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We identified three types of monosynaptic cholinergic inputs spatially arranged onto medial substantia nigra dopaminergic neurons in male and female mice: cotransmitted acetylcholine (ACh)/GABA, GABA-only, and ACh only. There was a predominant GABA-only conductance along lateral dendrites and soma-centered ACh/GABA cotransmission. In response to repeated stimulation, the GABA conductance found on lateral dendrites decremented less than the proximally located GABA conductance, and was more effective at inhibiting action potentials. While soma-localized ACh/GABA cotransmission showed depression of the GABA component with repeated stimulation, ACh-mediated nicotinic responses were largely maintained. We investigated whether this differential change in inhibitory/excitatory inputs leads to altered neuronal excitability. We found that a depolarizing current or glutamate preceded by cotransmitted ACh/GABA was more effective in eliciting an action potential compared with current, glutamate, or ACh/GABA alone. This enhanced excitability was abolished with nicotinic receptor inhibitors, and modulated by T- and L-type calcium channels, thus establishing that activity of multiple classes of ion channels integrates to shape neuronal excitability.SIGNIFICANCE STATEMENTOur laboratory has previously discovered a population of substantia nigra dopaminegic neurons (DA) that receive cotransmitted ACh and GABA. This study used subcellular optogenetic stimulation of cholinergic presynaptic terminals to map the functional ACh and GABA synaptic inputs across the somatodendritic extent of substantia nigra DA neurons. We determined spatially clustered GABA-only inputs on the lateral dendrites while cotransmitted ACh and GABA clustered close to the soma. We have shown that the action of GABA and ACh in cotransmission spatially clustered near the soma play a critical role in enhancing glutamate-mediated neuronal excitability through the activation of T- and L-type voltage-gated calcium channels.

Activation of cannabinoid 1 receptors (CB1 R) modulates multiple behaviours, including exploration, motor coordination and response to psychostimulants. It is known that CB1 R expressed by either excitatory or inhibitory neurons mediates different behavioural responses to CB1 R activation, yet the involvement of CB1 R expressed by medium spiny neurons (MSNs), the neuronal subpopulation that expresses the highest level of CB1 R in the CNS, remains unknown. We report a new genetically modified mouse line that expresses functional CB1 R in MSN on a CB1 R knockout (KO) background (CB1 R(MSN) mice). The absence of cannabimimetic responses measured in CB1 R KO mice was not rescued in CB1 R(MSN) mice, nor was decreased spontaneous locomotion, impaired instrumental behaviour or reduced amphetamine-triggered hyperlocomotion measured in CB1 R KO mice. Significantly, reduced novel environment exploration of an open field and absence of amphetamine sensitization (AS) measured in CB1 R KO mice were fully rescued in CB1 R(MSN) mice. Impaired motor coordination in CB1 R KO mice measured on the Rotarod was partially rescued in CB1 R(MSN) mice. Thus, CB1 R expressed by MSN control exploration, motor coordination, and AS. Our study demonstrates a new functional roles for cell specific CB1 R expression and their causal link in the control of specific behaviors.

The use and abuse of cannabis can be associated with significant pathophysiology, however, it remains unclear whether (1) acute administration of Δ-9-tetrahydrocannabinol (THC) during early adulthood alters the cannabinoid type 1 (CB1 ) receptor localization and expression in cells of the brain, and (2) THC produces structural brain changes. Here we use electron microscopy and a highly sensitive pre-embedding immunogold method to examine CB1 receptors in the hippocampus cornu ammonis subfield 1 (CA1) 30 min after male mice were exposed to a single THC injection (5 mg/kg). The findings show that acute exposure to THC can significantly decrease the percentage of CB1 receptor immunopositive terminals making symmetric synapses, mitochondria, and astrocytes. The percentage of CB1 receptor-labeled terminals forming asymmetric synapses was unaffected. Lastly, CB1 receptor expression was significantly lower at terminals of symmetric and asymmetric synapses as well as in mitochondria. Structurally, CA1 dendrites were significantly larger, and contained more spines and mitochondria following acute THC administration. The area of the dendritic spines, synaptic terminals, mitochondria, and astrocytes decreased significantly following acute THC exposure. Altogether, these results indicate that even a single THC exposure can have a significant impact on CB1 receptor expression, and can alter CA1 ultrastructure, within 30 min of drug exposure. These changes may contribute to the behavioral alterations experienced by young individuals shortly after cannabis intoxication.

The cannabinoid CB1 receptor localizes to the glutamatergic parallel fiber (PF) terminals of the cerebellar granule cells and participates in synaptic plasticity, motor control and learning that are impaired in CB1 receptor knockout (CB 1 -KO) mice. However, whether ultrastructural changes at the PF-Purkinje cell (PC) synapses occur in CB 1 -KO remains unknown. We studied this in the vermis of the spinocerebellar lobule V and the vestibulocerebellar lobule X of CB 1 -KO and wild-type (CB 1 -WT) mice by electron microscopy. Lobule V, but not lobule X, of CB 1 -KO had significantly less and longer synapses than in CB 1 -WT. PF terminals were significantly larger in both lobules of CB 1 -KO with no changes in PC dendritic spines. The PF terminals in lobule V of CB 1 -KO contained less synaptic vesicles and lower vesicle density; by contrast, vesicle density in lobule X of CB 1 -KO remained unchangeable relative to CB 1 -WT. There were as many vesicles in lobule V of CB 1 -KO as in CB 1 -WT, but their distribution decreased drastically at 300 nm of the active zone. In lobule X of CB 1 -KO, less vesicles were found within 150 nm from the presynaptic membrane; however, no vesicles were at 450-600 nm of the active zone. A significant higher amount of synaptic vesicles close to the active zone in lobule V and X of CB 1 -KO was observed. In conclusion, the absence of CB1 receptors strikingly and distinctively impacts on the ultrastructural architecture of the PF-PC synapses located in cerebellar lobules that differ in vulnerability to damage and motor functions.

The impact of pannexin-1 (Panx1) channels on synaptic transmission is poorly understood. Here, we show that selective block of Panx1 in single postsynaptic hippocampal CA1 neurons from male rat or mouse brain slices causes intermittent, seconds long increases in the frequency of sEPSC following Schaffer collateral stimulation. The increase in sEPSC frequency occurred without an effect on evoked neurotransmission. Consistent with a presynaptic origin of the augmented glutamate release, the increased sEPSC frequency was prevented by bath-applied EGTA-AM or TTX. Manipulation of a previously described metabotropic NMDAR pathway (i.e., by preventing ligand binding to NMDARs with competitive antagonists or blocking downstream Src kinase) also increased sEPSC frequency similar to that seen when Panx1 was blocked. This facilitated glutamate release was absent in transient receptor potential vanilloid 1 (TRPV1) KO mice and prevented by the TRPV1 antagonist, capsazepine, suggesting it required presynaptic TRPV1. We show presynaptic expression of TRPV1 by immunoelectron microscopy and link TRPV1 to Panx1 because Panx1 block increases tissue levels of the endovanilloid, anandamide. Together, these findings demonstrate an unexpected role for metabotropic NMDARs and postsynaptic Panx1 in suppression of facilitated glutamate neurotransmission.SIGNIFICANCE STATEMENTThe postsynaptic ion and metabolite channel, pannexin-1, is regulated by metabotropic NMDAR signaling through Src kinase. This pathway suppresses facilitated release of presynaptic glutamate during synaptic activity by regulating tissue levels of the transient receptor potential vanilloid 1 agonist anandamide.

