Polysaccharide glucomannan isolated from Heterodermia obscurata attenuates acute and chronic pain in mice.

Glucomannan (GM) is a polysaccharide obtained from Heterodermia obscurata lichens. The present study was conducted to elucidate the antinociceptive effect of GM in behavioural models of acute and chronic pain in mice. GM reduced mechanical allodynia and the levels of interleukin 1-ß (IL-1ß) in spinal cord and nerve in the partial sciatic nerve ligation (PSNL) model. Systemic treatment with GM inhibited the nociception induced by intraplantar injection of glutamate and by intrathecal injection of N-methyl-d-aspartic acid (NMDA), (±)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD), tumour necrosis factor α (TNF-α) and IL-1ß. Taken together, our data demonstrate that GM has significant antinociceptive effect in acute and chronic pain, suggesting a potential interest in the development of new clinically relevant drugs for the management of pain.

Glutamate-induced temporomandibular joint pain in healthy individuals is partially mediated by peripheral NMDA receptors.

AIM: To determine if glutamate injected into the healthy temporomandibular joint (TMJ) evokes pain through peripheral N-methyl-D-aspartate (NMDA) receptors and if such pain is influenced by sex or sex steroid hormones. METHODS: Sixteen healthy men and 36 healthy women were included and subjected to two randomized and double-blind intra-articular injections of the TMJ. Experimental TMJ pain was induced by injection of glutamate (1.0 mol/L) and NMDA block was achieved by co-injection of the NMDA antagonist ketamine (10 mmol/L). The TMJ pain intensity in the joint before and during a 25-minute postinjection period was continuously recorded on an electronic visual analog scale (0 to 10). Estradiol, progesterone, and testosterone levels in serum were analyzed. RESULTS: Glutamate-induced pain showed a median (25/75 percentile) duration of 8.3 (5.2/12.2) minutes. The peak pain intensity was 6.1 (4.2/8.2), the time to peak was 50 (30/95) seconds, and the area under the curve was 59 (29/115) arbitrary units. The women reported higher maximum pain intensity than the men and shorter time to peak. The sex hormone levels were not significantly related to the glutamate-induced TMJ pain. NMDA block significantly reduced the glutamate-induced TMJ pain, mainly in the women. There were no significant correlations between sex hormone levels and the effects of NMDA block for any pain variable. CONCLUSION: Glutamate evokes immediate pain in the healthy human TMJ that is partly mediated by peripheral NMDA receptors in the TMJ.

Early exposure of cultured hippocampal neurons to excitatory amino acids protects from later excitotoxicity.

Status epilepticus occurring in early postnatal development protects CA1 hippocampal neurons, the region most sensitive to seizure-induced injury in the developing brain. Here, we developed a "two hit" model in dissociated cultures of the rat hippocampus to test whether pre-exposure of immature neurons to high concentrations of glutamate, N-methyl-D-aspartic acid (NMDA) or alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) during a relatively resistant period prevents neurons from dying following a second exposure to the same chemicals after neurons mature and become highly vulnerable to excitatory amino acids (EAAs). Cultures were exposed to varied doses of glutamate, NMDA, or AMPA for 48 h at 5 DIV and again at 14 DIV for 5, 15, or 30 min. NeuN immunohistochemistry showed early exposure to glutamate (500 microM) killed approximately half of the neurons (52+/-8.6%) compared to the marked depletion that occurs after one exposure at 14 DIV (98+/-0.79%). When cultures were first challenged with moderate doses of glutamate (200 microM) followed by the high dose 7 days later, a significant population of neurons was spared (35.3+/-1.2%). Similarly, pre-exposure to maximal doses of NMDA (100 microM) increased the proportion of surviving cells following the second challenge. In contrast, AMPA (100 microM) was equally toxic after early or late applications and did not protect from the second exposure. GluR1 subunit expression was markedly decreased at 48 h after one or two exposures to 200 microM glutamate (by 44.57+/-3.6%, 45.07+/-3.69%) whereas GluR2 subunit expression was reduced by a lesser amount (25.7 57+/-3.8%). Confocal microscopy showed that one or two exposures to NMDA caused GluR2 protein to downregulate even further whereas parvalbumin (PV) was dramatically increased in the same neurons by over four-fold. On the other hand, calbindin (CB) immunoreactivity was nearly absent after the first exposure to 500 microM glutamate. These data indicate that early, transient exposure to certain EAAs at high doses can induce long-lasting neuroprotection. Alterations in the GluR1/GluR2 ratio as well as differential expression of specific calcium binding proteins may contribute to this neuroprotection.

