Decreased numerical density of kainate receptor-positive neurons in the orbitofrontal cortex of chronic schizophrenics

We utilised postmortem brain tissue to quantify sections of left and right orbitofrontal cortex (area 11) from nine schizophrenic and eight control patients from the Charing Cross Prospective Schizophrenia Study immunostained for the presence of the kainate receptor (GluR5/6/7). The numerical density of neurons immunopositive for kainate receptor was measured. Other sections from the same blocks were stained with cresyl violet to determine the total neuronal numerical density. All measurements were made blind: diagnoses were only revealed by a third party after measurements were completed. There was a significant reduction (21%) in numerical density of kainate receptor-positive neurons in both cortices in the schizophrenic group (488 cells/mm²) compared to that in the control group (618 cells/mm²) (P=0.033). Nissl-stained tissue showed no significant difference in total neuronal numerical density between control and schizophrenic groups. These observations suggest that there are actually fewer kainate receptor-positive neurons in schizophrenic orbitofrontal cortex. There was no correlation of reduced kainate receptor-positive cell number with age at death, postmortem interval, or other possibly confounding neuropathology. Our results support the concept of there being reduced glutamatergic activity in frontal cortex in schizophrenia.     

Involvement of glutamatergic receptors in the nucleus cuneiformis in modulating morphine-induced antinociception in rats

The nucleus cuneiformis (CnF), located just ventrolateral to the periaqueductal gray, is part of the descending pain modulatory system. Neurons in the CnF project to medullary nucleus raphe magnus (NRM), which plays an important role on pain modulation. In this study, we investigated the effect of microinjection of the non-competitive NMDA receptor antagonist MK-801, the competitive NMDA receptor antagonist AP-7, and the kainate/AMPA receptor antagonist DNQX, alone or in combination with morphine into the nucleus cuneiformis on morphine-induced analgesia to understand the role of glutamatergic receptors in the modulating activity of morphine. Antinociception was assessed with the tail-flick test. Morphine (10, 20, 40 microg in 0.5 microl saline) had an antinociceptive effect, increasing tail-flick latency in a dose-dependent manner. Microinjection of MK-801 (10 microg/0.5 microl saline) and AP7 (3 microg/0.5 microl saline) prior to morphine microinjection (10 microg/0.5 microl saline) attenuated the antinociceptive effects of morphine, whereas DNQX (0.5 microg/0.5 microl saline) showed a partial antinociceptive effect and potentiated the analgesic effect of morphine. These results indicated that the NMDA receptor partially potentiates the antinociceptive effect of morphine. Our results suggest that NMDA but not non-NMDA receptors are involved in the antinociception produced by morphine in the CnF. The non-NMDA receptors in this area may have a facilitatory effect on nociceptive transmission. The fact that morphine's effect was potentiated by NMDA receptor suggests that projection neurons within the CnF are under tonic, glutamatergic input and when the influence of this input is blocked, the descending inhibitory system is inactivated.     

Chronic ethanol upregulates NMDA and AMPA, but not kainate receptor subunit proteins in rat primary cortical cultures

The present study examined the effects of chronic ethanol exposure on the expression of N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxalone (AMPA) and kainate receptor subunit proteins in rat cortical neuronal cultures grown in media containing 2 mM (high) or 0.1 mM (low) glutamine. Immunoblot analysis of NMDA (NR1, NR2A, NR2B, and NR2D), AMPA (GluR1 and GluR2/3), and kainate (GluR6/7) subunit polypeptides in 3-, 5-, 8-, 10-, and 12 day-old-cultures showed that NMDA receptor subunits NR1, NR2A, and NR2B and AMPA receptor subunits GluR2/3 progressively increased as a function of time, whereas levels of NMDA subunit NR2D were high at day 3 and progressively declined to barely detectable levels by day 12. Levels of AMPA subunit GluR1 and the kainate subunit GluR6/7 remained stable throughout the time course. Replacing the culture media with low glutamine media at culture day 5 did not alter the levels of subunit proteins measured at culture days 9 and 13. However, exposure of low glutamine cultures to 100 mM ethanol for 4 days (starting at culture day 9) significantly increased the levels of NMDA receptor subunits (NR1, NR2A, and NR2B) and AMPA receptor subunits (GluR1 and GluR2/3), but had no effect upon kainate receptor subunits (GluR6/7) or the synapse-associated proteins synapsin I and PSD-95. In contrast, chronic ethanol did not alter the levels of any of these subunit proteins in cells grown in high glutamine. These data demonstrate that under certain experimental conditions, prolonged exposure to ethanol upregulates NMDA and AMPA receptor subunit proteins, but has no effect upon kainate receptor subunit proteins. Because we have previously shown that acute ethanol can inhibit NMDA and AMPA, but not kainate, receptor function in these cultures, the increase in subunit expression likely reflects an adaptive response to the inhibitory effects of ethanol and suggests that both NMDA and AMPA receptors may play an important role in adaptation of the CNS to chronic ethanol.     