Hippocampal slices are widely used for in vitro electrophysiological experiments to study underlying mechanisms for synaptic transmission and plasticity, and there is a growing appreciation for sex differences in synaptic plasticity. To date, several studies have shown that the process of making slices from male animals can induce synaptogenesis in cornu ammonis area 1 (CA1) pyramidal cells, but there is a paucity of data for females and other brain regions. In the current study we use microcrystals of the lipophilic carbocyanine dye DiI (1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate) to stain individual neurons in the CA1 and dentate gyrus (DG) hippocampal subfields of postnatal day 21 male and female rats. We show that the preparation of sections for electrophysiology produces significant increases in spines in sections obtained from females, similar to that observed in males. We also show that the procedures used for in vitro electrophysiology also result in significant spine increases in the DG and CA1 subfields. These results demonstrate the utility of this refined DiI procedure for staining neuronal dendrites and spines. They also show, for the first time, that in vitro electrophysiology slice preparations enhance spine numbers on hippocampal cells equivalently in both juvenile females and males.NEW & NOTEWORTHY This study introduces a new DiI technique that elucidates differences in spine numbers in juvenile female and male hippocampus, and shows that slice preparations for hippocampal electrophysiology in vitro may mask these differences.

International audience; Recent advances in neuroscience have positioned brain circuits as key units in controlling behavior, implying that their positive or negative modulation necessarily leads to specific behavioral outcomes. However, emerging evidence suggests that the activation or inhibition of specific brain circuits can actually produce multimodal behavioral outcomes. This study shows that activation of a receptor at different subcellular locations in the same neuronal circuit can determine distinct behaviors. Pharmacological activation of type 1 cannabinoid (CB1) receptors in the striatonigral circuit elicits both antinociception and catalepsy in mice. The decrease in nociception depends on the activation of plasma membrane-residing CB1 receptors (pmCB1), leading to the inhibition of cytosolic PKA activity and substance P release. By contrast, mitochondrial-associated CB1 receptors (mtCB1) located at the same terminals mediate cannabinoid-induced catalepsy through the decrease in intra-mitochondrial PKA-dependent cellular respiration and synaptic transmission. Thus, subcellular-specific CB1 receptor signaling within striatonigral circuits determines multimodal control of behavior.

Type 1 cannabinoid (CB1 ) receptors are widely distributed in the brain. Their physiological roles depend on their distribution pattern, which differs remarkably among cell types. Hence, subcellular compartments with little but functionally relevant CB1 receptors can be overlooked, fostering an incomplete mapping. To overcome this, knockin mice with cell-type-specific rescue of CB1 receptors have emerged as excellent tools for investigating CB1 receptors' cell-type-specific localization and sufficient functional role with no bias. However, to know whether these rescue mice maintain endogenous CB1 receptor expression level, detailed anatomical studies are necessary. The subcellular distribution of hippocampal CB1 receptors of rescue mice that express the gene exclusively in dorsal telencephalic glutamatergic neurons (Glu-CB1 -RS) or GABAergic neurons (GABA-CB1 -RS) was studied by immunoelectron microscopy. Results were compared with conditional CB1 receptor knockout lines. As expected, CB1 immunoparticles appeared at presynaptic plasmalemma, making asymmetric and symmetric synapses. In the hippocampal CA1 stratum radiatum, the values of the CB1 receptor-immunopositive excitatory and inhibitory synapses were Glu-CB1 -RS, 21.89% (glutamatergic terminals); 2.38% (GABAergic terminals); GABA-CB1 -RS, 1.92% (glutamatergic terminals); 77.92% (GABAergic terminals). The proportion of CB1 receptor-immunopositive excitatory and inhibitory synapses in the inner one-third of the dentate molecular layer was Glu-CB1 -RS, 53.19% (glutamatergic terminals); 2.30% (GABAergic terminals); GABA-CB1 -RS, 3.19% (glutamatergic terminals); 85.07% (GABAergic terminals). Taken together, Glu-CB1 -RS and GABA-CB1 -RS mice show the usual CB1 receptor distribution and expression in hippocampal cell types with specific rescue of the receptor, thus being ideal for in-depth anatomical and functional investigations of the endocannabinoid system. J. Comp. Neurol. 525:302-318, 2017. © 2016 Wiley Periodicals, Inc.

Comment in Sensory systems: the hungry sense. [Nat Rev Neurosci. 2014] Inhaling: endocannabinoids and food intake. [Nat Neurosci. 2014]; International audience; Hunger arouses sensory perception, eventually leading to an increase in food intake, but the underlying mechanisms remain poorly understood. We found that cannabinoid type-1 (CB1) receptors promote food intake in fasted mice by increasing odor detection. CB1 receptors were abundantly expressed on axon terminals of centrifugal cortical glutamatergic neurons that project to inhibitory granule cells of the main olfactory bulb (MOB). Local pharmacological and genetic manipulations revealed that endocannabinoids and exogenous cannabinoids increased odor detection and food intake in fasted mice by decreasing excitatory drive from olfactory cortex areas to the MOB. Consistently, cannabinoid agonists dampened in vivo optogenetically stimulated excitatory transmission in the same circuit. Our data indicate that cortical feedback projections to the MOB crucially regulate food intake via CB1 receptor signaling, linking the feeling of hunger to stronger odor processing. Thus, CB1 receptor-dependent control of cortical feedback projections in olfactory circuits couples internal states to perception and behavior.

The transient receptor potential vanilloid type 1 (TRPV1) is a non-selective cation channel that plays an important role in pain perception and modulates neurotransmitter release and synaptic plasticity in the brain. TRPV1 function must lay on its anatomical distribution in the peripheral and central nervous system regions involved in the physiological roles of the channel. However, the anatomical localization of TRPV1 is well established in the periphery, but in the brain it is a matter of debate. While some studies support the presence of TRPV1 in several brain regions, recent evidences suggest a restricted distribution of the channel in the central nervous system. To investigate to what extent central TRPV1 function stands on a precise brain distribution of the channel, we examined the mouse hippocampal dentate molecular layer (ML) where TRPV1 mediates long-term synaptic plasticity. Using pre-embedding immunocytochemistry for high resolution electron microscopy, we show that TRPV1 immunoparticles are highly concentrated in postsynaptic dendritic spines to asymmetric perforant path synapses in the outer 2/3 of the ML. However, TRPV1 is poorly expressed at the excitatory hilar mossy cell synapses in the inner 1/3 of this layer. Importantly, the TRPV1 pattern distribution disappeared in the ML of TRPV1-knockout mice. Taken together, these findings support the notion of the presence of TRPV1 in a brain region where the channel has been shown to have a functional role, such as the perforant path synapses in the hippocampal dentate ML.

