Assessment of Thy-1 mRNA levels as an index of retinal ganglion cell damage.
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PURPOSE: Thy-1 is primarily, if not entirely, expressed by the ganglion cells within the retina. This knowledge was used to index ganglion cell death after ischemia and excitotoxicity by studying changes in Thy-1 mRNA levels. METHODS: Insults to the rat retina were delivered either by elevation of intraocular pressure for 60 minutes or by intravitreal injection of N-methyl-D-aspartate (NMDA). After a defined period, changes in Thy-1 immunoreactivity and mRNA levels of Thy-1 and NR1 (NMDA receptor subunit) were used to index ganglion cell sensitivity to damage. Opsin mRNA levels were used as an internal control because photoreceptors lack NMDA receptors. RESULTS: Retinal Thy-1 immunoreactivity, associated with the ganglion cell and inner plexiform layers, is reduced by ischemia or intravitreal injections of NMDA in a dose-dependent manner. Using a semi-quantitative polymerase chain reaction (reverse transcription-polymerase chain reaction) methodology, the levels of total retinal Thy-1 and NR1 mRNAs were shown to be dramatically reduced after both transient ischemia and intravitreal injection of NMDA. The effect of NMDA was found to be both time- and dose-dependent. In contrast, no change occurred in the levels of opsin mRNA unless high levels of NMDA (200 nmoles) were administered. CONCLUSIONS: Ischemia and NMDA-induced excitotoxicity caused retinal ganglion cell destruction, but the photoreceptors were unaffected. Measurement of total retinal Thy-1 mRNA levels provides a useful way of following ganglion cell death especially when combined with immunohistochemical localization of Thy-1. Additionally, the effect on other retinal cell types such as the photoreceptors can be followed in concert using this technique. (+info)
Enhanced NR2A subunit expression and decreased NMDA receptor decay time at the onset of ocular dominance plasticity in the ferret.
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Enhanced NR2A subunit expression and decreased NMDA receptor decay time at the onset of ocular dominance plasticity in the ferret. The NMDA subtype of glutamate receptor is known to exhibit marked changes in subunit composition and functional properties during neural development. The prevailing idea is that NMDA receptor-mediated synaptic responses decrease in duration after the peak of cortical plasticity in rodents. Accordingly, it is believed that shortening of the NMDA receptor-mediated current underlies the developmental reduction of ocular dominance plasticity. However, some previous evidence actually suggests that the duration of NMDA receptor currents decreases before the peak of plasticity. In the present study, we have examined the time course of NMDA receptor changes and how they correlate with the critical period of ocular dominance plasticity in the visual cortex of a highly binocular animal, the ferret. The expression of NMDA receptor subunits NR1, NR2A, and NR2B was examined in animals ranging in age from postnatal day 16 to adult using Western blotting. Functional properties of NMDA receptors in layer IV cortical neurons were studied using whole cell patch-clamp techniques in an in vitro slice preparation of ferret primary visual cortex. We observed a remarkable increase in NR1 and NR2A, but not NR2B, expression after eye opening. The NMDA receptor-mediated synaptic currents showed an abrupt decrease in decay time concurrent with the increase in NR2A subunit expression. Importantly, these changes occurred in parallel with increased ocular dominance plasticity reported in the ferret. In conclusion, molecular changes leading to decreased duration of the NMDA receptor excitatory postsynaptic current may be a requirement for the onset, rather than the end, of the critical period of ocular dominance plasticity. (+info)
Glutamatergic projection to RVLM mediates suppression of reflex bradycardia by parabrachial nucleus.
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We investigated the role of glutamatergic projection from the parabrachial nucleus (PBN) complex to the rostral ventrolateral medulla (RVLM) in the PBN-induced suppression of reflex bradycardia in adult Sprague-Dawley rats that were maintained under pentobarbital anesthesia. Under stimulus conditions that did not appreciably alter the baseline systemic arterial pressure and heart rate, electrical (10-s train of 0.5-ms pulses, at 10-20 microA and 10-20 Hz) or chemical (L-glutamate, 1 nmol) stimulation of the ventrolateral regions and Koelliker-Fuse (KF) subnucleus of the PBN complex significantly suppressed the reflex bradycardia in response to transient hypertension evoked by phenylephrine (5 micrograms/kg iv). The PBN-induced suppression of reflex bradycardia was appreciably reversed by bilateral microinjection into the RVLM of the N-methyl-D-aspartate (NMDA)-receptor antagonist MK-801 (500 pmol) or the non-NMDA-receptor antagonist 6-cyano-7-nitroquinoxaline-2, 3-dione (50 pmol). Anatomically, most of the retrogradely labeled neurons in the ventrolateral regions and KF subnucleus of the ipsilateral PBN complex after microinjection of fast blue into the RVLM were also immunoreactive to anti-glutamate antiserum. These results suggest that a direct glutamatergic projection to the RVLM from topographically distinct regions of the PBN complex may participate in the suppression of reflex bradycardia via activation of both NMDA and non-NMDA receptors at the RVLM. (+info)
Generation of rhythmic patterns of activity by ventral interneurones in rat organotypic spinal slice culture.