Method for the construction and use of carbon fiber multibarrel electrodes for deep brain recordings in the alert animal.

Microiontophoresis of neuroactive substances during single unit recording in awake behaving animals can significantly advance our understanding of neural circuit function. Here, we present a detailed description of a method for constructing carbon fiber multibarrel electrodes suitable for delivering drugs while simultaneously recording single unit activity from deep structures, including brainstem nuclei and the cerebellum, in the awake behaving primate. We provide data that should aid in minimizing barrel resistance and the time required to fill long, thin multibarrel electrodes with solutions. We also show successful single unit recording from a variety of areas in the awake squirrel monkey central nervous system, including the vestibular nuclei, Interstitial Nucleus of Cajal, and the cerebellum. Our descriptions and data should be useful for investigators wishing to perform single unit recordings during microiontophoresis of neuroactive substances, particularly in deep structures of animals with chronically implanted recording chambers.

Effect of a metabotropic glutamate receptor 5 antagonist, MPEP, on the nociceptive response induced by intrathecal injection of excitatory aminoacids, substance P, bradykinin or cytokines in mice.

Metabotropic glutamate receptors (mGluRs) are expressed abundantly in the spinal cord and have been shown to play important roles in the modulation of nociceptive transmission and plasticity. In this study, the involvement of metabotropic glutamate receptor 5 (mGluRs) in the nociceptive response induced by intrathecal injection (i.t.) of excitatory aminoacids, substance P (SP), bradykinin (BK) and cytokines in mice was demonstrated. The administration of 2-methyl-6-(phenylethynyl)-pyridine (MPEP; 10-50 nmol/site, i.t.) caused a significant inhibition in the nociceptive response induced by glutamate and trans-ACPD with maximal inhibitory effects of 36 +/- 7% and 56 +/- 5%, respectively. MPEP completely failed to affect the nociception induced by alpha-amino-3-hydroxy-5-mehtyl-4-isoxazolepropionic acid (AMPA; 135 pmol/site), kainate (110 pmol/site) and N-methyl-D-aspartate (NMDA; 450 pmol/site). MPEP also reduced the nociceptive response induced by SP (135 ng/site, i.t.), BK (0.1 microg/site), tumor necrosis factor-alpha (TNF-alpha; 0.1 pg/site) and interleukin-1beta (IL-1beta; 1 pg/site) with maximal inhibitions of 29 +/- 5%, 37 +/- 5%, 83 +/- 3% and 88 +/- 1%, respectively. Together, these results indicate the involvement of mGluRs, more specifically of subtype-5, in the nociceptive response induced by i.t. injection of excitatory aminoacids, SP, BK and cytokines in mice.

Mapping of chemical trigger zones for reward.

Addictive drugs are thought to activate brain circuitry that normally mediates more natural rewards such as food or water. Drugs activate this circuitry at synaptic junctions within the brain; identifying the junctions at which this occurs provides clues to the neurochemical and anatomical characteristics of the circuitry. One approach to identifying the junctions at which drugs interact with this circuitry is to determine if animals will lever-press for site-specific microinjections of addictive drugs. This approach has identified GABAergic, dopaminergic, glutamatergic, and cholinergic trigger zones within meso-corticolimbic circuitry important for natural reward function.

Microglial expression of prostaglandin EP3 receptor in excitotoxic lesions in the rat striatum.

