Psychosocial Crowding Stress-Induced Changes in Synaptic Transmission and Glutamate Receptor Expression in the Rat Frontal Cortex.

This study demonstrates how exposure to psychosocial crowding stress (CS) for 3, 7, and 14 days affects glutamate synapse functioning and signal transduction in the frontal cortex (FC) of rats. CS effects on synaptic activity were evaluated in FC slices of the primary motor cortex (M1) by measuring field potential (FP) amplitude, paired-pulse ratio (PPR), and long-term potentiation (LTP). Protein expression of GluA1, GluN2B mGluR1a/5, VGLUT1, and VGLUT2 was assessed in FC by western blot. The body's response to CS was evaluated by measuring body weight and the plasma level of plasma corticosterone (CORT), adrenocorticotropic hormone (ACTH), and interleukin 1 beta (IL1B). CS 3 14d increased FP and attenuated LTP in M1, while PPR was augmented in CS 14d. The expression of GluA1, GluN2B, and mGluR1a/5 was up-regulated in CS 3d and downregulated in CS 14d. VGLUTs expression tended to increase in CS 7d. The failure to blunt the effects of chronic CS on FP and LTP in M1 suggests the impairment of habituation mechanisms by psychosocial stressors. PPR augmented by chronic CS with increased VGLUTs level in the CS 7d indicates that prolonged CS exposure changed presynaptic signaling within the FC. The CS bidirectional profile of changes in glutamate receptors' expression seems to be a common mechanism evoked by stress in the FC.

Neural substrates for the distinct effects of presynaptic group III metabotropic glutamate receptors on extinction of contextual fear conditioning in mice.

The group III metabotropic glutamate (mGlu) receptors mGlu7 and mGlu8 are receiving increased attention as potential novel therapeutic targets for anxiety disorders. The effects mediated by these receptors appear to result from a complex interplay of facilitatory and inhibitory actions at different brain sites in the anxiety/fear circuits. To better understand the effect of mGlu7 and mGlu8 receptors on extinction of contextual fear and their critical sites of action in the fear networks, we focused on the amygdala. Direct injection into the basolateral complex of the amygdala of the mGlu7 receptor agonist AMN082 facilitated extinction, whereas the mGlu8 receptor agonist (S)-3,4-DCPG sustained freezing during the extinction acquisition trial. We also determined at the ultrastructural level the synaptic distribution of these receptors in the basal nucleus (BA) and intercalated cell clusters (ITCs) of the amygdala. Both areas are thought to exert key roles in fear extinction. We demonstrate that mGlu7 and mGlu8 receptors are located in different presynaptic terminals forming both asymmetric and symmetric synapses, and that they preferentially target neurons expressing mGlu1α receptors mostly located around ITCs. In addition we show that mGlu7 and mGlu8 receptors were segregated to different inputs to a significant extent. In particular, mGlu7a receptors were primarily onto glutamatergic afferents arising from the BA or midline thalamic nuclei, but not the medial prefrontal cortex (mPFC), as revealed by combined anterograde tracing and pre-embedding electron microscopy. On the other hand, mGlu8a showed a more restricted distribution in the BA and appeared absent from thalamic, mPFC and intrinsic inputs. This segregation of mGlu7 and mGlu8 receptors in different neuronal pathways of the fear circuit might explain the distinct effects on fear extinction training observed with mGlu7 and mGlu8 receptor agonists. This article is part of a Special Issue entitled 'Metabotropic Glutamate Receptors'.

Regulation of connexin 36 expression during development.

In the mammalian CNS, the expression of neuronal gap junction protein, connexin 36 (Cx36), increases during the first 2 weeks of postnatal development and then decreases during the following 2 weeks. Recently we showed that the developmental increase in Cx36 expression is augmented by chronic (2 weeks) activation of group II metabotropic glutamate receptors (mGluR), prevented by chronic receptor inactivation, and the receptor-dependent increase in Cx36 expression is regulated via transcriptional control of the Cx36 gene activity. We demonstrate here that acute (60 min) activation of group II mGluRs in developing cortical neuronal cultures causes transient increase in Cx36 protein expression with decrease during the following 24h. However, there is no change in Cx36 mRNA expression. In addition, the data indicate that transient increase in Cx36 expression is due to new protein synthesis. The results suggest that, during development, acute activation of group II mGluRs causes up-regulation of Cx36 via post-transcriptional mechanisms. However, if the receptor activation is sustained, transcriptional activation of the Cx36 gene occurs.

Neuron-to-glia signaling mediated by excitatory amino acid receptors regulates ErbB receptor function in astroglial cells of the neuroendocrine brain.

