The circadian clock, located in the suprachiasmatic nucleus (SCN), receives a major afferent from the median raphe nucleus (MRN). In the Syrian hamster, only about 50% of the cells giving rise to this afferent contain serotonin. There is mixed evidence as to whether the serotonergic portion of this projection is involved in non-photic phase shifting of circadian locomotor rhythms. In order to better characterize the non-serotonergic projections, we conducted retrograde tract tracing using the beta subunit of cholera toxin combined with multi-label immunohistochemistry. Similar to previous findings, almost half of the retrogradely labeled cells contained serotonin. Additionally, approximately 30% of the retrogradely labeled cells contained vesicular glutamate transporter 3 (VGLUT3), but not serotonin. Surprisingly, some dorsal raphe cholera toxin labeling was also noted, particularly in animals with central-SCN injections. To determine if the non-serotonergic projections were important for non-photic phase shifts elicited by MRN stimulation, the MRN was electrically stimulated in animals pretreated with SCN injection of either the serotonin neurotoxin 5,7-dihydroxytryptamine or vehicle control. Intact animals phase advanced to midday electrical stimulation of the raphe while lesioned animals did not. Together, these results show that although some of the non-serotonergic raphe projections to the SCN contain VGLUT3, it is the serotonergic raphe innervation of the SCN that is critical for non-photic phase shifting elicited by MRN stimulation.

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.

We previously reported that about 80% of vesicular glutamate transporter 3 (VGLUT3)-positive cells displayed immunoreactivity for serotonin, but the others were negative in the rat midbrain raphe nuclei, such as the dorsal (DR) and median raphe nuclei (MnR). In the present study, to investigate the precise distribution of VGLUT3-expressing nonserotonergic neurons in the DR and MnR, we performed double fluorescence in situ hybridization for VGLUT3 and tryptophan hydroxylase 2 (TPH2). According to the distribution of VGLUT3 and TPH2 mRNA signals, we divided the DR into six subregions. In the MnR and the rostral (DRr), ventral (DRV), and caudal (DRc) parts of the DR, VGLUT3 and TPH2 mRNA signals were frequently colocalized (about 80%). In the lateral wings (DRL) and core region of the dorsal part of the DR (DRDC), TPH2-producing neurons were predominantly distributed, and about 94% of TPH2-producing neurons were negative for VGLUT3 mRNA. Notably, in the shell region of the dorsal part of the DR (DRDSh), VGLUT3 mRNA signals were abundantly detected, and about 75% of VGLUT3-expressing neurons were negative for TPH2 mRNA. We then examined the projection of VGLUT3-expressing nonserotonergic neurons in the DRDSh by anterograde and retrograde labeling after chemical depletion of serotonergic neurons. The projection was observed in various brain regions such as the ventral tegmental area, substantia nigra pars compacta, hypothalamic nuclei, and preoptic area. These results suggest that VGLUT3-expressing nonserotonergic neurons in the midbrain raphe nuclei are preferentially distributed in the DRDSh and modulate many brain regions with the neurotransmitter glutamate via ascending axons.

The third vesicular glutamate transporter (VGLUT3) is expressed in a subset of cholinergic and GABAergic neurons in the forebrain. In this study the distribution of VGLUT3 was mapped in relation to the receptor for substance P, neurokinin 1 (NK1), which has been independently reported within cholinergic and GABAergic neurons in a similar distribution. Dual immunofluorescence labeling techniques were used, sometimes in combination with triple labeling for the vesicular acetylcholine transporter (VAChT), to identify cholinergic cells. Virtually all cells immunolabeled for VGLUT3 in the nucleus accumbens core and shell regions, ventral pallidum, olfactory tubercle and caudate putamen were cholinergic and also contained immunolabeling for the NK1 receptor. In the hippocampal formation where VGLUT3 has been described in GABAergic neurons, colocalization between NK1 and VGLUT3 was also common but less complete. Cells double labeled for NK1 and VGLUT3 were most prevalent in stratum radiatum in the CA1 subfield. In the habenula VGLUT3 was also found within NK1 receptor immunolabeled neurons. However, there were some areas where neurons containing these two proteins were separate populations including the cerebral cortex and median raphe nucleus. These results reveal a trend for VGLUT3 to localize within neurons containing the NK1 receptor in several areas of the forebrain.