Prostaglandins (PG) are produced by the enzymatic activity of cyclooxygenase (COX). PGs and COX have been implicated in the pathophysiology of excitotoxicity and neurodegeneration in the central nervous system (CNS). The PGE2 receptor EP3 is the most abundantly expressed PGE2 receptor subtype in the brain. So far, in the innate rat brain EP3 receptors have been found exclusively in neurons. The aim of this study was to investigate whether EP3 expression in the brain changes under neurodegenerative circumstances such as an acute excitotoxic lesion. Intrastriatal injection of quinolinic acid (QUIN) resulted in a loss of EP3-positive striatal neurons, while simultaneously small glial-shaped EP3-positive cells appeared. Five days after lesioning, 63% of the glial-shaped EP3-positive cells could be identified as ED-1 expressing microglial cells. This percentage increased to 82% after 10 days, suggesting that most of the EP3-positive ED-1-negative cells on day 5 may be microglia which did not yet express ED-1. ED-1-positive microglia also expressed COX-1. These experiments show for the first time that activated microglial cells in excitotoxic lesions express in vivo the PGE2 receptor EP3 and the PGE2 synthesizing enzyme COX-1. Activation of EP3 receptor downregulates cAMP formation and may counteract the upregulation of cAMP formation via EP2 receptors, which has been linked to the anti-inflammatory effects of PGs. This change in EP3-receptor expression in microglia might participate in acute or chronic microglial activation in a variety of brain diseases such as ischemia or Alzheimer's disease (AD). Investigation of the expression of different PGE2 receptor subtypes might promote a better understanding of the pathophysiology of these diseases as well as leading to a modulation of microglial activation by a more specific interference with selective EP receptors than can be achieved by inhibiting global PG synthesis by selective or non-selective COX inhibitors.

Cholinergic modulation of neuron spike responses to dendritic and somatic application of excitatory amino acids.

The effects of acetylcholine on the spike discharges of neurons induced by iontophoretic application of excitatory amino acids to the bodies and dendrites of cells were studied in 98 neurons in living slices of guinea pig parietal cortex. Acetylcholine applied microiontophoretically to both the bodies and dendrites facilitated improvements in the parameters of responses induced by dendritic activation, with significant decreases in latent periods and increases in the intensity and duration of responses. Thee effects were stably induced at distances of 300 microm from the body and lasted 1 min after exposure to acetylcholine ended. Responses induced by application of excitatory amino acids directly to the cell body did not change significantly in the presence of acetylcholine regardless of the point on the membrane at which they were applied. It is concluded that the predominant effect of acetylcholine is on the efficiency of dendrosomatic conduction.

Estrogen-induced neurochemical and electrophysiological changes in the parabrachial nucleus of the male rat.

Estrogen has previously been shown to significantly change sympathetic and parasympathetic system output via an action within the central nuclei responsible for regulating autonomic tone. These estrogen-induced changes were observed within 30 min of systemic administration and could be blocked by the direct microinjection of the estrogen receptor antagonist, ICI 182780, into the parabrachial nucleus (PBN) of the pons. In the present investigation, we sought to determine the possible mechanism(s) by which estrogen produced these rapid changes in autonomic tone by determining if estrogen modulates neuronal excitability within the PBN. Male Sprague-Dawley rats were anaesthetized with Inactin (sodium thiobutabarbitol, 100 mg/kg) and instrumented for the intravenous injection of estrogen and placed in a stereotaxic frame for the insertion of a microdialysis probe or glass recording electrode into the PBN. In the first experiment, we sought to determine the local concentration of estrogen in the cerebrospinal fluid in the PBN following systemic injection of estrogen. In the second experiment, we sought to determine the functional significance of systemic estrogen injection on neuronal activity and amino acid neurotransmitter levels in the PBN. Systemic estrogen injection resulted in a significant increase in local estrogen concentration in the PBN which corresponded to a decrease in neuronal excitability and extracellular glutamate levels while increasing GABA levels in the PBN. These results suggest that estrogen decreases neuronal excitability in the PBN by modulating synaptic transmission via an increased release of GABA and a decreased release of glutamate.

17beta-estradiol inhibits excitatory amino acid-induced activity of neurons of the nucleus tractus solitarius.

