Effects of subthalamic nucleus lesions and stimulation upon corticostriatal afferents in the 6-hydroxydopamine-lesioned rat

Abnormalities of striatal glutamate neurotransmission may play a role in the pathophysiology of Parkinson's disease and may respond to neurosurgical interventions, specifically stimulation or lesioning of the subthalamic nucleus (STN). The major glutamatergic afferent pathways to the striatum are from the cortex and thalamus, and are thus likely to be sources of striatal neuronally-released glutamate. Corticostriatal terminals can be distinguished within the striatum at the electron microscopic level as their synaptic vesicles contain the vesicular glutamate transporter, VGLUT1. The majority of terminals which are immunolabeled for glutamate but are not VGLUT1 positive are likely to be thalamostriatal afferents. We compared the effects of short term, high frequency, STN stimulation and lesioning in 6-hydroxydopamine (6OHDA)-lesioned rats upon striatal terminals immunolabeled for both presynaptic glutamate and VGLUT1. 6OHDA lesions resulted in a small but significant increase in the proportions of VGLUT1-labeled terminals making synapses on dendritic shafts rather than spines. STN stimulation for one hour, but not STN lesions, increased the proportion of synapses upon spines. The density of presynaptic glutamate immuno-gold labeling was unchanged in both VGLUT1-labeled and -unlabeled terminals in 6OHDA-lesioned rats compared to controls. Rats with 6OHDA lesions+STN stimulation showed a decrease in nerve terminal glutamate immuno-gold labeling in both VGLUT1-labeled and -unlabeled terminals. STN lesions resulted in a significant decrease in the density of presynaptic immuno-gold-labeled glutamate only in VGLUT1-labeled terminals. STN interventions may achieve at least part of their therapeutic effect in PD by normalizing the location of corticostriatal glutamatergic terminals and by altering striatal glutamatergic neurotransmission.     

The role of glutamate receptors in antipsychotic drug action

Summary. It has recently been postulated that disturbances in glutamatergic neurotransmission may contribute to the pathophysiology of schizophrenia. Therefore the aim of the present study was to evaluate the role of glutamate NMDA and group II metabotropic receptors in the antipsychotic drug action. To this aim the influence of some well-known neuroleptics on cortical NMDA receptors was examined. Furthermore, their behavioral effects were compared with those of the novel agonist of group II glutamate metabotropic receptors, LY 354740, in some animal models of schizophrenic deficits. We found that long-term administration of the typical neuroleptic haloperidol and the atypical one clozapine increased the number of NMDA receptors labelled with [³H]CGP 39653 in different cortical areas. Long-, but not short-term, treatment with haloperidol and raclopride diminished the deficit of prepulse inhibition produced by phencyclidine, which is a model of sensorimotor gating deficit in schizophrenia. In contrast, neither short- nor long-term treatment with clozapine influenced the phencyclidine effect in that model. Acute treatment with LY 354740 reversed neither (1) the deficit of prepulse inhibition produced by phencyclidine or apomorphine, nor (2) the impairment in a delayed alternation task induced by MK-801, which is commonly used to model the frontal lobe deficits associated with schizophrenia. The present study suggests that an increase in the density of cortical NMDA receptors may be important to a longterm neuroleptic therapy. Conversely, the results do not support the role of group II metabotropic glutamate receptors in the antipsychotic drug action. Received August 31, 1999 Accepted September 20, 1999     

Novel localization of NMDA receptors within neuroendocrine gonadotropin-releasing hormone terminals