The brainstem raphe nuclei are typically assigned a role in serotonergic brain function. However, numerous studies have reported that a large proportion of raphe projection cells are nonserotonergic. The identity of these projection cells is unknown. Recent studies have reported that the vesicular glutamate transporter VGLUT3 is found in both serotonergic and nonserotonergic neurons in both the median raphe (MR) and dorsal raphe (DR) nuclei. We injected the retrograde tracer cholera toxin subunit B into either the dorsal hippocampus or the medial septum (MS) and used triple labeled immunofluorescence to determine if nonserotonergic raphe cells projecting to these structures contained VGLUT3. Consistent with previous studies, only about half of retrogradely labeled MR neurons projecting to the hippocampus contained serotonin, whereas a majority of the retrogradely labeled nonserotonergic cells contained VGLUT3. Similar patterns were observed for MR cells projecting to the MS. About half of retrogradely labeled nonserotonergic neurons in the DR contained VGLUT3. Additionally, a large number of retrogradely labeled cells in the caudal linear and interpeduncular nuclei projecting to the MS were found to contain VGLUT3. These data suggest the enigmatic nonserotonergic projection from the MR to forebrain regions may be glutamatergic. In addition, these results demonstrate a dissociation between glutamatergic and serotonergic MR afferent inputs to the MS and hippocampus suggesting divergent and/or complementary roles of these pathways in modulating cellular activity within the septohippocampal network. Synapse 63: 31-41, 2009.(c) 2008 Wiley-Liss, Inc.

The lateral septum (LS) plays a role in the adjustment of behavioral responses according to environmental demands. This is a complex integrative process wherein a variety of modulatory systems, i.e. cholinergic, dopaminergic and serotonergic projections forming pericellular baskets around LS neurons, are involved. Recently, vesicular glutamate transporter 3 (VGLUT3)-immunoreactive (-ir) structures outlining unlabeled somata and their proximal dendrites were described in the LS. However, the vesicular transporters for acetylcholine and GABA were not or only rarely co-expressed with VGLUT3 [Gras, C., Herzog, E., Bellenchi, G.C., Bernard, V., Ravassard, P., Pohl, M., Gasnier, B., Giros, B., El Mestikawy, S., 2002. A third vesicular glutamate transporter expressed by cholinergic and serotonergic neurons. J. Neurosci. 22, 5442-5451; Herzog, E., Gilchrist, J., Gras, C., Muzerelle, A., Ravassard, P., Giros, B., Gaspar, P., El Mestikawy, S., 2004. Localization of VGLUT3, the vesicular glutamate transporter type 3, in the rat brain. Neuroscience 123, 983-1002]. In this study, the morphology and distribution of these VGLUT3-ir structures were systematically analyzed revealing that (1) they form distinct pericellular baskets (PBs) displaying variable shapes,(2) they are arranged in a layer-like pattern similar to the terminals of other modulatory systems,(3) beside a few exceptions (e.g., choline acetyltransferase), they are generally not or very sparsely co-localized with other neurochemical markers characterizing major neuron populations or afferent systems of the LS, i.e. calcium-binding proteins, tyrosine hydroxylase, tryptophan hydroxylase, vesicular glutamate transporters 1 (VGLUT1) and 2 (VGLUT2) and the vesicular GABA transporter. Thus, in the LS, a separate population of neurons is covered by VGLUT3-ir PBs. The distribution pattern and the lack of co-localization indicate that the VGLUT3-expressing cells of origin are located in the brainstem and that they could be pure glutamatergic projection neurons-different from the well-defined canonical VGLUT1- and VGLUT2-expressing neurons. Alternatively, they could simultaneously express VGLUT3 and second transmitter, but use different release sites inside the LS for both.