The effects of 17beta-estradiol (17betaE2) on spontaneous and excitatory amino acid (EAA) induced nucleus tractus solitarius (NTS) neuronal activity were investigated by electrophysiological and immunohistochemical experiments in ovariectomized female Sprague-Dawley rats. Out of 62 NTS neurons tested, 42 were inhibited (68%) following iontophoretic application of 17betaE2 in a current-dependent manner. The averaged firing rate decreased from 3.06+/-0.40 to 0.78+/-0.17 Hz. The inhibitory responses were rapid in onset (within 1 min) and variable in duration (2-4 min). The inhibitory effects of 17betaE2 were blocked by simultaneously applied 17betaE2 antagonist ICI182,780, but not by GABA antagonist, bicuculline and phaclofen. L-Glutamate, AMPA or NMDA enhanced the activity of 71, 73 or 69% of NTS cells tested, respectively. The excitatory effects of EAA were significantly inhibited in the presence of 17betaE2. Fluorescent immunohistochemistry revealed that all subnuclei of the NTS contained high levels of estrogen receptors (ERs) immunoreactivity. These results suggest that 17betaE2 inhibits spontaneous and EAA-induced NTS neuronal activity through 17betaE2 activation of ERs.

Evidence for the involvement of glutamatergic receptors in the antinociception caused in mice by the sesquiterpene drimanial.

Drimanial, a new sesquiterpene isolated from the barks of the plant Drimys winteri (Winteraceae), given systemically, intraplantarly, or by spinal or supraspinal routes, produced pronounced antinociception against both phases of formalin-induced licking. The systemic injection of drimanial also inhibited, in a graded manner, the pain-related behaviours induced by intraplantar or intrathecal (i.t.) administration of glutamate. Moreover, drimanial also caused marked inhibition of the nociception induced by i.t. administration of a metabotropic glutamate agonist (1S,3R)-ACPD, without affecting nociceptive responses induced by ionotropic agonists (NMDA, kainate, AMPA) or by substance P. The antinociception caused by drimanial was not influenced by naloxone, nor did it interfere with the motor coordination of animals in the rota-rod test. Furthermore, drimanial caused graded inhibition of [(3)H]glutamate binding in cerebral cortical membranes from mice, with an IC(50) value of 4.39 micro M. Together, these results provide strong evidence indicating that the sesquiterpene drimanial produces antinociception in mice at peripheral, spinal and supraspinal sites. An interaction with metabotropic glutamate receptors seems to contribute to the mechanisms underlying its antinociceptive action.

Prospects for relevant glaucoma models with retinal ganglion cell damage in the rodent eye.

Retinal ganglion cell (RGC) death is the end result of practically all diseases of the optic nerve, including glaucomatous optic neuropathy. Understanding the factors determining susceptibility of the retina or the optic nerve to glaucomatous damage, and the means to prevent it, requires good animal models. Here we review the different, current models in rodents that have been used to study RGC damage, discuss their value, and their adequacy as models for human glaucoma.

Regulation of limbic information outflow by the subthalamic nucleus: excitatory amino acid projections to the ventral pallidum.

The subthalamic nucleus (STN), a component of the basal ganglia motor system, sends an excitatory amino acid (EAA)-containing projection to the ventral pallidum (VP), a major limbic system output region. The VP contains both NMDA and AMPA subtypes of EAA receptors. To characterize the physiology of the subthalamic pathway to the VP, and to determine the influence of EAA receptor subtypes, in vivo intracellular recordings, and in vivo extracellular recordings combined with microiontophoresis, were made from VP neurons in anesthetized rats. Of the intracellularly recorded neurons, 86% responded to STN stimulation, and these displayed EPSPs with an onset of 8.7 msec, consistent with a monosynaptic input. The EPSPs evoked in spontaneously firing neurons were nearly twice the amplitude of those in nonfiring cells (13.1 vs 6.8 mV, respectively). As neurons were depolarized by current injection, the latency for spiking decreased from 24.2 to 14.2 msec, although EPSP latency was unaffected. Eighty-seven percent of the extracellularly recorded VP neurons responded to STN stimulation with a rapid and robust enhancement of spiking; the response onset, like the EPSP onset, equaled 8.7 msec. Firing rate was enhanced by NMDA in 94% of the STN-excited cells, and AMPA increased firing in 94% as well. The NMDA-selective antagonist AP-5 attenuated 67% of the STN-evoked excitatory responses, and the AMPA-selective antagonist CNQX attenuated 52%. Both antagonists attenuated 33% of responses, and 78% were attenuated by at least one. This evidence suggests that a great majority of VP neurons are directly influenced by STN activation and that both NMDA and non-NMDA receptors are involved. Moreover, the VP response to STN stimulation appears to be strongly dependent on the depolarization state of the neuron.