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1. In the presence of certain excitatory substances the rat isolated spinal cord generates rhythmic oscillations believed to be an in-built locomotor programme (fictive locomotion). However, it is unknown whether a long-term culture of the same tissue can express rhythmic activity. Such a simplified model system would provide useful data on the minimal circuitry involved and the cellular mechanisms mediating this phenomenon. For this purpose we performed patch clamp recording (under whole-cell voltage or current clamp conditions) from visually identified ventral horn interneurones of an organotypic slice culture of the rat spinal cord. 2. Ventral horn interneurones expressed rhythmic bursting when the extracellular [K+] was raised from 4 to 6-7 mM. Under voltage clamp this activity consisted of composite synaptic currents grouped into bursts lasting 0.9 +/- 0.5 s (2.8 +/- 1.5 s period) and was generated at network level as it was blocked by tetrodotoxin or low-Ca2+-high-Mg2+ solution and its periodicity was unchanged at different potential levels. 3. In current clamp mode bursting was usually observed as episodes comprising early depolarizing potentials followed by hyperpolarizing events with tight temporal patterning. Bursting was fully suppressed by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and reduced in amplitude and duration by N-methyl-D-aspartate (NMDA) receptor antagonism without change in periodicity. Extracellular field recording showed bursting activity over a wide area of the ventral horn. 4. Regular, rhythmic activity similar to that induced by K+ also appeared spontaneously in Mg2+-free solution. The much slower rhythmic pattern induced by strychnine and bicuculline was also accelerated by high-K+ solution. 5. The fast and regular rhythmic activity of interneurones in the spinal organotypic culture is a novel observation which suggests that the oversimplified circuit present in this culture is a useful model for investigating spinal rhythmic activity. (+info)
Factors that enhance ethanol inhibition of N-methyl-D-aspartate receptors in cerebellar granule cells.
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The objective of this study was to identify factors that influence ethanol (EtOH) inhibition of the N-methyl-D-aspartate receptor (NMDAR) in primary cultured cerebellar granule cells. Several factors contributing to the inhibitory effects of EtOH on NMDAR function were assessed using both whole-cell and perforated patch-clamp recordings. The NMDAR subunit composition was examined by Western blot analysis using NR2 subunit-specific antibodies and pharmacological manipulation with the NR2B-specific antagonist infenprodil. Western blot analysis indicated that NMDAR subunit composition changed from a combination of NR2A and NR2B containing NMDARs to primarily NR2A with increasing days in vitro (DIV). Although the NR2B subunit was detectable until 21 DIV, there was a significant decrease in ifenprodil sensitivity after 7 DIV. EtOH sensitivity did not change with an increasing DIV. A high concentration of glycine reversed EtOH inhibition of steady-state, but not peak, NMDA-induced current during whole-cell recordings. Significant glycine reversal of effects of a low concentration of EtOH on peak current was observed under perforated patch-clamp conditions. A 30-s EtOH pretreatment significantly enhanced EtOH inhibition of NMDA-induced peak current. Collectively, these results indicate that EtOH sensitivity of the NMDAR in primary cultured cerebellar granule cells is not related to subunit composition nor ifenprodil sensitivity, involves a kinetic interaction with glycine, and can be enhanced by a slowly developing transduction mechanism that occurs within tens of seconds. (+info)
Zinc and ifenprodil allosterically inhibit two separate polyamine-sensitive sites at N-methyl-D-aspartate receptor complex.