About 1000 hypothalamic neurons synthesize and release gonadotropin-releasing hormone (GnRH), the master molecule of reproduction in all mammals. At the level of the median eminence at the base of the brain, where GnRH and other hypothalamic releasing hormones are secreted into the capillary system leading to the anterior pituitary gland, there is non-synaptic regulation of neurohormone release by a number of central neurotransmitters. For example, glutamate, the major excitatory amino acid in the brain, directly regulates GnRH release from nerve terminals via NMDA receptors (NMDARs). Moreover, the effects of glutamate action on GnRH secretion are potentiated by estrogens, and this relates to the physiologic control of ovulation by the hypothalamus. We sought to determine the ultrastructural relationship between GnRH neuroterminals and NMDARs, and this regulation by estradiol. Using immunofluorescent confocal microscopy, postembedding immunogold electron microscopy, fractionation, and Western blotting, we demonstrated: (i) GnRH is localized in large dense-core vesicles of neurosecretory profiles/terminals, (ii) the NMDAR1 subunit is found primarily on large dense-core vesicles of neurosecretory profiles/terminals, (iii) there is extensive colocalization of GnRH and NMDAR1 on the same vesicles, and (iv) estradiol modestly but significantly alters the distribution of NMDAR1 in GnRH neuroterminals by increasing expression of NMDAR1 on large dense-core vesicles. Western blots of fractionated median eminence support the presence of NMDAR1 in subcellular fractions containing large dense-core vesicles. These data are the first to show the presence of the NMDAR on neuroendocrine secretory vesicles, its co-expression with GnRH, and its regulation by estradiol. The results provide a novel anatomical site for the NMDAR and may represent a new mechanism for the regulation of GnRH release.     

Age and Visual Experience-dependent Expression of NMDAR1 Splice Variants in Rat Retina

The N-methyl-D-aspartate receptor (NMDAR) is a key molecule mediating brain plasticity related processes. Knowing that alternative splicing of the NMDAR1 (NR1) subunit offers molecular diversity to NMDAR, controls the forward trafficking of the NR1 protein and is important for placing NMDA receptors at synapses, we investigated herein the postnatal developmental expression and the influence of visual deprivation on NR1 subunit splice variants in rat retina. Real-time PCR was performed using oligonucleotide primers specific for N- terminal (NR1a, NR1b) and C-terminal splice variants (NR1-1, NR1-2, NR1-3, NR1-4). The developmental profiles of mRNA expression levels of all NR1 isoforms peaked at the end of the third week. Dark rearing led to reductions in both N- and C-terminal NR1 variants in several developmental ages and a significant interaction between age and visual experience was observed for NR1a, NR1-2 and NR1-4 expression. Our results have demonstrated a developmental and visual experience-dependent regulation of NR1 splicing in rat retina.     

Dystrophin splice variants are distinctly localized in the hippocampus

It has previously been demonstrated that Dp71, the most abundant dystrophin protein in the brain, is mainly localized in the postsynaptic densities. Here we show the localization of Dp71f, one of the splice variants of this protein, within the CA3 region of the hippocampus. Immunopositivity occurs in the postsynaptic density of small asymmetrical axospinous and axodendritic synapses, while it is absent in the postsynaptic densities of the axospinous synapses of the large mossy fiber terminals. Dp71f immunoreactivity was found to be attached to the membranes of the mossy fibers in the stratum lucidum of the CA3 area. In a certain population of thin myelinated axons the protein seems to be present within the axon proper. These data support the notion of a physiological role of Dp71f distinct from other dystrophin isoforms present in the central nervous system.     

Short-Range Functional Interaction Between Connexin35 and Neighboring Chemical Synapses

Auditory afferents terminating as mixed, electrical, and chemical, synapses on the goldfish Mauthner cells constitute an ideal experimental model to study the properties of gap junctions in the nervous system as well as to explore possible functional interactions with the other major form of interneuronal communication—chemically mediated synapses. By combining confocal microscopy and freeze-fracture replica immunogold labeling (FRIL), we found that gap junctions at these synapses contain connexin35 (Cx35), the fish ortholog of the neuron-specific human and mouse connexin36 (Cx36). Conductance of gap junction channels at these endings is known to be dynamically modulated by the activity of their co-localized chemically mediated glutamatergic synapses. By using simultaneous pre- and postsynaptic recordings at these single terminals, we demonstrate that such functional interaction takes place in the same ending, within a few micrometers. Accordingly, we also found evidence by confocal and FRIL double-immunogold labeling that the NR1 subunit of the NMDA glutamate receptor, proposed to be a key regulatory element, is present at postsynaptic densities closely associated with gap junction plaques containing Cx35. Given the widespread distribution of Cx35- and Cx36-mediated electrical synapses and glutamatergic synapses, our data suggest that the local functional interactions observed at these identifiable junctions may also apply to other electrical synapses, including those in mammalian brain.     