Effects of excitatory amino acids and nitric oxide on flight behavior elicited from the dorsolateral periaqueductal gray.

Microinjection of excitatory amino acids (EAA) into the dorsolateral periaqueductal gray (dlPAG) induces flight reactions while EAA antagonists show anxiolytic effects. Part of the effects mediated by NMDA receptors may involve an increase in nitric oxide (NO) production. We showed that nitric oxide synthase (NOS) inhibitors injected into the dlPAG induced anxiolytic effects. Conversely, SIN-1, a NO donor, produced orientated flight reactions that resemble stimulation of the medial hypothalamus. This compound also produced extensive Fos-like immunoreactivity in this region and in other areas related to defensive reactions such as the medial amygdala and cingulate cortex. Since part of the effects of NO involves increases in guanylate cyclase levels, we found that intra-dlPAG injection of 8-Br-cGMP induced a brief flight reaction followed by increased locomotion. In another experiment, we showed that single or repeated restraint stress produced an increased expression of neuronal NOS in the dlPAG and other areas related to defense, as measured by in situ hybridization, diaphorase histochemistry and immunocytochemistry. Together, these data suggest that NO may participate in the modulation of defensive responses in the dlPAG.

The role of nitric oxide and prostaglandin E2 on the hyperalgesia induced by excitatory amino acids in rats.

The present study was designed to investigate the role of nitric oxide (NO), N-methyl-D-aspartate (NMDA) receptor and prostaglandins on hyperalgesia induced in rats by excitatory amino acids and the possibility that prostaglandins may act as the retrograde messenger in the spinal cord like NO. Nomega-nitro-L-arginine methyl ester (L-NAME; 500 microg/paw, intraplantarly (i.pl.)), MK-801 (10 microg/paw, i.pl.) or indomethacin (300 microg/paw, i.pl.) reduced the duration of phase 2 of the biting/licking and scratching (B/L + S) response induced by formalin injection from 255.6+/-16.7 s to 155.6+/-16.9, 172.25+/-33.3 or 205.6+/-16.7 s, respectively. L-NAME (0.3 mg, i.th.), MK-801 (8 microg, i.th.) or indomethacin (20 microg, i.th) reduced the duration of phase 2 of the B/L + S response induced by saline injection from 288.5+/-7.7s to 207.7+/-19.2, 184.6+/-7.7 or 1923+/-38.5 s, respectively. L-NAME or indomethacin injected into the spinal cord of the rat significantly reduced the hyperalgesia induced by NMDA (1 microg, i.th.) from 43.8+/-4.6% to 12.3+/-3.1 and 19.2+/-2.3%, respectively. It is assumed that NO produced by excitatory amino acids may increase prostaglandin production by cyclooxygenase activation. L-NAME, MK-801 or indomethacin injected into the rat spinal cord significantly reduced the hyperalgesia induced by prostaglandin E2 (PGE2, 25 ng, i.th.) in the tail-flick test from 40.6+/-3.5% to 18.2+/-3.2, 18.8+/-1.8 or 17.6+/-4.1%, respectively, but had little effect on hyperalgesia in the paw pressure test (except for indomethacin). In conclusion, NO and PGE2 affect the hyperalgesia induced by excitatory amino acids. It is suggested that PGE2, like NO, may act as a retrograde messenger in the spinal cord.

Right hemisphere involvement in imprinting memory revealed by glutamate treatment.

The lateralized use of the forebrain hemispheres during recall of imprinting memory was investigated using unilateral intrahemispheric injections of glutamate. Administration of glutamate to the right hemisphere 1.3, or 6 h after exposure to the imprinting stimulus disrupted recall 8 h after the end of training, whereas the same treatment of the left hemisphere had no effect. Imprinted chicks treated with glutamate injected into the right hemisphere did not approach the imprinting stimulus in preference to an alternative, unfamiliar stimulus during a simultaneous choice test, whereas imprinted chicks treated with glutamate injected into the left hemisphere showed a preference for the imprinting stimulus. Thus, the left and right hemispheres are involved differentially in the recall of imprinting memory. Fear behavior or activity levels were not altered by glutamate treatment of either the right or left hemisphere, indicating that the effects of glutamate were specific to recall of imprinting memory. However, the amnestic effect of treatment of the right hemisphere with glutamate was transient: it was no longer evident by 48 h after the end of training. Also, glutamate had no effect when the chicks were treated 9 h after the end of training. These results suggest that regions in right hemisphere of the chick brain are involved in early (0-8 h after training) recall of imprinting memory.

