous CR-immunoreactive fibers, as well as some nNOS-, CR- or CB-positive cells. In contrast, the superficial region of the posterior medial amygdala appears to be non-GABA immunoreactive. This band in the posterior medial amygdala matches a Tbr1-expressing territory. Our results also show differences between dorsal and ventral parts of the postnatal and adult posterior medial amygdala. Especially, a compact cell aggregate of nNOS immunoreactive cells was found in the ventral portion of the medial amygdala, whereas the dorsal part is occupied by scattered weakly stained cells. Comparison of our results with gene expression patterns and fiber-tracing studies, let us to propose that the superficial band is a pallial derivative, whereas the dorsal and ventral nuclei of the posterior medial amygdala receive each neurons from different subpallial compartments, and the latter one a subset of excitatory, pallial projection neurons.

Vesicular glutamate transporter 2 protein and mRNA containing neurons in the hypothalamic suprachiasmatic nucleus of the rat

The hypothalamic suprachiasmatic nucleus is the key structure of the control of circadian rhythms and has a rich glutamatergic innervation. Besides the presence of glutamatergic afferents, several findings also suggest the existence of glutamatergic efferents from the suprachiasmatic nucleus to its target neurons in various prominent hypothalamic cell groups. However, there is no direct neuromorphological evidence for the presence of glutamatergic neurons in the suprachiasmatic nucleus. Therefore, the purpose of the present investigations was to try to clarify this question. Immunocytochemistry was used at the light and electron microscopy level to identify vesicular glutamate transporter type 2 (VGluT2) immunopositive (presumed glutamatergic) neurons in the rat suprachiasmatic nucleus. In addition VGluT2 mRNA expression in neurons of the nucleus was also addressed with radioisotopic in situ hybridization. Both at the light and electron microscopy level we detected VGluT2 positive neurons, which did not contain GABA, vasoactive intestinal polypeptide or vasopressin. Further, we demonstrated the expression of VGluT2 mRNA in a few cells within the suprachiasmatic nucleus; these glutamatergic cells were distinct from somatostatin mRNA expressing neurons. As VGluT2 is a selective marker of glutamatergic neuronal elements, the present observations provide direct neuromorphological evidence for the presence of glutamatergic neurons in the cell group.

Impaired cannabinoid receptor type 1 signaling interferes with stress-coping behavior in mice

Dysregulation of the endocannabinoid system is known to interfere with emotional processing of stressful events. Here, we studied the role of cannabinoid receptor type 1 (CB1) signaling in stress-coping behaviors using the forced swim test (FST) with repeated exposures. We compared effects of genetic inactivation with pharmacological blockade of CB1 receptors both in male and female mice. In addition, we investigated potential interactions of the endocannabinoid system with monoaminergic and neurotrophin systems of the brain. Naive CB1 receptor-deficient mice (CB1-/-) showed increased passive stress-coping behaviors as compared to wild-type littermates (CB1+/+) in the FST, independent of sex. These findings were partially reproduced in C57BL/6N animals and fully reproduced in female CB1+/+ mice by pharmacological blockade of CB1 receptors with the CB1 receptor antagonist SR141716. The specificity of SR141716 was confirmed in female CB1-/- mice, where it failed to affect behavioral performance. Sensitivity to the antidepressants desipramine and paroxetine was preserved, but slightly altered in female CB1-/- mice. There were no genotype differences between CB1+/+ and CB1-/- mice in monoamine oxidase A and B activities under basal conditions, nor in monoamine content of hippocampal tissue after FST exposure. mRNA expression of vesicular glutamate transporter type 1 was unaffected in CB1-/- mice, but mRNA expression of brain-derived neurotrophic factor (BDNF) was reduced in the hippocampus. Our results suggest that impaired CB1 receptor function promotes passive stress-coping behavior, which, at least in part, might relate to alterations in BDNF function.

Abnormal mGluR2/3 expression in the perforant path termination zones and mossy fibers of chronically epileptic rats

Epilepsy is characterized by hyperexcitability of hippocampal networks, excessive release of glutamate, and progressive neurodegeneration. Presynaptic group II metabotropic receptors (mGluR2 and mGluR3) are among different mechanisms that modulate presynaptic release of glutamate, especially at the mossy fibers in the hippocampus. Here, we explore whether mGluR2/3 expression is affected in a rat model of temporal lobe epilepsy obtained via pilocarpine-induced status epilepticus (SE). Immunohistochemical assays were performed in age-matched controls and two groups of epileptic rats sacrificed at 25–35 days (1 month post-SE) and at 55–65 days (2 months post-SE) following SE onset. A dramatic lessening of mGluR2/3 immunofluorescence was observed at CA1 and CA3 stratum lacunosum/molecular (SLM) declining to 60% and 68% of control values in 1-month and 2-month post-SE, respectively. Additionally, thickness of mGluR2/3-stained SLM layer narrowed up to 70% of controls indicating atrophy at this branch of the perforant path. Epileptic rats exhibited a marked and progressive down-regulation of mGluR2/3 expression in mossy fiber at hilus and CA3 stratum lucidum in contrast with an enhanced expression of vesicular glutamate transporter type 1 (VGluT1) at the mossy fibers. Intense VGluT1 punctated staining was detected at the inner third molecular layer indicating glutamatergic sprouting. In the molecular layer, mGluR2/3 labeling slightly declined in the 1-month post-SE group but then increased in the 2-month post-SE group although it was diffusely distributed. Down-regulation of mGluR2/3 at the mossy fibers and the SLM may render epileptic hippocampal networks hyperexcitable and susceptible to glutamate-mediated excitotoxicity and neurodegeneration.