Hypothalamic astroglial erbB tyrosine kinase receptors are required for the timely initiation of mammalian puberty. Ligand-dependent activation of these receptors sets in motion a glia-to-neuron signaling pathway that prompts the secretion of luteinizing hormone-releasing hormone (LHRH), the neuropeptide controlling sexual development, from hypothalamic neuroendocrine neurons. The neuronal systems that may regulate this growth factor-mediated back signaling to neuroendocrine neurons have not been identified. Here we demonstrate that hypothalamic astrocytes contain metabotropic receptors of the metabotropic glutamate receptor 5 subtype and the AMPA receptor subunits glutamate receptor 2 (GluR2) and GluR3. As in excitatory synapses, these receptors are in physical association with their respective interacting/clustering proteins Homer and PICK1. In addition, they are associated with erbB-1 and erbB-4 receptors. Concomitant activation of astroglial metabotropic and AMPA receptors results in the recruitment of erbB tyrosine kinase receptors and their respective ligands to the glial cell membrane, transactivation of erbB receptors via a mechanism requiring metalloproteinase activity, and increased erbB receptor gene expression. By facilitating erbB-dependent signaling and promoting erbB receptor gene expression in astrocytes, a neuron-to-glia glutamatergic pathway may represent a basic cell-cell communication mechanism used by the neuroendocrine brain to coordinate the facilitatory transsynaptic and astroglial input to LHRH neurons during sexual development.

Developmental expression of the group III metabotropic glutamate receptor mGluR4a in the medial nucleus of the trapezoid body of the rat.

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.

Dopamine D1 receptor modulation of glutamate receptor messenger RNA levels in the neocortex and neostriatum of unilaterally 6-hydroxydopamine-lesioned rats.

The effect of treatment with the D1 dopamine receptor agonist SKF 38393 on the expression of metabotropic glutamate receptor 1, 3, 4 and 5 receptor subtypes and of the glutamate N-methyl-D-aspartate ionotropic receptor subunits NRI, NR2A and NR2B was analysed using in situ hybridization. We studied the neocortex and neostriatum of normal rats and of rats unilaterally treated with 6-hydroxydopamine, a neurotoxin that, after intracerebral injection into the ventral tegmental area, causes selective degeneration of the ascending dopamine pathway. In the 6-hydroxydopamine-lesioned rats, metabotropic glutamate receptor subtype 3 messenger RNA levels were ipsilaterally increased in the neocortex and neostriatum, while the levels of metabotropic glutamate receptor subtype 4 messenger RNA were bilaterally increased in both regions. When administered to the 6-hydroxydopamine-lesioned rats, the D1 receptor agonist SKF 38393 (3 x 20 mg/kg, s.c.) produced a bilateral decrease in the expression of the metabotropic glutamate receptor subtype 1 and 5 receptor messenger RNA levels in the neocortex and neostriatum. In the neostriatum, SKF 38393 attenuated the ipsilateral increase in the expression of striatal metabotropic glutamate receptor subtype 3 messenger RNA produced by the 6-hydroxydopamine lesion. Furthermore, SKF 38393 produced a bilateral decrease in the levels of NRI receptor subunit messenger RNA and, in contrast, an increase in the striatal NR2B messenger RNA levels. All of these effects were abolished by the D1 receptor antagonist SCH 23360. These results indicate a differential D1 receptor-mediated modulation of the expression of some glutamate receptor subtypes in the neostriatum and neocortex, in agreement with the idea of a functional coupling between dopamine and excitatory amino acid systems in both regions. Thus, pharmacological targeting of excitatory amino acid systems could provide alternative or complementary treatment strategies for diseases involving dopaminergic systems in the striatum (e.g., Parkinson's disease) and cortex (e.g., schizophrenia).

Immortalized hypothalamic neurons express metabotropic glutamate receptors positively coupled to cyclic AMP formation.