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In this study, we investigated the hypothesis that inhibition of the N-methyl-D-aspartate (NMDA) receptor complex by zinc involves a polyamine-sensitive regulatory site. We found that the specific binding of the open channel ligand [3H]MK-801 to rat hippocampal membranes 1) was inhibited by low concentrations of Zn2+ (IC50 = 5.5 microM) by 65%. 2) This high-affinity component of inhibition was reversed by the polyamine spermine to an extent that could be reconciled with competitive interaction between Zn2+ and spermine. 3) Partial inhibition by Zn2+ was additive with partial inhibition by ifenprodil, an inhibitor of the NMDA receptor complex supposed to act at a polyamine-sensitive regulatory site, and 4) in membranes prepared from several other brain regions, inhibition of [3H]MK-801 binding by Zn2+ and by ifenprodil was either less than additive, or superadditive. Our observation that ifenprodil, at concentrations saturating its high-affinity component of inhibition, prevented spermine from reversing the inhibition by Zn2+ indicates that spermine did not increase [3H]MK-801 binding by competition with Zn2+ but rather via another polyamine regulatory site not sensitive to zinc but sensitive to ifenprodil. We conclude that Zn2+ reduces channel opening of the NMDA receptor complex by allosteric inhibition of a polyamine-sensitive regulatory site different from that inhibited by ifenprodil and that these two allosteric sites influence each other in a manner dependent on the brain region investigated. The different proportions of zinc/ifenprodil inhibition in different regions could reflect different percentages of various NMDA receptor subtypes. (+info)
Characterization of MALS/Velis-1, -2, and -3: a family of mammalian LIN-7 homologs enriched at brain synapses in association with the postsynaptic density-95/NMDA receptor postsynaptic complex.
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Protein assembly at the postsynaptic density (PSD) of neuronal synapses is mediated in part by protein interactions with PSD-95/discs large/zona occludens-1 (PDZ) motifs. Here, we identify MALS-1, -2, -3, a family of small synaptic proteins containing little more than a single PDZ domain. MALS-1, -2, and -3 are mammalian homologs LIN-7, a Caenorhabditis elegans protein essential for vulval development. In contrast to functions for LIN-7 in epithelial cells, MALS-1 and -2 are selectively expressed in specific neuronal populations in brain and are enriched in PSD fractions. In cultured hippocampal neurons, MALS proteins are clustered together with PSD-95 and NMDA type glutamate receptors, consistent with a postsynaptic localization for MALS proteins. Immunoprecipitation and affinity chromatography studies readily identify association of MALS with PSD-95 and an NMDA receptor subunit. The PDZ domain of MALS selectively binds to peptides terminating in E-T/S-R/X-V/I/L, which corresponds to the C terminus of NMDA type 2 receptors and numerous other ion channels at the PSD. This work suggests a role for MALS proteins in regulating recruitment of neurotransmitter receptors to the PSD. (+info)
During cortical development, embryonic neurons migrate from germinal zones near the ventricle into the cortical plate, where they organize into layers. Mechanisms that direct neuronal migration may include molecules that act as chemoattractants. In rats, GABA, which localizes near the target destination for migrating cortical neurons, stimulates embryonic neuronal migration in vitro. In mice, glutamate is highly localized near the target destinations for migrating cortical neurons. Glutamate-induced migration of murine embryonic cortical cells was evaluated in cell dissociates and cortical slice cultures. In dissociates, the chemotropic effects of glutamate were 10-fold greater than the effects of GABA, demonstrating that for murine cortical cells, glutamate is a more potent chemoattractant than GABA. Thus, cortical chemoattractants appear to differ between species. Micromolar glutamate stimulated neuronal chemotaxis that was mimicked by microM NMDA but not by other ionotropic glutamate receptor agonists (AMPA, kainate, quisqualate). Responding cells were primarily derived from immature cortical regions [ventricular zone (vz)/subventricular zone (svz)]. Bromodeoxyuridine (BrdU) pulse labeling of cortical slices cultured in NMDA antagonists (microM MK801 or APV) revealed that antagonist exposure blocked the migration of BrdU-positive cells from the vz/svz into the cortical plate. PCR confirmed the presence of NMDA receptor expression in vz/svz cells, whereas electrophysiology and Ca2+ imaging demonstrated that vz/svz cells exhibited physiological responses to NMDA. These studies indicate that, in mice, glutamate may serve as a chemoattractant for neurons in the developing cortex, signaling cells to migrate into the cortical plate via NMDA receptor activation. (+info)