To study the effect of adrenal steroids on neuropeptide Y (NPY) synthesis in the hypothalamic-pituitary system, we examined NPY expression in rats treated with dexamethasone (a synthetic glucocorticoid) by in situ hybridization and immunohistochemistry. Rats were injected daily with dexamethasone (0.2mg/100g/day for 10 days, sc) or sesame oil (vehicle control), or non-injected (intact control). Relative staining area for corticotropin-releasing hormone or neurophysin II, a vasopressin carrier protein, was increased in the external zone of the median eminence in vehicle control, but was equivalent to that of intact control in the dexamethasone-injected group. Density of NPY-stained fiber varicosities was drastically increased in the external, but not the internal, zone of dexamethasone-injected group, coinciding with the increased NPY hybridization signal level in the arcuate nucleus. Dual-labeling experiments revealed no colocalization of NPY with hypophysiotropic or other peptides examined in single fibers of the median eminence. In the dexamethasone-injected group, expressions of NPY mRNA and peptide were detectable in a few pituitary cells, with some being corticotropes. These results suggest that NPY plays hormonal roles in the hypothalamic-pituitary-adrenal axis.

In the light of the various neurobiological effects of glutamate in brain development, although some embryonic cells are a probable source of glutamate involved in the development of precursor cells and/or immature neurons, little is known about when and where glutamate plays its crucial roles during corticogenesis. To investigate these roles, we focused on the developmental expression of vesicular glutamate transporter (VGLUT)1 and VGLUT2, which are regarded as the best markers for verifying glutamatergic neuron identity, especially the spatiotemporal distributions of their transcripts and proteins in the developing mouse cortex and hippocampus. In situ hybridization studies revealed that VGLUT1 mRNA is expressed in preplate and marginal zone cells at embryonic day (E)10 and in subplate cells by E13, whereas VGLUT2 mRNA is expressed in preplate and marginal zone cells at E10 and in cells of the subventricular zone by E13. Reverse transcriptase-polymerase chain reaction analysis detected full-length VGLUT1 and VGLUT2 gene transcripts in the embryonic brain. By dual labeling combined with immunostaining for microtubule-associated protein 2 (MAP2) or reelin, we showed that MAP2-positive preplate and marginal zone neurons and subplate neurons express VGLUT1, while reelin-positive preplate and marginal zone cells and MAP2-negative subventricular zone cells express VGLUT2. The present study is the first to provide morphologically reliable evidence showing that Cajal-Retzius cells and subplate neurons are glutamatergic, and that the two cells differentially express VGLUT1 and VGLUT2, respectively, as the specific transport system of glutamate in some events orchestrated by these cells during the cortical development of mice.

Three distinct subtypes of vesicular glutamate transporters (VGLUTs) have been identified to date that are expressed basically in a cell type-specific manner. We have found a splice variant of VGLUT1 mRNA that is expressed almost exclusively in photosensitive tissues, i.e. the retina and the pineal gland. The variant mRNA, termed VGLUT1v, contains an additional 75 base pair sequence derived from part of a second intron (designated as exon IIa) between exons 2 and 3. The variant accounted for approximately 70% and 25%of VGLUT1 mRNA in the adult retina and pineal gland, respectively. The expression of VGLUT1v was developmentally regulated in both tissues. Organ culture showed that expression of the variant in the retina increased in association with the development of rod cells, suggesting that VGLUT1v is expressed in rod cells. In situ hybridization with variant-specific probes showed expression of VGLUT1v in the inner segment layer of photoreceptor cells. On the other hand, variant expression did not parallel the development of rhodopsin-positive cells in the pineal gland. As rod cells and pinealocytes are known to release glutamate continuously at ribbon synapses, it is possible that the variant has some functional advantage over the wild-type transporter in such a specialized manner of glutamate release.

A second vesicular glutamate transporter (VGLUT2) is detected in magnocellular neurons in the rat hypothalamus. The present study revealed what phenotype of neurons express VGLUT2 mRNA by the histological method. We found that most vasopressin (VP) neurons and several oxytocin (OT) neurons express VGLUT2 mRNA. VGLUT2 gene expression in VP and OT neurons is enhanced with osmotic challenges. In the neurohypophysis, VGLUT2-staining in OT terminals was reduced with osmotic stimulation. These results indicate that VGLUT2 is principally expressed in VP neurons and also in some OT neurons and that VGLUT2 in VP and OT neurons is involved in osmotic regulation.