Alternative Splicing Controls Neuronal Expression of v-ATPase Subunit a1 and Sorting to Nerve Terminals

Vacuolar proton ATPase accumulates protons inside various intracellular organelles such as synaptic vesicles; its membrane domain V0 could also be involved in membrane fusion. These different functions could require vacuolar proton ATPases possessing different V0 subunit a isoforms. In vertebrates, four genes encode isoforms a1-a4, and a1 variants are also generated by alternative splicing. We identified a novel a1 splice variant a1-IV and showed that the two a1 variants containing exon C are specifically expressed in neurons. Single neurons coexpress a2, a1-I, and a1-IV, and these subunit a isoforms are targeted to different membrane compartments. Recombinant a2 was accumulated in the trans-Golgi network, and a1-I was concentrated in axonal varicosities, whereas a1-IV was sorted to both distal dendrites and axons. Our results indicate that alternative splicing of exon N controls differential sorting of a1 variants to nerve terminals or distal dendrites, whereas exon C regulates their neuronal expression.

Interaction between the vesicular glutamate transporter type 1 and endophilin A1, a protein essential for endocytosis

In the nerve terminal, neurotransmitter is actively packaged into synaptic vesicles before its release by Ca2+-dependent exocytosis. The three vesicular glutamate transporters (VGLUT1, -2 and -3) are highly conserved proteins that display similar bioenergetic and pharmacological properties but are expressed in different brain areas. We used the divergent C-terminus of VGLUT1 as a bait in a yeast two-hybrid screen to identify and map the interaction between a proline-rich domain of VGLUT1 and the Src homology domain 3 (SH3) domain of endophilin. We further confirmed this interaction by using different glutathione-S-transferase-endophilin fusion proteins to pull down VGLUT1 from rat brain extracts. The expression profiles of the two genes and proteins were compared on rat brain sections, showing that endophilin is most highly expressed in regions and cells expressing VGLUT1. Double immunofluorescence in the rat cerebellum shows that most VGLUT1-positive terminals co-express endophilin, whereas VGLUT2-expressing terminals are often devoid of endophilin. However, neither VGLUT1 transport activity, endophilin enzymatic activity nor VGLUT1 synaptic targeting were altered by this interaction. Overall, the discovery of endophilin as a partner for VGLUT1 in nerve terminals strongly suggests the existence of functional differences between VGLUT1 and -2 terminals in their abilities to replenish vesicle pools.

Glia re‐sealed particles freshly prepared from adult rat brain are competent for exocytotic release of glutamate

Glial subcellular re-sealed particles (referred to as gliosomes here) were purified from rat cerebral cortex and investigated for their ability to release glutamate. Confocal microscopy showed that the glia-specific proteins glial fibrillary acidic protein (GFAP) and S-100, but not the neuronal proteins 95-kDa postsynaptic density protein (PSD-95), microtubule-associated protein 2 (MAP-2) and beta-tubulin III, were enriched in purified gliosomes. Furthermore, gliosomes exhibited labelling neither for integrin-alphaM nor for myelin basic protein, which are specific for microglia and oligodendrocytes respectively. The Ca2+ ionophore ionomycin (0.1-5 microm) efficiently stimulated the release of tritium from gliosomes pre-labelled with [3H]d-aspartate and of endogenous glutamate in a Ca(2+)-dependent and bafilomycin A1-sensitive manner, suggesting the involvement of an exocytotic process. Accordingly, ionomycin was found to induce a Ca(2+)-dependent increase in the vesicular fusion rate, when exocytosis was monitored with acridine orange. ATP stimulated [3H]d-aspartate release in a concentration- (0.1-3 mm) and Ca(2+)-dependent manner. The gliosomal fraction contained proteins of the exocytotic machinery [syntaxin-1, vesicular-associated membrane protein type 2 (VAMP-2), 23-kDa synaptosome-associated protein (SNAP-23) and 25-kDa synaptosome-associated protein (SNAP-25)] co-existing with GFAP immunoreactivity. Moreover, GFAP or VAMP-2 co-expressed with the vesicular glutamate transporter type 1. Consistent with ultrastructural analysis, several approximately 30-nm non-clustered vesicles were present in the gliosome cytoplasm. It is concluded that gliosomes purified from adult brain contain glutamate-accumulating vesicles and can release the amino acid by a process resembling neuronal exocytosis.