Glutamate, kainate and quisqualate enhance GABA-dependent chloride uptake in cortex

Several lines of evidence indicate a possible interaction between the major inhibitory and excitatory cortical neurotransmitters, GABA and glutamate. To assess the neurochemical basis for such an interaction, we examined the effects of glutamate and several analogs on GABA-dependent chloride uptake in a mouse cortical synaptoneurosome preparation. L-Glutamate and the specific receptor subtype ligands kainate and quisqualate led to a small but significant enhancement in chloride uptake in the presence, but not the absence, of the GABA analog muscimol (5 microM). Enhancement was seen at excitatory amino acid (EAA) concentrations of 2-10 microM, but not at higher concentrations. D-Glutamate, NMDA, the NMDA-related antagonists APV and MK801, and the kainate/quisqualate antagonist CNQX, had no effect on chloride uptake. However, CNQX (50 microM) but not APV (50 microM) blocked the increase in chloride uptake due to kainate or quisqualate (10 microM). In addition, depolarization of synaptoneurosomes using high potassium (40 mM KC1) or ouabain pretreatment (5 microM) blocked the effects of kainate and quisqualate. Glutamate, kainate, and quisqualate had no effect on binding at the benzodiazepine, TBPS, or GABA sites on the GABAA receptor complex.     

Phosphorylated neurofilament expression and resistance to kainate toxicity

Antibodies directed against phosphorylated neurofilaments, which are major proteins of the neuronal cytoskeleton, usually do not label neuronal cell bodies except in some neurological diseases. In the present study, we show that in rat cortical cell cultures exposed to kainate there is an inverse relation between neuronal survival and the proportion of neuronal cell bodies stained by a monoclonal antibody (clone SMI₃₁) that recognizes extensively phosphorylated neurofilament proteins (150 kDa and 200 kDa). The immunoblot analysis also revealed an increase in 150-kDa phosphorylated neurofilament expression in kainate-treated cell cultures. Furthermore, the direct quantification of viable neurons SMI₃₁-immunopositive or immunonegative in perikarya showed that the majority of neurons resistant to kainate toxicity expressed phosphorylated neurofilaments in their cell bodies. The percentage of viable neurons displaying SMI₃₁-immunoreactivity in their cell bodies increased from 14.7% in control cultures to 30.0% in cultures treated with 10 μM kainate. These data suggest that phosphorylated neurofilament expression is associated with a reduced cell vulnerability to excitotoxicity induced by kainate.     

Susceptibility of Flinders sensitive and resistant rats to pharmacologically induced seizures

The Flinders sensitive (FSL) and Flinders resistant (FRL) line rats have been selectively bred for hyper- and hyposensitivity to the hypothermie effect of cholinergic agonists respectively. In this study, pilocarpine (250 mg/kg) and physostigmine (0.8 mg/kg) doses that are subconvulsant to outbread Sprague-Dawley rats were systemically injected to the FSL and FRL rats and a heterogenous F2 cross. All of the FRL rats developed severe motor limbic seizures in response to pilocarpine, while none of the FSL animals did. The F2 crosses showed intermediate reaction. The FRL rats were also more affected by physostigmine than the other two groups. However, the FSL rats were confirmed to be more sensitive to the hypothermic effects of pilocarpine (20 mg/kg) and physostigmine (0.6 mg/kg). Picrotoxin and kainic acid produced similiar responses in the both lines, i.e., induced clonk convulsions in some animals when applied in subtreshold doses (2 and 10 mg/kg, respectively). Thus, the normally cholinergicinsensitive rats are more sensitive to the convulsant effects of high doses of cholinergic agonists, but this increased sensitivity does not extend to noncholinergic convulsants.     