The failure to undergo remyelination is a critical impediment to recovery in multiple sclerosis. Chondroitin sulfate proteoglycans (CSPGs) accumulate at demyelinating lesions creating a nonpermissive environment that impairs axon regeneration and remyelination. Here, we reveal a new role for 2-arachidonoylglycerol (2-AG), the major CNS endocannabinoid, in the modulation of CSPGs deposition in a progressive model of multiple sclerosis, the Theiler's murine encephalomyelitis virus-induced demyelinating disease. Treatment with a potent reversible inhibitor of the enzyme monoacylglycerol lipase, which accounts for 85% of the 2-AG degradation in the mouse CNS, modulates neuroinflammation and reduces CSPGs accumulation and astrogliosis around demyelinated lesions in the spinal cord of Theiler's murine encephalomyelitis virus-infected mice. Inhibition of 2-AG hydrolysis augments the number of mature oligodendrocytes and increases MBP, leading to remyelination and functional recovery of mice. Our findings establish a mechanism for 2-AG promotion of remyelination with implications in axonal repair in CNS demyelinating pathologies., This work was supported by Ministry of the Economy and Competition (MINECO) Grants SAF2013-42784-R and SAF2016-76449-R, Comunidad de Madrid Grant S2011/BMD-2308, and Red Española de Esclerosis Múltiple Grants RD12/0032/0008 and RD16/0015/0021 (sponsored by the Fondo de Investigación Sanitaria) to C.G., Basque Government Grant IT764-13, MINECO/FEDER Grant SAF2015-65034-R, and University of the Basque Country Grant UPV/EHU UFI11/41 to P.G., and MINECO Grant SAF2013-48271-C2-1-R to M.L.-R. None of the funding bodies played any role in the study design, data collection and analysis, the decision to publish, or the preparation of the manuscript. A.F. is a predoctoral fellow supported by MINECO Grant BES-2014-068459. We thank Laura Ramos for excellent technical support.

1,2-diacylglycerol lipase alpha (DAGLα) is responsible for the biosynthesis and release of 2-arachidonoyl-glycerol (2-AG), the most abundant endocannabinoid in the brain. Although its expression has been detected in discrete regions, we showed here an integrated description of the distribution of DAGLα mRNA and protein in the rat forebrain using in situ hybridization histochemistry and immunohistochemistry. As novelty, we described the distribution of DAGLα protein expression in the olfactory system, the rostral migratory stream, neocortex, septum, thalamus, and hypothalamus. Similar DAGLα immunostaining pattern was also found in the brain of wild-type, but not of DAGLα knockout mice. Immunohistochemical data were correlated by the identification of DAGLα mRNA expression, for instance, in the somata of specific cells in olfactory structures, rostral migratory stream and neocortex, cells in some septal-basal-amygdaloid areas and the medial habenula, and magnocellular cells of the paraventricular hypothalamic nucleus. This widespread neuronal distribution of DAGLα is consistent with multiple roles for endocannabinoids in synaptic plasticity, including presynaptic inhibition of neurotransmitter release. We discuss our comparative analysis of the forebrain expression patterns of DAGLα and other components of the endocannabinoid signaling system, including the CB1 receptor, monoacylglyceride lipase (MAGL), and fatty acid amide hydrolase (FAAH), providing some insight into the potential physiological and behavioral roles of this system. © 2011 IBRO.

Similar to dopamine (DA), cannabinoids strongly influence prefrontal cortical functions, such as working memory, emotional learning, and sensory perception. Although endogenous cannabinoid receptors (CB1Rs) are abundantly expressed in the prefrontal cortex (PFC), very little is known about endocannabinoid (eCB) signaling in this brain region. Recent behavioral and electrophysiological evidence has suggested a functional interplay between the dopamine and cannabinoid receptor systems, although the cellular mechanisms underlying this interaction remain to be elucidated. We examined this issue by combining neuroanatomical and electrophysiological techniques in PFC of rats and mice (both genders). Using immunoelectron microscopy, we show that CB1Rs and dopamine type 2 receptors (D2Rs) colocalize at terminals of symmetrical, presumably GABAergic, synapses in the PFC. Indeed, activation of either receptor can suppress GABA release onto layer 5 pyramidal cells. Furthermore, coactivation of both receptors via repetitive afferent stimulation triggers eCB-mediated long-term depression of inhibitory transmission (I-LTD). This I-LTD is heterosynaptic in nature, requiring glutamate release to activate group I metabotropic glutamate receptors. D2Rs most likely facilitate eCB signaling at the presynaptic site as disrupting postsynaptic D2R signaling does not diminish I-LTD. Facilitation of eCB–LTD may be one mechanism by which DA modulates neuronal activity in the PFC and regulates PFC-mediated behaviorin vivo.

The mature calyx of Held ending on principal neurons of the medial nucleus of the trapezoid body (MNTB) has very specialized morphological and molecular features that make it possible to transmit auditory signals with high fidelity. In a previous work we described an increased localization of the ionotropic α-amino-3-hydroxy-5-methyl-4 isoxazolepropionic acid (AMPA) glutamate receptor (GluA) subunits at postsynaptic sites of the calyx of Held-principal cell body synapses from postnatal development to adult. The aim of the present study was to investigate whether the pattern of the synaptic distribution of GluA2/3/4c and -4 in adult MNTB principal cell bodies correlated with preferential subcellular domains (stalks and swellings) of the calyx. We used a postembedding immunocytochemical method combined with specific antibodies to GluA2/3/4c and GluA4 subunits. We found that the density of GluA2/3/4c in calyceal swellings (19 ± 1.54 particles/μm) was higher than in stalks (10.93 ± 1.37 particles/μm); however, the differences for GluA4 were not statistically significant (swellings: 13.84 ± 1.39 particles/μm; stalks: 10.42 ± 1.24 particles/μm). Furthermore, GluA2/3/4c and GluA4 labeling co-localized to some extent in calyceal stalks and swellings. Taking these data together, the distribution pattern of GluA subunits in postsynaptic specializations are indicative of a spatial compartmentalization of AMPA subunits in mature calyx-principal neuron synapses that may support the temporally precise transmission required for sound localization in the auditory brainstem. J. Comp. Neurol. 518:163–174, 2010. © 2009 Wiley-Liss, Inc.

The endocannabinoid system is involved in multiple physiological functions including reward. Cannabinoids potently control the activity of midbrain dopamine cells, but the contribution of cortical projections in this phenomenon is unclear. We show that the bed nucleus of the stria terminalis (BNST) efficient relays cortical excitation to dopamine neurons of the ventral tegmental area (VTA). Anatomical andin vivoelectrophysiological evidence demonstrate that excitatory projections arising exclusively from the infralimbic cortex converge on BNST neurons, which in turn project to and excite >80% VTA dopamine cells. At the ultrastructural level, cannabinoid type 1 receptors are detected within the BNST on axon terminals arising from the infralimbic cortex. We found that intra-BNST infusion of a cannabinoid agonist inhibits the firing of dopamine cells evoked by stimulation of the infralimbic cortex. Our data identify a new neuronal substrate for the actions of cannabinoids in the reward pathway.