Effects of chronic lithium treatment on ornithine decarboxylase induction and excitotoxic neuropathology in the rat.

Young adult rats were chronically treated with lithium (2.5 mmol/kg/day) for 16 days. The day after the last lithium administration, rats were injected s.c. with the excitotoxic convulsant kainic acid (10 mg/kg). As compared to saline controls, lithium-treated rats had no apparent attenuation of convulsions. Furthermore, the induction of brain ornithine decarboxylase and the consequent increase of putrescine levels, an index related to the convulsant effects of kainic acid, were similar in saline- and lithium-treated rats. Other rats were unilaterally injected with ibotenic acid into the nucleus basalis magnocellularis: no differences were measured in cortical choline acetyltransferase (ChAT) decrease among saline- and lithium-treated rats. In both the above experiments, apoptotic cell death was monitored in relevant brain regions of saline- or lithium-treated rats through a specific in situ labeling method for fragmented DNA. Whilst morphological evidence for a reduced damage in the olfactory cortex and hippocampus of kainic acid-injected rats was not obtained, lithium-treated rats showed a lower decrease of specific neurochemical markers: [3H]D-aspartate uptake and glutamate decarboxylase. This result suggests that mechanisms of recovery, absent in saline-treated animals, are elicited by the excitotoxic insult in lithium-treated rats.

In vivo electrochemical studies of the dynamic effects of locally applied excitatory amino acids in the striatum of the anesthetized rat.

The actions of locally applied excitatory amino acids (EAAs) on dopamine nerve terminals in the striatum of the urethane-anesthetized rat were investigated. Rapid (5 Hz) in vivo electrochemical recording was used to measure the amplitude and duration of dopamine (DA) overflow elicited by the local application of glutamate, N-methyl-D- aspartate (NMDA), kainate, quisqualate, quinolinate and DL- alpha-amino-3-hydroxy-5-methylisoxazolepropionic acid. EAAs were pressure ejected into the striatum via a pipette positioned 300 +/- 30 mum away from the electrochemical working electrode. Brief (5s) applications of the EAA agonists directly elicited DA-like electrochemical signals with amplitudes averaging about 2 microM. In some instances, putative increases in ascorbic acid-like signals were detected. Repeated applications of glutamate agonists in the same brain site resulted in diminished electrochemical responses, compared to the complete reproducibility seen after repeated applications of 120 mM potassium. Low doses of NMDA (10 mM barrel conc), which did not cause a detectable increase in the electrochemical baseline signal, significantly potentiated (50%) potassium-evoked DA overflow. These results indicate that low levels of endogenously released glutamate may modulate overflow when DA nerve terminals are depolarized, while higher concentrations of glutamate may directly elicit increases in extracellular levels of DA and/or ascorbic acid.

Effects of repeated cold stress on aversive responses produced by intrathecal excitatory amino acids in rats.

We previously demonstrated the involvement of spinal glutamatergic system in repeated cold stress (RCS)-induced hyperalgesia. In the present experiments, to estimate the involvement of an enhancement of responsiveness to endogenously released glutamate in RCS-induced hyperalgesia, we examined the effects of RCS on behavioral nociceptive responses (biting or licking the hind paws and the tail) produced by intrathecal injections of selective agonists at subtypes of glutamate receptor, N-methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) and kainate, in rats. The exposure of rats to RCS significantly intensified the behavioral responses produced by intrathecal NMDA (1 nmol/rat) in comparison to the control rats. The increase in the behavioral response of the RCS rats to AMPA was significant at a dose of 1 nmol/rat of AMPA as compared to the control rats. A significant increase in aversive response over control rats was not seen when kainate (0.3--nmol/rat) was injected into the spinal subarachnoid space of the RCS rats. These results suggest that RCS induces an enhancement of transmission mediated by endogenously released glutamate through NMDA and non-NMDA (especially AMPA) receptors in the spinal dorsal horn.