We have characterized the expression pattern and pharmacological profile of activation of metabotropic glutamate receptors (mGluRs) in immortalized, gonadotropin releasing hormone (GnRH)-secreting GT1-7 cells, which represent a homogeneous cellular population of hypothalamic origin. These cells are known to respond to the mGluR agonist (1S,3R)-cyclopentanedicarboxylic acid (1S,3R-ACPD) with increased GnRH release. To establish which specific mGluR subtypes are expressed by GT1-7 cells, we used polyclonal antibodies raised against non-conserved regions of the carboxy-terminal domains of individual subtypes. The selectivity of these antibodies was tested in HEK 293 cells transiently transfected with each mGluR subtype. GT1-7 cells stained positively for the subtypes mGluR1a, -1b and -5 (belonging to group I mGluR2/3 (group II) and mGluR7 (group III). Agonists of group I mGluRs, including 1S,3R-ACPD, activated phosphoinositide hydrolysis in GT1-7 cells. This effect, however, was manifested only when cell density was low, and it disappeared when cells reached confluence. Stimulation of phosphoinositide hydrolysis could not therefore have been related to hormone secretion because 1S,3R-ACPD effectively released GnRH in confluent cultures. We then focused on group II and III mGluRs, which in transfected cells are negatively linked to adenylate cyclase activity. Unexpectedly, however, agonist which preferentially activate group II and III mGluRs increased both basal and forskolin-stimulated cAMP accumulation in GT1-7 cells. Stimulation of cAMP accumulation by mGluR agonists was not prevented by enzymatic depletion of endogenous adenosine, but was obliterated when cells were incubated with agonists of receptors positively coupled to adenylate cyclase, such as beta-adrenergic and prostaglandin E2 receptors. These results suggest that GT1-7 cells express a novel mGluR subtype positively coupled to adenylate cyclase, which shares the same transduction pathway of other classical receptors coupled with a Gs-type of GTP-binding protein.

Palmitoylation of the GluR6 kainate receptor.

The G-protein-coupled metabotropic glutamate receptor mGluR1 alpha and the ionotropic glutamate receptor GluR6 were examined for posttranslational palmitoylation. Recombinant receptors were expressed in baculovirus-infected insect cells or in human embryonic kidney cells and were metabolically labeled with [3H]palmitic acid. The metabotropic mGluR1 alpha receptor was not labeled whereas the GluR6 kainate receptor was labeled after incubation with [3H]palmitate. The [3H]palmitate labeling of GluR6 was eliminated by treatment with hydroxylamine, indicating that the labeling was due to palmitoylation at a cysteine residue via a thioester bond. Site-directed mutagenesis was used to demonstrate that palmitoylation of GluR6 occurs at two cysteine residues, C827 and C840, located in the carboxyl-terminal domain of the molecule. A comparison of the electrophysiological properties of the wild-type and unpalmitoylated mutant receptor (C827A, C840A) showed that the kainate-gated currents produced by the unpalmitoylated mutant receptor were indistinguishable from those of the wild-type GluR6. The unpalmitoylated mutant was a better substrate for protein kinase C than the wild-type GluR6 receptor. These data indicate that palmitoylation may not modulate kainate channel function directly but instead affect function indirectly by regulating the phosphorylation state of the receptor.

A novel metabotropic glutamate receptor expressed in the retina and olfactory bulb.

A novel metabotropic glutamate receptor, mGluR8, was identified by screening a mouse retina cDNA library. This receptor is most related to mGluR4, mGluR7, and mGluR6 (74%, 74%, and 70% identical amino acid residues, respectively). Similar to these receptors, stimulation by L-glutamate or L-2-amino-4-phosphonobutyrate (L-APB) of Chinese hamster ovary (CHO) cells stably transfected with mGluR8 result in the inhibition of forskolin-stimulated adenylyl cyclase. In situ hybridization studies revealed a strong expression of the mGluR8 gene in the olfactory bulb, accessory olfactory bulb, and mammillary body. A weaker expression was found in the retina, and in scattered cells in the cortex and hindbrain. During development, the distribution of mGluR8 expression was more widespread. These results extend the diversity of metabotropic glutamate receptors in the CNS. Because at least two APB receptors are expressed in the retina, the use of this drug to block selectively the ON pathway needs to be reconsidered. The pharmacology and expression of mGluR8 in mitral/tufted cells suggest it could be a presynaptic receptor modulating glutamate release by these cells at their axon terminals in the entorhinal cortex.

Molecular properties of the glutamate receptor mediating synaptic excitation in rat hypothalamic neurons.

AMPA-type glutamate receptors (GluRs) mediate synaptic excitation in networks of cultured rat hypothalamic neurons [18, 25]. Under voltage clamp the agonists quisqualate and AMPA induce current responses which consist of a maintained and/or transient component depending on the concentrations applied. The current-voltage relationship for both components is linear. The biphasic response patterns are due to receptor desensitization which is fast and does not require intracellular second messengers for its activation. Several GluR-subtype-encoding transcripts were found in these neurons using polymerase chain reaction (PCR) methods. While mRNAs encoding the GluR2 and 3 flip forms are expressed early, mRNAs encoding the GluR1, 2 and 3 flop forms and the GluR4 flip form appear only in cultures older than 3 weeks. By comparison to recombinant receptors, the properties of the native receptor can be accommodated by a heteromeric receptor containing GluR2 as one of the subunits.