BACKGROUND: Nociceptive behaviors might attenuate pain sensation. Phosphorylation of extracellular signal-regulated kinase (pERK) was recently reported to be induced by noxious stimuli in dorsal horn neurons. We investigated, in a formalin test, whether pERK of the dorsal horn is affected by licking. METHODS: Twenty-four adult male rats were divided into four groups: control, formalin test, restricted control, and restricted formalin test. Ten percent formalin was injected subcutaneously into the left rear paw of the formalin test and restricted formalin test groups. The control and formalin test group rats were kept in a clear plastic chamber, whereas the restricted control and restricted formalin test group rats were kept in a modified-restraint, pipe-shaped chamber. All rats were killed after 25 min. Twelve sections of the lumbar spinal cord were processed for p-ERK immunohistochemistry using the avidin-biotin peroxidase method. RESULTS: The number of p-ERK positive cells in the restricted formalin test group was significantly higher than in the other three groups in the ipsilateral-side superficial dorsal horn (P < 0.05). However, there was no significant difference between the formalin test group and the two control groups in pERK expression. CONCLUSION: Licking decreased pERK of the spinal cord of the formalin test group. The findings suggested that licking attenuated the pain of the formalin test.

In this study, we generated mice lacking the gene for G-substrate, a specific substrate for cGMP-dependent protein kinase uniquely located in cerebellar Purkinje cells, and explored their specific functional deficits. G-substrate-deficient Purkinje cells in slices obtained at postnatal weeks (PWs) 10-15 maintained electrophysiological properties essentially similar to those from WT littermates. Conjunction of parallel fiber stimulation and depolarizing pulses induced long-term depression (LTD) normally. At younger ages, however, LTD attenuated temporarily at PW6 and recovered thereafter. In parallel with LTD, short-term (1 h) adaptation of optokinetic eye movement response (OKR) temporarily diminished at PW6. Young adult G-substrate knockout mice tested at PW12 exhibited no significant differences from their WT littermates in terms of brain structure, general behavior, locomotor behavior on a rotor rod or treadmill, eyeblink conditioning, dynamic characteristics of OKR, or short-term OKR adaptation. One unique change detected was a modest but significant attenuation in the long-term (5 days) adaptation of OKR. The present results support the concept that LTD is causal to short-term adaptation and reveal the dual functional involvement of G-substrate in neuronal mechanisms of the cerebellum for both short-term and long-term adaptation.

The developmental process of prolactin (PRL) cells in the fetal pituitary gland was studied in mice. While PRL cells were hardly detectable in the pituitary gland of intact fetuses, a treatment with 17beta-estradiol in vitro induced a number of PRL cells that varied drastically in number depending on the stage of gestation with a peak at E15. This effect was specific to E2, with epidermal growth factor, Insulin and forskolin failing to induce PRL cells. Although both estrogen receptor (ER) alpha and ERbeta were expressed in the fetal pituitary gland, the results from ER knockout models showed that only ERalpha mediates E2 action on PRL cells. A few PRL cells were observed in ERalpha-deficient mice as well as in their control littermates, suggesting that estrogen is not required for the phenotype determination of PRL cells. Unexpectedly, the effect of E2 on the induction of PRL cells in vitro was diminished after E15. Present results suggest that the exposure of fetal PRL cells to glucocorticoids (GCs) results in a reduction of sensitivity to E2. The mechanism underlying the down-regulation of estrogen sensitivity by GCs was found not to be down-regulation of ER levels, induction of annexin 1, a GCs-inducible inhibitor of PRL secretion, and a decrease in the number of PRL precursors by apoptosis. The effect of GCs appeared within 2 h, and did not require a de novo protein synthesis. GCs are considered to be involved in the mechanisms of silencing pituitary PRL in gestation possibly through a novel mechanism.

Recent studies have disclosed the molecular mechanisms responsible for the phenotype determination of the anterior pituitary cell types. However, as far as growth hormone (GH) cells are concerned, particular extra-cellular cues are required for the initiation of GH and GH-releasing hormone (GHRH)-receptor gene production in addition to the expression of the cell type specific transcription factor, pit-1. The glucocorticoids play a principal role in the functional maturation of nascent GH cells in the fetal pituitary glands in rodents, inducing GH and GHRH-receptor gene expression, and establish the GH secretory system regulated by the brain in late gestation. Research supporting this role for glucocorticoid in the development of GH cells is discussed.