Deep cerebellar dentate nuclei are in a key position to control motor planning as a result of an integration of cerebropontine inputs and hemispheric Purkinje neurons signals, and their influence through synaptic outputs onto extracerebellar hubs. GABAergic dentate neurons exhibit broader action potentials and slower afterhyperpolarization than non-GABAergic (presumably glutamatergic) neurons. Specific potassium channels may be involved in these distinct firing profiles, particularly, Kv3.1 and Kv3.3 subunits which rapidly activate at relatively positive potentials to support the generation of fast action potentials. To investigate the subcellular localization of Kv3.1b and Kv3.3 in GAD− and GAD+ dentate neurons of glutamic acid decarboxylase 67–green fluorescent protein (GAD67-GFP) knock-in mice a preembedding immunocytochemical method for electron microscopy was used. Kv3.1b and Kv3.3 were in membranes of cell somata, dendrites, axons and synaptic terminals of both GAD− and GAD+ dentate neurons. The vast majority of GAD− somatodendritic membrane segments domains labeled for Kv3.1b and Kv3.3 (96.1% and 84.7%, respectively) whereas 56.2% and 69.8% of GAD− axonal membrane segments were immunopositive for these subunits. Furthermore, density of Kv3.1b immunoparticles was much higher in GAD− somatodendritic than axonal domains. As to GAD+ neurons, only 70.6% and 50% of somatodendritic membrane segments, and 53.3% and 59.5% of axonal membranes exhibited Kv3.1b and Kv3.3 labeling, respectively. In contrast to GAD− cells, GAD+ cells exhibited a higher density labeling for both Kv3 subunits at their axonal than at their somatodendritic membranes. Taken together, Kv3.1b and Kv3.3 potassium subunits are expressed in both GAD− and GAD+ cells, albeit at different densities and distribution. They likely contribute to the distinct biophysical properties of both GAD− and GAD+ neurons in the dentate nucleus.

Alterations of long-term synaptic plasticity have been proposed to participate in the development of addiction. To preserve synaptic functions, homeostatic processes must be engaged after exposure to abused drugs. At the mouse cortico-accumbens synapses, a singlein vivoinjection of Δ9-tetrahydrocannabinol (THC) suppresses endocannabinoid-mediated long-term depression. Using biochemical and electrophysiological approaches, we now report that 1 week of repeatedin vivoTHC treatment reduces the coupling efficiency of cannabinoid CB1 receptors (CB1Rs) to Gi/otransduction proteins, as well as CB1R-mediated inhibition of excitatory synaptic transmission at the excitatory synapses between the prefrontal cortex and the nucleus accumbens (NAc). Nonetheless, we found that cortico-accumbens synapses unexpectedly express normal long-term depression because of a reversible switch in its underlying mechanisms. The present data show that, in THC-treated mice, long-term depression is expressed because a presynaptic mGluR2/3 (metabotropic glutamate receptor 2/3)-dependent mechanism replaces the impaired endocannabinoid system. Thus, in the NAc, a novel form of presynaptic homeostasis rescues synaptic plasticity from THC-induced deficits.

Several studies indicate that metabotropic glutamate receptors (mGluRs) participate in the transmission of visual stimuli in optic layers of the superior colliculus (SC). We examined the cellular and subcellular distribution of the group III mGluR4a in superficial layers of the rat SC by means of a specific antiserum and a preembedding immunogold method for electron microscopy. Deposits of mGluR4a immunoparticles were mostly observed on presynaptic membranes of large synaptic terminals, which made asymmetrical synapses and contained abundant spherical, clear synaptic vesicles and numerous electron translucent mitochondria. These characteristic ultrastructural features correspond to retinocollicular synaptic terminals. Also, chains of synaptic retinal terminals along dendrites were labeled for mGluR4a. About 70% of morphologically identified retinal terminals were mGluR4a immunopositive. Furthermore, mGluR4a immunoreactivity in SC greatly disappeared following retinal ablation. About 28% of cortical terminals identified by anterograde tracing showed mGluR4a labeling, whereas only 2% of collicular GABAergic profiles were labeled for mGluR4a. These results reveal that retinal terminals are the major contributors to the mGluR4a immunoreactivity observed in the superior collicular circuitry.

During the last few years a variety of genetically encodable optical probes that monitor physiological parameters such as local pH, Ca 2 + Cl - , or transmembrane voltage have been developed. These sensors are based on variants of green-fluorescent protein (GFP) and can be synthesized by mammalian cells after transfection with cDNA. To use these sensor proteins in intact brain tissue, specific promoters are needed that drive protein expression at a sufficiently high expression level in distinct neuronal subpopulations. Here we investigated whether the promoter sequence of a particular potassium channel may be useful for this purpose. We produced transgenic mouse lines carrying the gene for enhanced yellow-fluorescent protein (EYFP), a yellow-green pH- and Cl - sensitive variant of GFP, under control of the Kv3.1 K + channel promoter (pKv3.1). Transgenic mouse lines displayed high levels of EYFP expression, identified by confocal microscopy, in adult cerebellar granule cells, interneurons of the cerebral cortex, and in neurons of hippocampus and thalamus. Furthermore, using living cerebellar slices we demonstrate that expression levels of EYFP are sufficient to report intracellular pH and Cl - concentration using imaging techniques and conditions analogous to those used with conventional ion-sensitive dyes. We conclude that transgenic mice expressing GFP-derived sensors under the control of cell-type specific promoters, provide a unique opportunity for functional characterization of defined subsets of neurons.

The existence of a neuronal-glial signalling through the activation of neurotransmitter receptors expressed in glia is well-documented. In excitatory synapses, glutamate released from presynaptic terminals activates not only postsynaptic receptors, but also ionotropic and metabotropic glutamate receptors localized in the glia ensheathing the synapses. The medial nucleus of the trapezoid body of the auditory system is involved in the localization of sounds in the space. In this nucleus, the large excitatory synaptic terminals formed by the calyces of Held on the principal globular cell bodies are wrapped by astrocytic processes. Since these synapses are functional from early postnatal days, glia receiving excitatory synaptic signals from the calyces may participate in modulating the maturation and development of the system. Groups I and II of metabotropic glutamate receptors (mGluRs) have been localized in glial cells in different brain regions. To investigate whether group II mGluRs are present in the medial nucleus of the trapezoid body, we have studied the pattern of expression of mGluR2/3 in the developing and mature nucleus by means of immunocytochemichal methods. The most remarkable finding was the switch in the occurrence of mGluR2/3 from glial to neuronal compartments. Thus, a preferential localization of mGluR2/3 immunoreactivity was observed in astrocytic processes surrounding the calyces of Held during the early postnatal development. In contrast, the main feature in adult rats was the presence of the group II mGluRs in presynaptic calyces of Held and postsynaptic principal globular cells. From these observations we suggest a role for group II mGluRs in neuronal-glial signalling in the calyx of Held-principal globular neuron synapses. Activation of these receptors might be relevant to the maturation and modulation of synaptic transmission in the medial nucleus of the trapezoid body.