Short-range functional interaction between connexin35 and neighboring chemical synapses

Auditory afferents terminating as mixed, electrical, and chemical, synapses on the goldfish Mauthner cells constitute an ideal experimental model to study the properties of gap junctions in the nervous system as well as to explore possible functional interactions with the other major form of interneuronal communication--chemically mediated synapses. By combining confocal microscopy and freeze-fracture replica immunogold labeling (FRIL), we found that gap junctions at these synapses contain connexin35 (Cx35), the fish ortholog of the neuron-specific human and mouse connexin36 (Cx36). Conductance of gap junction channels at these endings is known to be dynamically modulated by the activity of their co-localized chemically mediated glutamatergic synapses. By using simultaneous pre- and postsynaptic recordings at these single terminals, we demonstrate that such functional interaction takes place in the same ending, within a few micrometers. Accordingly, we also found evidence by confocal and FRIL double-immunogold labeling that the NR1 subunit of the NMDA glutamate receptor, proposed to be a key regulatory element, is present at postsynaptic densities closely associated with gap junction plaques containing Cx35. Given the widespread distribution of Cx35- and Cx36-mediated electrical synapses and glutamatergic synapses, our data suggest that the local functional interactions observed at these identifiable junctions may also apply to other electrical synapses, including those in mammalian brain.     

Prolonged inhibition of glutamate reuptake down-regulates NMDA receptor functions in cultured cerebellar granule cells

In the present study, we have examined the effects of prolonged (up to 72 h) inhibition of high-affinity glutamate reuptake by L-trans-pyrrolidine-2,4-dicarboxylate (PDC; 100 microM) on glutamate receptor functions in primary cultures of rat cerebellar granule neurons. This was done by comparing the effects of various glutamate receptor agonists on neuronal 45Ca2+ uptake, free cytoplasmic Ca2+ concentration ([Ca2+]i), and cell viability. We also determined the parameters of[3H]MK-801 binding as well as the expression of the NMDAR1 subunit protein in control and PDC-exposed cultures. The blockade of glutamate reuptake by PDC led to a gradual increase of ambient glutamate to concentrations that are neurotoxic when applied acutely to control cells. In PDC-exposed cells, however, the acute glutamate-induced NMDA receptor-mediated calcium fluxes were strongly diminished and no toxicity was observed. The down-regulation of the functional effects of glutamate was dependent on the duration of PDC exposure and was accompanied by a reduced NMDAR1 subunit expression and decreased [3H]MK-801 binding, indicative of a pronounced structural rearrangement of NMDA receptors. The possibility that the decrease of NMDA glutamate receptor sensitivity can be explained on the basis of a reduced density or altered subunit composition of NMDA receptors is discussed.     

Rapid bidirectional switching of synaptic NMDA receptors

Synaptic NMDA-type glutamate receptors (NMDARs) play important roles in synaptic plasticity, brain development, and pathology. In the last few years, the view of NMDARs as relatively fixed components of the postsynaptic density has changed. A number of studies have now shown that both the number of receptors and their subunit compositions can be altered. During development, the synaptic NMDARs subunit composition changes, switching from predominance of NR2B-containing to NR2A-containing receptors, but little is known about the mechanisms involved in this developmental process. Here, we report that, depending on the pattern of NMDAR activation, the subunit composition of synaptic NMDARs is under extremely rapid, bidirectional control at neonatal synapses. This switching, which is at least as rapid as that seen with AMPARs, will have immediate and dramatic consequences on the integrative capacity of the synapse.     