Ca2+-dependent activator protein for secretion 2 (CAPS2/CADPS2) is a secretory granule-associated protein that is abundant at the parallel fiber terminals of granule cells in the mouse cerebellum and is involved in the release of neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF), both of which are required for cerebellar development. The human homolog gene on chromosome 7 is located within susceptibility locus 1 of autism, a disease characterized by several cerebellar morphological abnormalities. Here we report that CAPS2 knock-out mice are deficient in the release of NT-3 and BDNF, and they consequently exhibit suppressed phosphorylation of Trk receptors in the cerebellum; these mice exhibit pronounced impairments in cerebellar development and functions, including neuronal survival, differentiation and migration of postmitotic granule cells, dendritogenesis of Purkinje cells, lobulation between lobules VI and VII, structure and vesicular distribution of parallel fiber-Purkinje cell synapses, paired-pulse facilitation at parallel fiber-Purkinje cell synapses, rotarod motor coordination, and eye movement plasticity in optokinetic training. Increased granule cell death of the external granular layer was noted in lobules VI-VII and IX, in which high BDNF and NT-3 levels are specifically localized during cerebellar development. Therefore, the deficiency of CAPS2 indicates that CAPS2-mediated neurotrophin release is indispensable for normal cerebellar development and functions, including neuronal differentiation and survival, morphogenesis, synaptic function, and motor leaning/control. The possible involvement of the CAPS2 gene in the cerebellar deficits of autistic patients is discussed.

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.

The brainstem raphe nuclei are typically assigned a role in serotonergic brain function. However, numerous studies have reported that a large proportion of raphe projection cells are nonserotonergic. The identity of these projection cells is unknown. Recent studies have reported that the vesicular glutamate transporter VGLUT3 is found in both serotonergic and nonserotonergic neurons in both the median raphe (MR) and dorsal raphe (DR) nuclei. We injected the retrograde tracer cholera toxin subunit B into either the dorsal hippocampus or the medial septum (MS) and used triple labeled immunofluorescence to determine if nonserotonergic raphe cells projecting to these structures contained VGLUT3. Consistent with previous studies, only about half of retrogradely labeled MR neurons projecting to the hippocampus contained serotonin, whereas a majority of the retrogradely labeled nonserotonergic cells contained VGLUT3. Similar patterns were observed for MR cells projecting to the MS. About half of retrogradely labeled nonserotonergic neurons in the DR contained VGLUT3. Additionally, a large number of retrogradely labeled cells in the caudal linear and interpeduncular nuclei projecting to the MS were found to contain VGLUT3. These data suggest the enigmatic nonserotonergic projection from the MR to forebrain regions may be glutamatergic. In addition, these results demonstrate a dissociation between glutamatergic and serotonergic MR afferent inputs to the MS and hippocampus suggesting divergent and/or complementary roles of these pathways in modulating cellular activity within the septohippocampal network. Synapse 63: 31-41, 2009.(c) 2008 Wiley-Liss, Inc.