Metabotropic glutamate receptors (mGluRs) modulate somatosensory, autonomic, and motor functions at spinal levels. mGluR postsynaptic actions over spinal neurons display the pharmacologic characteristics of type I mGluRs; however, the spinal distribution of type I mGluR isoforms remains poorly defined. In this study, the authors describe a differential distribution of immunoreactivity to various type I mGluR isoforms (mGluR1a, mGluR5a,b, and mGluR1b) that suggests a correlation between specific isoforms and particular aspects of spinal cord function. Two different antisera raised against mGluR5a,b detected intense immunoreactivity within nociceptive afferent terminal fields (laminae I and II) and also in autonomic regions (parasympathetic and sympathetic). In contrast, two of three anti-mGluR1a antibodies did not immunostain lamina I or II. Laminae I and II immunostaining by a third anti-mGluR1a antibody was competed by a peptide sequence obtained from a homologous region in mGluR5, suggesting possible cross reactivity in fixed tissue. Autonomic neurons did not express mGluR1a immunoreactivity. All anti-mGluR1a antibodies strongly and specifically immunolabeled dendritic and somatic membranes of neurons in the deep dorsal horn (lamina III-V) and the ventral horn (lamina VI-IX). Somatic motoneurons expressed mGluR1a immunoreactivity but little or no mGluR5 immunoreactivity. Phrenic and pudendal motoneurons expressed the highest level of mGluR1a immunoreactivity in the spinal cord. Intense mGluR1b immunoreactivity was restricted to a few scattered neurons and a prominent group of neurons in lamina X. Lamina II neurons expressed low levels of mGluR1b immunoreactivity. Ultrastructurally, type I mGluR immunoreactivity was found mostly at extrasynaptic sites on the plasma membrane, but it was also found perisynaptically, in the body of the postsynaptic regions or in relation to intracytoplasmic structures.

We used affinity purified antisera specific for group II metabotropic glutamate receptors in combination with a pre-embedding immunocytochemical method for light microscopy to investigate the localization of mGluR2/3 receptors in the enteric nervous system of the rat small intestine. In the submucosal plexuses of the jejunum and ileum, strongly mGluR2/3 immunoreactive cells were distributed between the circular muscular layer and the muscularis mucosa. In addition, oval or rounded immunostained ganglion cells appeared located in the myenteric plexuses of both intestinal regions. Nerve fibers intensely stained were observed extending between adjacent myenteric ganglia. The localization of mGluR2/3 receptors in enteric neurons might have functional implications in the physiology and pathology of the gut.

We report in this study with a pre-embedding immunogold method, the clustering of the group III metabotropic glutamate receptor 4a (mGluR4a) along the presynaptic membrane of parallel fiber synaptic terminals in the cerebellar molecular layer. The mGluR4a clusters were homogeneously distributed and interspaced by about 60 nm. These results suggest a particular arrangement of mGluR4a which might help to a rapid and effective activation of this receptor by glutamate.

A preembedding immunocytochemical method for light microscopy was used to study the postnatal development of expression of the group III metabotropic glutamate receptor mGluR4a in the medial nucleus of the trapezoid body (MNTB) of the rat. Immunoreactivity for mGluR4a was localized in axonal endings wrapping the principal globular neurons in MNTB, known as calyces of Held. The percentage of calyces of Held immunoreactive for mGluR4a increased progressively from postnatal day 3 (PND3), showing the highest density of labeled calyces by PND9. From this postnatal age on, a gradual reduction in the number of mGluR4a-immunopositive calyces of Held was observed, reaching the lowest level of labeled profiles in adult tissue. The developmental expression of mGluR4a in calyces of Held correlates well with previous studies in young animals showing a modulation of synaptic neurotransmission by group III mGluRs in these giant excitatory synapses made on MNTB principal neurons. All these observations together suggest that the expression of mGluR4a mainly between PND7 and PND12 might be relevant to the maturation and modulation of synaptic transmission at the calyces of Held.

Alternative splicing has been shown to occur at the metabotropic glutamate receptor 1 (mGluR1) gene. Three main isoforms that differ in their carboxy-termini have been described so far and named mGluR1alpha, mGluR1beta and mGluR1c. These variants when expressed in recombinant systems all activate phospholipase C, although the [Ca2+] signals generated have different kinetics. Tissue distribution studies of specific mGluR1 splice variants are limited to the mGluR1alpha isoform. In the present work, we examined the localization of mGluR1beta in the adult rat and mouse forebrain by using a specific antipeptide antibody. Furthermore, the mGluR1beta immunostaining was compared with that obtained with antibodies specific for mGluR1alpha or with a pan-mGluR1 antibody which recognizes all isoforms. mGluR1beta-like immunoreactivity (LI) was found confined to the neuropil and neuronal perikarya and appeared discretely distributed in the rodent forebrain. Differential cellular distribution between mGluR1alpha and mGluR1beta was observed. In the hippocampus, mGluR1alpha-LI was restricted to non-principal neurons in all fields, whereas mGluR1beta-LI was strongest in principal cells of the CA3 field and dentate granule cells but absent in CA1. We have also shown that the vast majority of neurons in the striatum express mGluR1. The predominant form appeared to be mGluR1beta, with a distribution pattern reflecting the patch-matrix organization of the striatum. The specificity of the immunoreactivity described for mGluR1 splice variants was confirmed in mGluR1-deficient mice. The observation of a different cellular and regional distribution of mGluR1 splice variants, in particular in the hippocampus, suggests that they may mediate different roles in synaptic transmission.

The midbrain periaqueductal gray is a key structure for the mediation of an integrated defence behaviour. Although a prominent role for glutamate in PAG mechanisms is supported by both behavioural and morphological studies, whether PAG afferents conveying somatosensory information constitute a source of glutamatergic input to the PAG remains unknown. Here, we have compared the projection pattern of orthogradely-labelled spinoannular fibres with the distribution of glutamate-like immunoreactivity in the PAG at the light microscopic level. Transaxonal labelling was observed throughout the whole rostrocaudal axis of the PAG except for the dorsolateral regions. Cell-processes and terminal-reminiscent puncta were strongly immunoreactive in all PAG regions, including the dorsolateral areas. To ascertain whether glutamate-immunoreactive puncta observed at light microscopy indeed constituted axon terminals of the spinoannular system, glutamate-like immunoreactivity was assessed in orthogradely-labelled synaptic terminals using a post-embedding immunogold procedure for electron microscopy. Quantitative analysis of gold particle densities revealed over twice as strong an immunoreactivity in anatomically-identified spinoannular axon terminals as in dendrites postsynaptic to them, perikarya and inhibitory Gray II synapses, as well as an over 5-fold heavier immunolabelling than in glial profiles. These findings reveal that glutamate is accumulated in synaptic terminals of the spinoannular system, supporting a neurotransmitter role for this acidic amino acid in spinofugal afferents to the PAG.