Presynaptic Glutamate Receptors Modulate Dopamine Release from Striatal Synaptosomes

The wide-ranging neuronal actions of glutamate are thought to be mediated by postsynaptic N-methyl-D-aspartate (NMDA) and non-NMDA receptors. The present report demonstrates the existence of presynaptic glutamate receptors in isolated striatal dopaminergic nerve terminals (synaptosomes). Activation of these receptors, by NMDA in the absence of Mg2+ and presence of glycine and by non-NMDA agonists in the presence of Mg2+, results in Ca(2+)-dependent release of dopamine from striatal synaptosomes. The release stimulated by NMDA is blocked by Mg2+ and by selective NMDA antagonists, whereas the release stimulated by selective non-NMDA agonists is blocked by a non-NMDA antagonist but not by Mg2+ or NMDA antagonists. Thus, these presynaptic glutamate receptors, localized on dopaminergic terminals in the striatum, appear to be pharmacologically similar to both the NMDA and the non-NMDA postsynaptic receptors. By modulating the release of dopamine, these presynaptic receptors may play an important role in transmitter interactions in the striatum.     

CaMKII binding to GluN2B is critical during memory consolidation

Memory is essential for our normal daily lives and our sense of self. Ca(2+) influx through the NMDA-type glutamate receptor (NMDAR) and the ensuing activation of the Ca(2+) and calmodulin-dependent protein kinase (CaMKII) are required for memory formation and its physiological correlate, long-term potentiation (LTP). The Ca(2+) influx induces CaMKII binding to the NMDAR to strategically recruit CaMKII to synapses that are undergoing potentiation. We generated mice with two point mutations that impair CaMKII binding to the NMDAR GluN2B subunit. Ca(2+)-triggered postsynaptic accumulation is largely abrogated for CaMKII and destabilized for TARPs, which anchor AMPA-type glutamate receptors (AMPAR). LTP is reduced by 50% and phosphorylation of the AMPAR GluA1 subunit by CaMKII, which enhances AMPAR conductance, impaired. The mutant mice learn the Morris water maze (MWM) as well as WT but show deficiency in recall during the period of early memory consolidation. Accordingly, the activity-driven interaction of CaMKII with the NMDAR is important for recall of MWM memory as early as 24 h, but not 1-2 h, after training potentially due to impaired consolidation.     

Acute knockdown of AMPA receptors reveals a trans-synaptic signal for presynaptic maturation

Newly formed glutamatergic synapses often lack postsynaptic AMPA-type glutamate receptors (AMPARs). Aside from 'unsilencing' the postsynaptic site, however, the significance of postsynaptic AMPAR insertion during synapse maturation remains unclear. To investigate the role of AMPAR in synapse maturation, we used RNA interference (RNAi) to knockdown AMPARs in cultured hippocampal neurons. Surprisingly, loss of postsynaptic AMPARs increased the occurrence of presynaptically inactive synapses without changing the release probability of the remaining active synapses. Additionally, heterologous synapses formed between axons and AMPAR-expressing HEK cells develop significantly fewer inactive presynaptic terminals. The extracellular domain of the AMPAR subunit GluA2 was sufficient to reproduce this effect at heterologous synapses. Indeed, the retrograde signalling by AMPARs is independent of their channel function as RNAi-resistant AMPARs restore synaptic transmission in neurons lacking AMPARs despite chronic receptor antagonist treatment. Our findings suggest that postsynaptic AMPARs perform an organizational function at synapses that exceeds their standard role as ionotropic receptors by conveying a retrograde trans-synaptic signal that increases the transmission efficacy at a synapse.     

Enzyme-linked sensitive fluorometric imaging of glutamate release from cerebral neurons of chick embryos

This paper describes a method for imaging the endogenous release of glutamate from cerebral neurons. This method is based on the reactions of glutamate oxidase and peroxidase, and on the detection of hydrogen peroxide by a fluorescent substrate of peroxidase. Glutamate has been sensitively measured in vitro in the range of 20 nM to 1 microM. We used two types of Ca(2+) channel inhibitors, MK-801 and omega-Conotoxin GVIA, which act to suppress Ca(2+) transport at postsynaptic and presynaptic neurons, respectively. MK-801 did not inhibit the increase in glutamate release after KCl stimulation, while there was no increase in glutamate release after KCl stimulation when omega-Conotoxin GVIA was used, probably due to the inhibition of voltage-activated Ca(2+) channels in the presynapse. Glutamate release and Ca(2+) flow in the synaptic regions were imaged using a laser confocal fluorescence microscope. KCl-evoked glutamate release was localized around cell bodies linked to axon terminals. This procedure allows imaging that can be sensitively detected by the fluorometric enzymatic assay of endogenous glutamate release in synapses.     