The lateral septum (LS) plays a role in the adjustment of behavioral responses according to environmental demands. This is a complex integrative process wherein a variety of modulatory systems, i.e. cholinergic, dopaminergic and serotonergic projections forming pericellular baskets around LS neurons, are involved. Recently, vesicular glutamate transporter 3 (VGLUT3)-immunoreactive (-ir) structures outlining unlabeled somata and their proximal dendrites were described in the LS. However, the vesicular transporters for acetylcholine and GABA were not or only rarely co-expressed with VGLUT3 [Gras, C., Herzog, E., Bellenchi, G.C., Bernard, V., Ravassard, P., Pohl, M., Gasnier, B., Giros, B., El Mestikawy, S., 2002. A third vesicular glutamate transporter expressed by cholinergic and serotonergic neurons. J. Neurosci. 22, 5442-5451; Herzog, E., Gilchrist, J., Gras, C., Muzerelle, A., Ravassard, P., Giros, B., Gaspar, P., El Mestikawy, S., 2004. Localization of VGLUT3, the vesicular glutamate transporter type 3, in the rat brain. Neuroscience 123, 983-1002]. In this study, the morphology and distribution of these VGLUT3-ir structures were systematically analyzed revealing that (1) they form distinct pericellular baskets (PBs) displaying variable shapes,(2) they are arranged in a layer-like pattern similar to the terminals of other modulatory systems,(3) beside a few exceptions (e.g., choline acetyltransferase), they are generally not or very sparsely co-localized with other neurochemical markers characterizing major neuron populations or afferent systems of the LS, i.e. calcium-binding proteins, tyrosine hydroxylase, tryptophan hydroxylase, vesicular glutamate transporters 1 (VGLUT1) and 2 (VGLUT2) and the vesicular GABA transporter. Thus, in the LS, a separate population of neurons is covered by VGLUT3-ir PBs. The distribution pattern and the lack of co-localization indicate that the VGLUT3-expressing cells of origin are located in the brainstem and that they could be pure glutamatergic projection neurons-different from the well-defined canonical VGLUT1- and VGLUT2-expressing neurons. Alternatively, they could simultaneously express VGLUT3 and second transmitter, but use different release sites inside the LS for both.

Recent studies have demonstrated a functional interaction between group I metabotropic glutamate (mGlu) receptors and the cannabinoid system in the modulation of synaptic transmission. By using antisera directed against mGlu1alpha and CB1 cannabinoid receptors, we examined their distribution in the CA1 region of rat organotypic hippocampal slice cultures. Immunoreactive mGlu1alpha and CB1 elements were localized in non-principal cells, with a labeling distribution that was very similar to the pattern previously observed in the adult rat brain. Double-immunofluorescence staining and confocal microscopy showed that a subset of interneurons, mainly located in the stratum radiatum, was double-labeled for both mGlu1alpha and CB1 receptors. Co-localization of the two receptor subtypes was confirmed in hippocampal sections from adult rat brain. By using the "mirror technique" in adjacent sections, we observed that the double-labeled cells for mGlu1alpha and CB1 receptors were also immunopositive for the cholecystokinin peptide. Quantitative analysis revealed that in the stratum radiatum the majority (92%) of the CB1-positive cells and 19% of the mGlu1alpha-positive cells expressed both receptors. Triple immunofluorescence staining showed partial co-labeling of mGlu1alpha- and CB1-immunopositive cells with the vesicular glutamate transporter 3 or calbindin. Our results demonstrate that mGlu1alpha and CB1 receptors co-exist in a subpopulation of inhibitory neurons in the stratum radiatum of the hippocampus that is suggestive of the Schaffer collateral-associated interneurons. Hence, additional functional mechanisms underlying the cooperation between these two receptor subtypes may exist.

Substance P (SP) and glutamate are implicated in cardiovascular regulation by the nucleus tractus solitarii (NTS). Our earlier studies suggest that SP, which acts at neurokinin 1 (NK1) receptors, is not a baroreflex transmitter while glutamate is. On the other hand, our recent studies showed that loss of NTS neurons expressing NK1 receptors leads to loss of baroreflex responses and increased blood pressure lability. Furthermore, studies have suggested that SP may interact with glutamate in the NTS. In this study, we sought to test the hypothesis that NK1 receptors colocalize with glutamate receptors, either N-methyl-d-aspartate (NMDA) receptors or AMPA receptors or both in the NTS. We performed double-label immunofluorescent staining for NK1 receptors and either N-methyl-d-aspartate receptor subunit 1 (NMDAR1) or AMPA specific glutamate receptor subunit 2 (GluR2) in the rat NTS. Because vesicular glutamate transporter 2 (VGLUT2) containing fibers are prominent in portions of the NTS where cardiovascular afferent fibers terminate, we also performed double-label immunofluorescent staining for NK1 receptors and VGLUT2. Confocal microscopic images showed that NK1 receptors-immunoreactivity (IR) and NMDAR1-IR colocalized in the same neurons in many NTS subnuclei. Almost all NTS neurons positive for NK1 receptor-IR also contained NMDAR1-IR, but only 53.4% to 74.8% of NMDAR1-IR positive neurons contained NK1 receptors-IR. NK1 receptor-IR and GluR2-IR also colocalized in many neurons in NTS subnuclei. A majority of NK1 receptor-IR positive NTS neurons also contained GluR2-IR, but only 45.8% to 73.9% of GluR2-IR positive NTS neurons contained NK1 receptors-IR. Our results also showed that fibers labeled for VGLUT2-IR were in close apposition to fibers and neurons labeled for NK1 receptor-IR. The data support our hypothesis, provide an anatomical framework for glutamate and SP interactions, and may explain the loss of baroreflexes when NTS neurons, which could respond to glutamate as well as SP, are killed.