To study the contribution of retinal and cortical afferents to the patterns of glutamate- and homocysteate-like immunoreactivities in dorsal lateral geniculate, combined retinal and cortical ablations were performed in rats. In controls, glutamate immunoreactivity was in terminal-like dots and neurons. Homocysteate immunoreactivity was in small puncta. In lesioned animals, most glutamate-immunoreactive dots disappeared. In contrast, abundant puncta resembling parts of glial cells were immunoreactive for homocysteate.

NMDA receptors (NMDARs) are classically known as coincidence detectors for the induction of long-term synaptic plasticity and have been implicated in hippocampal CA3 cell-dependent spatial memory functions that likely rely on dynamic cellular ensemble encoding of space. The unique functional properties of both NMDARs and mossy fiber projections to CA3 pyramidal cells place mossy fiber NMDARs in a prime position to influence CA3 ensemble dynamics. By mimicking presynaptic and postsynaptic activity patterns observed in vivo, we found a burst timing-dependent pattern of activity that triggered bidirectional long-term NMDAR plasticity at mossy fiber-CA3 synapses in rat hippocampal slices. This form of plasticity imparts bimodal control of mossy fiber-driven CA3 burst firing and spike temporal fidelity. Moreover, we found that mossy fiber NMDARs mediate heterosynaptic metaplasticity between mossy fiber and associational-commissural synapses. Thus, bidirectional NMDAR plasticity at mossy fiber-CA3 synapses could substantially contribute to the formation, storage and recall of CA3 cell assembly patterns.

To study the distribution of L-homocysteate in the rat retina, specific polyclonal and monoclonal anti-homocysteate antibodies have been used in combination with a highly sensitive postembedding method for light microscopic immunocytochemistry. In central and peripheral retina, the most strongly immunoreactive cell bodies lay in the inner nuclear layer. They represented about 17% of the total neuronal cell population of the layer and were identified as bipolar cells (19-20% of cells in the outer half of the inner nuclear layer) and amacrine cells (15% of cells in the inner half of the inner nuclear layer). A third cell type showing heavy homocysteate-like immunoreactivity was identified as Müller glial cells. Characteristically, their descending processes formed three immunoreactive bands in the inner plexiform layer. Furthermore, the outer and inner limiting membranes as well as glia around and between ganglion cell axons and in the vicinity of blood vessels were labelled intensely. Photoreceptors and their terminals, and ganglion cells, were not immunostained. These findings indicate the presence of homocysteate in some bipolar and amacrine cells of the inner nuclear layer and support a role for this sulphur-containing excitatory amino acid as a neurotransmitter candidate in the retina.

Antibodies were raised against C-terminal peptides of the splice variants a, b and c of the rat mGluR1 metabotropic glutamate receptor. Affinity purified antibodies each specifically reacting with mGluR1a, mGluR1b and mGluR1c were used to study the cellular localization of these receptors in rat cerebellum. The mGluR1a antibody strongly labelled Purkinje cells at their cells bodies, portions of their dendritic trees and numerous small punctate elements reminiscent of dendritic spines. Also labelled were some stellate, basket, Golgi and Lugaro cells. Granule cells were devoid of staining. The mGluR1b antibody strongly labelled Purkinje cell bodies and their dendrites at levels which varied within the same lobule of the vermis or the hemispheres. No significant labelling was observed at stellate, basket, Golgi and granule cells, while occasionally a fraction of basket cells and cerebellar glomeruli was moderately immunoreactive. The mGluR1c antibody strongly labelled cell bodies and thick principal dendrites of Purkinje cells but not dendritic spines. Immunonegaative Purkinje cells were intermingled with strongly labelled ones in lobules 4–10, while in lobules 1, 2 and 3, no stained Purkinje cells were detected. The mGluR1c antibody also labelled stellate, basket, some Golgi and some Lugaro cells as well as granule cells.

International audience; The reason why neurons synthesize more than one endocannabinoid (eCB) and how this is involved in the regulation of synaptic plasticity in a single neuron is not known. We found that 2-arachidonoylglycerol (2-AG) and anandamide mediate different forms of plasticity in the extended amygdala of rats. Dendritic L-type Ca(2+) channels and the subsequent release of 2-AG acting on presynaptic CB1 receptors triggered retrograde short-term depression. Long-term depression was mediated by postsynaptic mGluR5-dependent release of anandamide acting on postsynaptic TRPV1 receptors. In contrast, 2-AG/CB1R-mediated retrograde signaling mediated both forms of plasticity in the striatum. These data illustrate how the eCB system can function as a polymodal signal integrator to allow the diversification of synaptic plasticity in a single neuron.

Olfactory sensory neurons (OSNs) expressing the same odorant receptor converge in specific glomeruli where they transmit olfactory information to mitral cells. Surprisingly, synaptic mechanisms underlying mitral cell activation are still controversial. Using patch-clamp recordings in mouse olfactory bulb slices, we demonstrate that stimulation of OSNs produces a biphasic postsynaptic excitatory response in mitral cells. The response was initiated by a fast and graded monosynaptic input from OSNs and followed by a slower component of feedforward excitation, involving dendro-dendritic interactions between external tufted, tufted and other mitral cells. The mitral cell response occasionally lacked the fast OSN input when few afferent fibers were stimulated. We also show that OSN stimulation triggers a strong and slow feedforward inhibition that shapes the feedforward excitation but leaves unaffected the monosynaptic component. These results confirm the existence of direct OSN to mitral cells synapses but also emphasize the prominence of intraglomerular feedforward pathways in the mitral cell response.

Activation of cannabinoid type-1 receptors (CB(1)) is universally recognized as a powerful endogenous orexigenic signal, but the detailed underlying neuronal mechanisms are not fully understood. Using combined genetic and pharmacological approaches in mice, we found that ventral striatal CB(1) receptors exerted a hypophagic action through inhibition of GABAergic transmission. Conversely, brain CB(1) receptors modulating excitatory transmission mediated the well-known orexigenic effects of cannabinoids.

The position of neurotransmitter receptors relative to active neurotransmitter release sites may be a major factor influencing neuronal responses. The location of the metabotropic glutamate receptor subtype mGluR2/3 was investigated in synaptic structures in the rat superficial spinal dorsal horn laminae by using a pre-embedding immunogold technique. Immunostaining for mGluR2/3 occurred in laminae I through III. Gold particles were encountered both in the cytosol and along the plasma membrane. Distinctive plasmalemmal immunodeposits were detected in vesicle-containing profiles, where they were located to membrane compartments distant from active release sites rather than in the close vicinity of synaptic specialisations. No distinct immunolabelling was observed in profiles meeting characteristics of primary afferent terminals. The extrasynaptic occurrence of mGluR2/3 suggests a presynaptic heteroreceptor role for these receptor subtypes in the spinal dorsal horn.