Lack of correlation between glutamate-induced depletion of ATP and neuronal death in primary cultures of cerebellum

The aim of this work was to identify, using primary cultures of cerebellar neurons, the receptors involved in glutamate-induced depletion of ATP and to assess whether there is a correlation between glutamate-induced ATP depletion and neuronal death. Glutamate induced a rapid depletion of ATP (40% decrease at 5 min). After 60 min incubation with 1 mM glutamate ATP content decreased by 60–70%. Similar effects were induced by glutamate, NMDA and kainate while quisqualate, AMPA or trans-ACPD did not affect significantly ATP content. The EC₅₀ were 6, 25 and 30 μM for glutamate, NMDA and kainate, respectively. DNQX and AP-5, competitive antagonists of kainate and NMDA receptors, respectively, prevented in a dose-dependent manner the glutamate-induced depletion of ATP. These results indicate that glutamate-induced depletion of ATP is mediated by activation of kainate and NMDA receptors. Glutamate-induced neuronal death was prevented by MK-801, calphostin C, H7, carnitine, nitroarginine and W7. However, only MK-801 and W7 prevented glutamate-induced depletion of ATP, while calphostin C, H7, carnitine and nitroarginine did not. This indicates that there is not a direct correlation between ATP depletion and neuronal death.     

Intracellular acidification and Ca2+ transients in cultured rat cerebellar astrocytes evoked by glutamate agonists and noradrenaline

The effect of different neurotransmitters on the intracellular pH (pH_i) and intracellular calcium (Ca²⁺_i) was studied in cultured astrocytes from neonatal rat cerebellum, using the fluorescent dyes 2,7′-bis(carboxyethyl)-5,6-carboxy-fluorescein (BCECF) and Fura-2. Application of glutamate or kainate (100 μM) in a HEPES-buffered, CO₂/HCO₃^?-free saline induced a decrease in pH_i and an increase in Ca²⁺_i. Amplitude and time course of the pH_i and Ca²⁺_i transients were different. Glutamate and kainate evoked a mean acidification of 0.22 ± 0.05 (n = 29) and 0.20 ± 0.04 (n = 12) pH units, respectively. The changes in pH_i and Ca²⁺_i induced by kainate, but not by glutamate, were inhibited by 6-cyano-7-dinitroquinozalin-2,3-dion (CNQX; 50 μM). In order to elucidate the mechanism of the agonist-induced acidification, whether the pH_i changes were secondary to the Ca²⁺ rises was tested. In the absence of extracellular Ca²⁺, the kainate-induced Ca²⁺_i transient was suppressed, while the intracellular acidification was only reduced by 13%. Removal of extracellular Ca²⁺ reduced the glutamate-induced pH_i change by 8%, while the second component of the Ca²⁺_i transient was abolished. Application of trans-(±)-1-amino-(1S,3R)-cyclopentadicarboxylic acid (t-ACPD, 100 μM), a metabotropic glutamate receptor agonist, and of noradrenaline (20 μM) evoked a Ca²⁺_i increase, but no change of pH_i. D-aspartate, which has a low affinity to glutamate receptors, but is known to be transported by the glutamate uptake system in some astrocytes, evoked an intracellular acidification, similar to that induced by glutamate, but no Ca²⁺_i transient. The results suggest that the kainate-induced acidification is only partly due to the concomitant Ca²⁺_i rise, while the glutamate/aspartate-induced acidification is mainly due to the activation of the glutamate uptake system. © 1995 Wiley-Liss, Inc.