Contrary to the widespread assumption, the filum terminale in the rat possesses a precise glial and neuronal organization. The processes of glial fibrillary acidic protein-stained astrocytes form a rich, three dimensional array. The crescent shaped white matter could be outlined with antibody detecting oligodendrocytes. The neurons in the filum terminale, labeled with neuron-specific nuclear protein, are distributed in a small midline group (dorsal nucleus) dorsal to and in two symmetrical clusters at both sides of the central canal (lateral nuclei). Nitric oxide synthase-, calretinin-, choline acetyltransferase-, substance P- and neurokinin receptor-1-immunoreactive neurons were detected in the lateral nuclei. Axons were classified based on their course and termination. Small number of calcitonin gene-related peptide-immunoreactive fibers was found exclusively in the dorsal nucleus. Nitric oxide synthase-, substance P-, and neurokinin receptor-1-stained axon arborizations were detected mainly in the lateral nucleus. A dense array of extremely fine vesicular glutamate transporter 2- and fine, synaptophysin-immunoreactive varicosities covered densely the lateral nuclei. Fine glycine-transporter 2-immunoreactive axon arborization like structures were seen also in the lateral nucleus. Vesicular glutamate transporter 1- and choline acetyltransferase-immunoreactive axons arborized in the entire gray matter. Serotonin- and enkephalin-immunoreactive fibers congregated in the dorsolateral portion of the white matter, called "shoulder region", while calretinin- and thick, varicose neurokinin receptor-1-stained axons were also seen in the same area of the white matter. Synaptophysin-immunoreactive fine varicosities colocalized only with vesicular glutamate transporter 2 immunoreaction. Substance P and glycine-transporter 2-immunoreactive puncta were found in close contact with neurokinin receptor-1-immunostained perikarya and dendrites.

It has been reported that mu-opioid agonists depress glutamate release in some neurons but the specific receptor subtype mediating this effect is unclear. The purpose of the present study was to examine whether a particular mu-opioid receptor (MOR) splice-variant, MOR(1C), is expressed in rat central nervous system (CNS) by terminals expressing the vesicular glutamate transporter2 (VGLUT2), a marker of glutamatergic neurons. Several MOR splice variants have been identified in mice and MOR(1C) appears mainly to be localized to fibers and terminals, from which most neurotransmitter release would be expected. In addition, VGLUT2 has been found in the CNS and antibodies to it are reliable markers for glutamatergic terminals. Using fluorescence immunohistochemistry and confocal microscopy to examine spatial relationships between MOR(1C) and VGLUT2, we found that MOR(1C) and VGLUT2 puncta were widely distributed throughout the rat CNS; moreover, many regions contained terminals that expressed both. Thus, it appears that coexpression of MOR(1C) and VGLUT2 is common in the rat CNS. We hypothesize that activation of MOR(1C) by mu-opioid agonists at some glutamatergic terminals may be a mechanism by which glutamate release is inhibited. J. Comp. Neurol. 508:542-564, 2008.(c) 2008 Wiley-Liss, Inc.