The pattern of expression of ionotropic glutamate receptor (GluR) subunits 1–4 in the medial nucleus of the trapezoid body (MNTB) has been reported to change during development. The aim of this study was to compare the distribution of the GluR1–4 subunits in the MNTB at postnatal day (P) 9, before high-frequency signal transmission in the auditory system has developed, with that observed in mature adult rats. GluR1–4 subunits were studied by preembedding and postembedding immunocytochemical methods. Increased levels of GluR1, 2/3, and 4 associated with development were evident only at postsynaptic sites of MNTB principal cell bodies receiving calyces of Held synapses, whereas receptor density at nonsynaptic sites was found to remain unaltered. Taken together, the expression pattern of GluR subunits and the density of immunoparticles in postsynaptic specializations are indicative of a compartmentalization of alpha-amino-3-hydroxy-5-methyl-4 isoxazolepropionic acid (AMPA) subunits upon development. These developmental changes provide a morphological basis for establishment of the postsynaptic properties needed for high-frequency synaptic transmission of auditory signals to MNTB principal neurons. J. Comp. Neurol. 495:624–634, 2006. © 2006 Wiley-Liss, Inc.

We investigated the mechanisms by which activation of group I metabotropic glutamate receptors (mGluRs) and CB1 cannabinoid receptors (CB1Rs) leads to inhibition of synaptic currents at the calyx of Held synapse in the medial nucleus of the trapezoid body (MNTB) of the rat auditory brainstem. In ∼50% of the MNTB neurons tested, activation of group I mGluRs by the specific agonist (s)-3,5-dihydroxyphenylglycine (DHPG) reversibly inhibited AMPA receptor- and NMDA receptor-mediated EPSCs to a similar extent and reduced paired-pulse depression, suggestive of an inhibition of glutamate release. Presynaptic voltage-clamp experiments revealed a reversible reduction of Ca2+currents by DHPG, with no significant modification of the presynaptic action potential waveform. Likewise, in ∼50% of the tested cells, the CB1 receptor agonist (R)-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl)pyrrolo[1,2,3-de]-1,4-benzoxazin-6-yl]-1-naphthalenylmethanone (WIN) reversibly inhibited EPSCs, presynaptic Ca2+currents, and exocytosis. For a given cell, the amount of inhibition by DHPG correlated with that by WIN. Moreover, the inhibitory action of DHPG was blocked by the CB1R antagonistN-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) and occluded by WIN, indicating that DHPG and WIN operate via a common pathway. The inhibition of EPSCs by DHPG, but not by WIN, was abolished after dialyzing 40 mmBAPTA into the postsynaptic cell, suggesting that DHPG activated postsynaptic mGluRs. Light and electron microscopy immunolabeling indicated a presynaptic expression of CB1Rs and postsynaptic localization of mGluR1a. Our data suggest that activation of postsynaptic mGluRs triggers the Ca2+-dependent release of endocannabinoids that activate CB1 receptors on the calyx terminal, which leads to a reduction of presynaptic Ca2+current and glutamate release.

A pre-embedding immunocytochemical method was used to study the subcellular distribution of the voltage-dependent potassium channel Kv3.1b in the medial nucleus of the trapezoid body (MNTB) in developing and adult rat. The main finding was the localization of the channel in specific membrane compartments of the calyces of Held and principal globular neurons. Thus, at postnatal day (P) 9 immunoparticles were densely localized in plasma membranes of globular cell bodies and their main dendrites. At P16, a strong Kv3.1b labeling was still observed in these globular cell compartments, but the most remarkable feature was the presence of immunoparticles in synaptic terminal membranes of the calyces of Held. However, the presynaptic and postsynaptic specializations of the calyx of Held–globular cell synapses were virtually devoid of immunoparticles. This same subcellular distribution of Kv3.1b was seen in adult, with membranes of calycine terminals more uniformly labeled. The developmental profile of Kv3.1b expression in MNTB coincides with the functional maturation of the calyx of Held–principal globular neuron synapse. The presence of the channel in this system is crucial for the high-frequency synaptic transmission of auditory signals.

Studies indicate that metabotropic glutamate receptors (mGluRs) may play a role in spinal sensory transmission. We examined the cellular and subcellular distribution of the mGluR subtype 4a in spinal tissue by means of a specific antiserum and immunocytochemical techniques for light and electron microscopy. A dense plexus of mGluR4a-immunoreactive elements was seen in the dorsal horn, with an apparent accumulation in lamina II. The immunostaining was composed of sparse immunoreactive fibres and punctate elements. No perikaryal staining was seen. Immunostaining for mGluR4a was detected in small to medium-sized cells but not in large cells in dorsal root ganglia. At the electron microscopic level, superficial dorsal horn laminae demonstrated numerous immunoreactive vesicle-containing profiles. Labelling was present in the cytoplasmic matrix, but accretion of immunoreaction product to presynaptic specialisations was commonly observed. Axolemmal labelling was confirmed by using a preembedding immunogold technique, which revealed distinctive deposits of gold immunoparticles along presynaptic thickenings with an average centre-to-centre distance of 41 nm (41.145 +/- 13.59). Immunoreactive terminals often formed synaptic contacts with dendritic profiles immunonegative for mGluR4a. Immunonegative dendritic profiles were observed in apposition to both mGluR4a-immunoreactive and immunonegative terminals. Diffuse immunoperoxidase reaction product was also detected in dendritic profiles, some of which were contacted by mGluR4a-immunoreactive endings, but only occasionally were they observed to accumulate immunoreaction product along the postsynaptic density. Terminals immunoreactive for mGluR4a also formed axosomatic contacts. The present results reveal that mGluR4a subserves a complex spinal circuitry to which the primary afferent system seems to be a major contributor.

This study evaluates the localization of the metabotropic glutamate receptor mGluR4a in the piriform cortex of rats using preembedding immunocytochemical methods. At the light microscopic level, punctate labeling was evident in layers Ia and Ib of the piriform cortex, and immunolabeled fibers were present in layers II and III. Following bilateral destruction of the olfactory bulb, the density of labeled puncta in layer Ia decreased. These results suggest that the receptor is present on the terminals of the lateral olfactory tract (LOT). Electron microscopic evaluation of layers Ia and Ib revealed that mGluR4a was localized in synaptic terminals in layers Ia and Ib. The terminals had clear, round synaptic vesicles and terminated on asymmetric synapses on dendritic spines and shafts. There was also immunolabeling of some dendritic profiles in layers Ia and Ib that were postsynaptic to unlabeled presynaptic terminals. These observations suggest that mGluR4a is present on presynaptic terminals in the layers of the piriform cortex that receive LOT and associational synapses. This is the same area in which previous studies have revealed the presence of mGluR7 and mGluR8, suggesting that all three receptors may be colocalized.