Pre- and postsynaptic effects of dopamine antagonists on dopaminergic synaptic transmission in Helix pomatia

The rate of transmitter mobilization in identified dopaminergic synapses was decreased by the dopamine antagonists pimozide, chlorpromazine, haloperidol, cis-flupenthixol, curare, clozapine and high concentrations of ergometrine. The depolarizing postsynaptic potential was inhibited by pimozide, chlorpromazine, haloperidol, cis-flupenthixol, curare, clozapine, (+)-butaclamol and high concentrations of ergometrine. The hyperpolarizing synaptic potential was inhibited by naloxone, methysergide, (+)-butaclamol, haloperidol, 6-hydroxydopamine and low concentrations of ergometrine, while pimozide, cis-flupenthixol, trans-flupenthixol, curare, clozapine, promethazine, chlorpromazine and (-)-butaclamol had no clear effect. The presynaptic receptors involved in modulation of the mobilization rate showed similarities with the dopamine receptors mediating depolarizations. The dopamine antagonists changed dynamics of synaptic transmission.     

Kinetics and subunit composition of NMDA receptors in respiratory-related neurons

NMDA receptors are involved in a variety of brainstem functions. The excitatory postsynaptic NMDA currents of pre-Botzinger complex interneurons and hypoglossal motoneurons, which are located in the medulla oblongata, show remarkably fast deactivation kinetics of approximately 30 ms compared with NMDA receptors in other types of neurons. Because structural heterogeneity might be the basis for physiological properties, we examined the expression of six NMDA receptor subunits (NMDAR1, NR2A-2D, and NR3A) plus eight NMDR1 splice variants in pre-Botzinger complex, hypoglossal and, for comparison, neurons from the nucleus of the solitary tract in young rats using single cell multiplex RT-PCR. Expression of NR2A, NR2B, and NR2D was observed in all three cell types while NR3A was much more abundant in pre-Botzinger complex interneurons, which belong to the rhythm generator of respiratory activity. In hypoglossal neurons, the NMDAR1 splice variants NMDAR1-4a and NMDAR1-4b were found. In neurons of the nucleus of the solitary tract, instead of NMDAR1-4b, the NMDAR1-2a splice variant was detected. This differential expression of modulatory splice variants might be the molecular basis for the characteristic functional properties of NMDA receptors, as neurons expressing a special NMDAR1 splice variant at the mRNA level show fast kinetics compared with neurons lacking this splice variant.     

Clozapine but not haloperidol suppresses the changes in the levels of neuropeptides in MK-801-treated rat brain regions

Noncompetitive NMDA receptor antagonist (+)MK-801 is known to induce neurotoxicity and schizophrenia-like symptomatology where atypical neuroleptic clozapine is effective in contrast to typical neuroleptic, haloperidol. Although neuropeptides are implicated in memory and cognition, their roles in schizophrenia are not well understood. In the present study, we therefore examined the possible roles of neuropeptides, cholecystokinin (CCK) and somatostatin (SS) in the posterior cingulate/retrosplenial cortices (PC/RSC), frontal cortex, and hippocampus of a MK-801-induced schizophrenia-like model rat brain. This study further investigated the pretreated effect of atypical versus typical neuroleptics on the peptidergic system. SS mRNA and peptide levels significantly decreased in the PC/RSC and hippocampus but not in the frontal cortex 3 days after 0.5 mg/kg MK-801 treatment whereas CCK mRNA and peptide levels significantly decreased in all of the brain regions examined. Pretreatment with clozapine but not haloperidol completely recovered the changes in both mRNA and peptide levels of SS and CCK in those brain regions. These data suggest that peptidergic system in the brain presumably plays an important role in the control of negative schizophrenia.     