The entorhinal cortex of the rat (EC) contains a dense fiber plexus that expresses the calcium-binding protein calretinin (CR). Some CR fibers contain vesicular glutamate transporter 2 (VGluT2, associated with glutamatergic neurotransmission). CR-VGluT2 coexpressing fibers may have an extrinsic origin, for instance, the midline thalamic nucleus reuniens. Alternatively, they may belong to cortical interneurons. We studied the first possibility with anterograde and retrograde neuroanatomical tracing methods combined with CR and VGluT2 immunofluorescence and confocal laser scanning. The alternative possibility was studied with in situ hybridization fluorescence histochemistry for VGluT2 mRNA combined with CR immunofluorescence. In the anterograde tracing experiments, we observed many labeled reuniens fibers in EC expressing CR. Some of these labeled fibers contained immunoreactivity for VGluT2 and CR. In the complementary retrograde tracing experiments, we found retrogradely labeled cell bodies in nucleus reuniens of the thalamus that coexpressed CR. We also examined the colocalization of VGluT2 and CR in the entorhinal cortex by using in situ hybridization and CR immunofluorescence. In these experiments, we observed CR-immunopositive cortical neurons that coexpressed VGluT2. For the same sections, with CR as the principal marker and parvalbumin as a control marker, we found that parvalbumin neurons were negative for VGluT2 mRNA. Thus, CR-VGluT2-expressing axon terminals in EC belong to two sources: projection fibers from the thalamus and axon collaterals of local interneurons. VGluT2 expression is linked to the synaptic transmission of the excitatory neurotransmitter glutamate, so these thalamic CR-VGluT2 projection neurons and entorhinal CR-VGluT2 interneurons should be regarded as excitatory. J. Comp. Neurol. 506:359-370, 2008.(c) 2007 Wiley-Liss, Inc.

Electrophysiological, microdialysis and behavioral studies support a modulatory role for corticotropin-releasing factor (CRF) in regulating the dorsal raphe nucleus (DRN)-serotonin (5-HT) system. CRF and 5-HT are implicated in the pathophysiology of depression, thus neuroanatomical substrates of CRF-DRN-5-HT interactions are of interest. Identification of co-transmitters within DRN CRF axon terminals is important for elucidating the complex effects underlying CRF afferent regulation of DRN neurons. This study investigated whether CRF-labeled axon terminals within the DRN contain immunoreactivity for vesicular glutamate transporters (isoforms vGlut1 and vGlut2) indicative of the excitatory neurotransmitter glutamate. Dual immunohistochemistry for CRF and either vGlut1 or vGlut2 was conducted within the same tissue section and immunofluorescence results indicated patterns of immunoreactivity consistent with previous reports. Abundant vGlut1- and vGlut2-immunoreactivity was found in puncta exhibiting a largely uniform distribution, whereas CRF-immunoreactivity was localized to topographically distributed varicose processes within the DRN. Profiles containing both CRF- and either vGlut1- or vGlut2-immunoreactivity were apparent in the DRN. Electron microscopy confirmed that immunoreactivity for CRF and vGlut1 was localized primarily to separate axon terminals in the DRN, with a subset co-localizing CRF and vGlut1. Examination of CRF and vGlut2 immunoreactivities in the DRN indicated that CRF and vGlut2 were found within the same axon terminal more frequently than CRF and vGlut1. Overall, these anatomical findings suggest that CRF may function, in part, with the excitatory neurotransmitter glutamate in the modulation of neuronal activity in the DRN.

Objective To examine the vesicular glutamate transporters (VGluTs: VGluT1-VGluT3) in the peripheral vestibular system. Methods The vestibular structures, including Scarpa's ganglion (vestibular ganglion, VG), maculae of utricle and saccule, and ampullary cristae, from normal Sprague-Dawley rats were processed immunohistochemically for VGluTs, by avidin-biotinylated peroxidase complex method, with 3-3'-diaminobenzidine (DAB) as chromogen. Results (1) VGluT1 was localized to partial neurons of VG and to the putative primary afferent fibers innervating vestibular end-organs.(2) Intense VGluT3 immunoreactivity was detected in large number of sensory epithelia cells, and weak labeling of VGluT3-positive afferent fibers was in the maculae and ampullary cristae.(3) No or very weak VGluT2 immunoreactivity was observed in the VG and acoustic maculae. Conclusion These results provide the morphological support that glutamate exists in the peripheral vestibular system, and it may play an important role in the centripetal vestibular transmission.