The mGluR1 metabotropic glutamate receptor is a G-protein-coupled receptor that exists as different C-terminal splice variants. When expressed in mammalian cells, the mGluR1 splice variants exhibit diverse transduction mechanisms and also slightly differ in their apparent agonist affinities. In the present study, we used an affinity-purified antiserum, specifically reactive to the mGluRlb splice variant, in combination with a highly sensitive preembedding immunocytochemical method for light microscopy to investigate the distribution of this receptor in the rat hypothalamus. An intense immunoreactivity for mGluRlb was observed in distinct hypothalamic nuclei. Thus, neuronal cell bodies and dendrites were stained in the preoptic area, suprachiasmatic nucleus, dorsal hypothalamus, lateral hypothalamus, dorsomedial nucleus, tuberomammilary nucleus, and lateral mammilary body. The ventromedial nucleus exhibited neuropil immunostaining but neuronal cell bodies were not labeled. Strong mGluRlb immunoreactivity was observed in magnocellular neurons of the neuroendocrine supraoptic, paraventricular, and arcuate nuclei. Also, neuronal cell bodies were heavily labeled in the retrochiasmatic nucleus, anterior commissural nucleus, and periventricular nucleus. These immunocytochemical observations, together with previous studies, suggest that mGluRlb is coexpressed with other class I mGluRs in some nuclei throughout the hypothalamus. However, mGluRlb is so far the only receptor of this class strongly expressed in the supraoptic, paraventricular, and arcuate nuclei, which might have relevant implications in the physiological control of the neuroendocrine hypothalamic-pituitary system.

The periaqueductal grey matter (PAG) is known to adjust somatic and neurovegetative elements of the defence behaviour. We have used specific polyclonal antibodies to examine the distribution of the metabotropic glutamate receptor subtype 5 (mGluR5) in this region. Immunolabelling for mGluR5 displayed a net preference for dorsolateral areas at rostral and intermediate levels. Electronmicroscopic examination revealed that mGluR5 is expressed in neuronal perikarya and in dendrites receiving synaptic contacts of Gray I type. To investigate the possible relevance of mGluR5 to integration of somatosensory information, spinoannular (SA) neurones were peroxidase-labelled and their relationship with mGluR5-expressing PAG neurones was examined at the ultrastructural level. A number of synaptic terminals of the SA pathway established synaptic contact of asymmetric type onto mGluR5-immunoreactive dendrites. It is suggested that mGluR5 might be involved in the temporal integration of somatosensory inputs to the PAG.

We have investigated the pattern of glutamate-like immunoreactivity in the superficial layers of the rat superior colliculus by means of postembedding immunocytochemical methods for light and electron microscopy. At the light microscopic level, labelling was faintly to moderately intense in most perikarya of the stratum zonale, stratum griseum superficiale and stratum opticum. Furthermore, strong glutamate-immunoreactive terminal-like elements were accumulated most densely in stratum zonale, stratum griseum superficiale and stratum opticum. At the electron microscopic level, a postembedding immunogold method revealed that the vast majority of those labelled elements corresponded to retinal and visual cortical terminals. These profiles were about twice as heavily labelled as their postsynaptic partners. To determine the contribution of retinal and cortical afferents to the pattern of glutamate-like immunoreactivity, rats were subjected to right retinal ablation, left cortical ablation or combined right retinal and left cortical ablations. After retinal ablation, strongly labelled perikarya were observed in the retinorecipient layers. Furthermore, a prominent loss of glutamate-immunoreactive terminal-like elements occurred in stratum zonale and stratum griseum superficiale. Ipsilateral superior colliculus to cortical ablation exhibited subtle changes characterized by a moderate increase in perikaryal immunostaining in stratum zonale, stratum griseum superficiale and stratum opticum and by an apparent discrete reduction of labelled dots in stratum griseum superficiale and stratum opticum. In cases with combined lesions, strongly immunoreactive cell bodies and dendrites were accompanied by a massive disappearance of labelled terminal-like elements in stratum zonale, stratum griseum superficiale and stratum opticum. The effect of retinal and visual cortical ablations on the pattern of glutamate-like immunoreactivity suggests that these afferents are the major sources for glutamate-immunoreactive terminals in the rat superior colliculus. In addition, these findings provide further evidence for glutamate as neurotransmitter in the visual pathways studied.

Anti-homocysteate antibodies with postembedding immunohistochemistry for light microscopy were used to localize homocysteate-like immunoreactivity in human multiform glioblastoma. The most remarkable stained elements (6.8% of the total tumoral tissue) corresponded to distinct astrocytic cell bodies, intermingled fibrous processes and puncta of diverse size, some of them closely apposed to capillaries. In not affected peritumoral tissue, on the other hand, numerous labelled dots resembling portions of glial processes were observed in the neuropil and around blood vessels (2.7% of the total peritumoral tissue). However, glial and neuronal cell bodies could not be detected. These observations extend to a human brain tumor of glial nature the knowledge on the preferential localization of homocysteate in glia.

l-Homocysteate, a sulphur-containing l-glutamate analogue has recently been proposed as a neurotransmitter candidate. However, the cellular localization of this excitatory amino acid remained to be determined. By means of immunocytochemistry, the localization of homocysteate was accomplished in the cerebellar cortex of rats. Cerebellar glia could be defined as the major store of this compound. Homocysteate, thus, may not be a classical neurotransmitter but rather a member of another class of intercellular messengers that might be termed ‘gliotransmitters’.

To investigate the relation between perforant path and the pattern of glutamate-like immunoreactivity in its target regions in the rat hippocampal formation, unilateral lesions of various size and location were placed to interrupt certain contingents of these afferent fibers. Postembedding immunohistochemistry at the level of light microscopy yielded the same pattern of immunoreactivity in the hippocampal formation contralateral to the lesion as in untreated animals. On the ipsilateral side, however, extensive transections of the perforant path led to a drastic loss of glutamate-immunoreactive terminal-like elements in the outer part of the dentate molecular layer. More restricted lesions induced a loss of punctate glutamate-like immunoreactivity in narrower bands within this zone. The width and the location of the affected bands appeared to depend on the extent of the transections and their topographical relation to the perforant path fiber system. These results and those obtained using a postembedding immunogold method at the level of electron microscopy strongly indicate that perforant path terminals in the dentate molecular layer of the rat contain high levels of glutamate and, thus, provide further support for an already well-documented role of this excitatory amino acid as neurotransmitter in this system.

The pattern of glutamate-like immunoreactivity was investigated in the medial nucleus of the trapezoid body of adult rats. A monoclonal 'anti-glutamate' antibody was combined with postembedding immunohistochemistry involving a silver-intensified peroxidase-antiperoxidase procedure for light microscopy or an immunogold method for electron microscopy. The most conspicuous glutamate-immunoreactive elements were labelled calyces of Held. Because of the outstanding size of these terminals they may provide an attractive model for studying the details of glutamatergic neurotransmission at a synapse of the mammalian CNS.