Pre‐synaptic GABAB receptors inhibit glutamate release through GIRK channels in rat cerebral cortex

Neuronal G protein‐gated inwardly rectifying potassium (GIRK) channels mediate the slow inhibitory effects of many neurotransmitters post‐synaptically. However, no evidence exists that supports that GIRK channels play any role in the inhibition of glutamate release by GABA_B receptors. In this study, we show for the first time that GABA_B receptors operate through two mechanisms in nerve terminals from the cerebral cortex. As shown previously, GABA_B receptors reduces glutamate release and the Ca²⁺ influx mediated by N‐type Ca²⁺ channels in a mode insensitive to the GIRK channel blocker tertiapin‐Q and consistent with direct inhibition of this voltage‐gated Ca²⁺ channel. However, by means of weak stimulation protocols, we reveal that GABA_B receptors also reduce glutamate release mediated by P/Q‐type Ca²⁺ channels, and that these responses are reversed by the GIRK channel blocker tertiapin‐Q. Consistent with the functional interaction between GABA_B receptors and GIRK channels at nerve terminals we demonstrate by immunogold electron immunohistochemistry that pre‐synaptic boutons of asymmetric synapses co‐express GABA_B receptors and GIRK channels, thus suggesting that the functional interaction of these two proteins, found at the post‐synaptic level, also occurs at glutamatergic nerve terminals.     

Ultrastructure and morphometric parameters of glutamatergic synapses on nigrothalamic and unidentified neurons of the reticular zone of theSubstantia nigra in cats

Nigrothalamic neurons were identified into thesubstantia nigra by their retrograde labelling with horseradish peroxidase. Axon terminals that contain glutamate (the excitatory transmitter) were revealed immunocytochemically with an immunogold electron microscopic technique. Ultrastructural parameters (the large and small diameters of axon terminals, area of their profiles, coefficient of form of profiles, large and small diameters of synaptic vesicles) were analyzed in all 240 synapses under study. Synaptic contacts localized on both nigrothalamic and unidentified neurons belonged to three morphologically specific groups. Synapses of the groups I and III, according to classification by Rinvik and Grofova, were characterized by a symmetric type of synaptic contact and contained polymorphic synaptic vesicles. Contacts in group-II synapses were asymmetric, and respective terminals contained round vesicles. Among the studied synapses, 65.8% were classified as group-I contacts, 25.0% belonged to group II, and 9.2% belonged to group III. Glutamate-positive axon terminals formed predominantly group-II synapses; such connections constituted 70% of this group's synapses. Sixty percent of glutamate-positive synapses were localized on the distal dendrites and 23% on the proximal dendrites, while 17% of such synapses were distributed on the somata of nigral neurons. Such a pattern of distribution of glutamate-positive synapses was observed on both nigrothalamic and unidentified nigral neurons. About 7% of glutamate-positive synapses were formed by very large axon terminals containing round synaptic vesicles; yet, the contacts of these terminals were of a symmetric type. Twenty percent of group-I synapses, i.e., synapses considered inhibitory connections, were found to manifest a weak immune reaction to glutamate.Neirofiziologiya/Neurophysiology, Vol. 28, No. 6, pp. 285–295, November–December, 1996.     

AMPA and NMDA receptors: similarities and differences in their synaptic distribution

Recent technical developments, including antigen-retrieval and electron microscopic immunogold methods, are making it possible to determine some of the basic principles governing the subcellular distribution of ionotropic glutamate receptors. Distinct AMPA and NMDA receptor subtypes are selectively targeted to functionally different synapses of a single cell, resulting in an input-selective fine-tuning and regulation of the postsynaptic responses. The amount, density and variability of AMPA receptors at a given glutamatergic synapse is governed by both pre- and postsynaptic factors, resulting in functionally distinct glutamatergic connections that display characteristic patterns of receptor expression.