Glutamate-mediated transmission, alcohol, and alcoholism

Glutamate-mediated neurotransmission may be involved in the range of adaptive changes in brain which occur after ethanol administration in laboratory animals, and in chronic alcoholism in human cases. Excitatory amino acid transmission is modulated by a complex system of receptors and other effecters, the efficacy of which can be profoundly affected by altered gene or protein expression. Local variations in receptor composition may underlie intrinsic regional variations in susceptibility to pathological change. Equally, ethanol use and abuse may bring about alterations in receptor subunit expression as the essence of the adaptive response. Such considerations may underlie the regional localization characteristic of the pathogenesis of alcoholic brain damage, or they may form part of the homeostatic change that constitutes the neural substrate for alcohol dependence. (C) 2000 Elsevier Science Ltd. All rights reserved.

Effects of age and alcohol exposure during early life on pyramidal cell numbers in the CA1-CA3 region of the rat hippocampus

We have previously shown that exposing rats to a relatively high dose of ethanol during early postnatal life can result in an alteration in spatial learning ability. The hippocampal formation is known to be involved in the control of this ability. The purpose of the present study was to determine whether exposure of rats to ethanol during early postnatal life had either immediate or delayed effects on the numbers of pyramidal cells in the CA1-CA3 subregion of the hippocampus. Wistar rats were exposed to a relatively high daily dose of ethanol at postnatal day 10-15 by placing them for 3 h/day in a chamber containing ethanol vapor. Groups of ethanol-treated (ET), separation control (SC), and mother-reared control (MRC) rats were anesthetized and killed at 16 and 30 days of age by perfusion with phosphate-buffered 2.5% glutaraldehyde. The Cavalieri principle was used to determine the volumes of the CA1 and CA2+CA3 regions. The physical disector method was used to estimate the numerical density of neurons in each of the subdivisions. The total number of pyramidal cells was calculated by multiplying the appropriate estimates of the numerical density by the volume. There were significant age-related reductions in the total numbers of pyramidal cells at 16-30 days of age irrespective of the groups examined. Ethanol treated rats were found to have slightly but significantly fewer pyramidal cell neurons than either the MRC or SC groups. These observations indicate that pyramidal cells in the hippocampus may be vulnerable to a relatively high dose of ethanol exposure during this short period of early postnatal life. (C) 2003 Wiley-Liss, Inc.

Gabaergic mechanisms of hypothalamic nuclei in the expression of conditioned fear

The amygdala, the dorsal periaqueductal gray (dPAG), and the media] hypothalamus have long been recognized to be a neural system responsible for the generation and elaboration of unconditioned fear in the brain. It is also well known that this neural substrate is under a tonic inhibitory control exerted by GABA mechanisms. However, whereas there is a growing body of evidence to suggest that the amygdala and dPAG are also able to integrate conditioned fear, it is still unclear, however, how the distinct hypothalamic nuclei participate in fear conditioning. In this work we aimed to examine the extent to which the gabaergic mechanisms of this brain region are involved in conditioned fear using the fear-potentiated startle (FPS). Muscimol, a GABA-A receptor agonist, and semicarbazide, an inhibitor of the GABA synthesizing enzyme glutamic acid decarboxylase (GAD), were used as an enhancer and inhibitor of the GABA mechanisms, respectively. Muscimol and semicarbazide were injected into the anterior hypothalamus (AHN). the dorsomedial part of the ventromedial nucleus (VMHDM), the dorsomedial (DMH) or the dorsal premammillary (PMD) nuclei of male Wistar rats before test sessions of the fear conditioning paradigm. The injections into the DMH and PMD did not produce any significant effects on FPS. On the other hand, muscimol injections into the AHN and VMHDM caused significant reduction in FPS. These results indicate that injections of muscimol and semicarbazide into the DMH and PMD fail to change the FPS, whereas the enhancement of the GABA transmission in the AHN and VMHDM produces a reduction of the conditioned fear responses. On the other hand, the inhibition of this transmission led to an increase of this conditioned response in the AHN. Thus, whereas DMH and PMD are known to be part of the caudal-most region of the medial hypothalamic defensive system, which integrates unconditioned fear, systems mediating conditioned fear select the AHN and VMHDM nuclei that belong to the rostral-most portion of the hypothalamic defense area. Thus, distinct subsets of neurons in the hypothalamus could mediate different aspects of the defensive responses. (C) 2008 Elsevier Inc. All rights reserved.

Selective involvement of GABAergic mechanisms of the dorsal periaqueductal gray and inferior colliculus on the memory of the contextual fear as assessed by the fear potentiated startle test

The inferior colliculus (IC) together with the dorsal periaqueductal gray (dPAG), the amygdala and the medial hypothalamus make part of the brain aversion system, which has mainly been related to the organization of unconditioned fear. However, the involvement of the IC and dPAG in the conditioned fear is still unclear. It is certain that GABA has a regulatory role on the aversive states generated and elaborated in these midbrain structures. In this study, we evaluated the effects of injections of the GABA-A receptor agonist muscimol (1.0 and 2.0 nmol/0.2 mu L) into the IC or dPAG on the freezing and fear-potentiated startle (FPS) responses of rats submitted to a context fear conditioning. Intra-IC injections of muscimol did not cause any significant effect on the FPS or conditioned freezing but enhanced the startle reflex in non-conditioned animals. In contrast, intra-dPAG injections of muscimol caused significant reduction in FPS and conditioned freezing without changing the startle reflex in non-conditioned animals. Thus, intra-dPAG injections of muscimol produced the expected inhibitory effects on the anxiety-related responses, the FPS and the freezing whereas these injections into the IC produced quite opposite effects suggesting that descending inhibitory pathways from the IC, probably mediated by GABA-A mechanisms, exert a regulatory role on the lower brainstem circuits responsible for the startle reflex. (C) 2008 Elsevier Inc. All rights reserved.

Association between a 15q25 gene variant, smoking quantity and tobacco-related cancers among 17 000 individuals

Methods We performed a detailed analysis of one 15q single nucleotide polymorphism (SNP) (rs16969968) with smoking behaviour and cancer risk in a total of 17 300 subjects from five LC studies and four upper aerodigestive tract (UADT) cancer studies. Results Subjects with one minor allele smoked on average 0.3 cigarettes per day (CPD) more, whereas subjects with the homozygous minor AA genotype smoked on average 1.2 CPD more than subjects with a GG genotype (P < 0.001). The variant was associated with heavy smoking (> 20 CPD) [odds ratio (OR) = 1.13, 95% confidence interval (CI) 0.96-1.34, P = 0.13 for heterozygotes and 1.81, 95% CI 1.39-2.35 for homozygotes, P < 0.0001]. The strong association between the variant and LC risk (OR = 1.30, 95% CI 1.23-1.38, P = 1 x 10(-18)), was virtually unchanged after adjusting for this smoking association (smoking adjusted OR = 1.27, 95% CI 1.19-1.35, P = 5 x 10(-13)). Furthermore, we found an association between the variant allele and an earlier age of LC onset (P = 0.02). The association was also noted in UADT cancers (OR = 1.08, 95% CI 1.01-1.15, P = 0.02). Genome wide association (GWA) analysis of over 300 000 SNPs on 11 219 subjects did not identify any additional variants related to smoking behaviour. Conclusions This study confirms the strong association between 15q gene variants and LC and shows an independent association with smoking quantity, as well as an association with UADT cancers.

Association Between Polymorphisms in GRIK2 Gene and Obsessive-Compulsive Disorder: A Family-Based Study

Several studies support a genetic influence on obsessive-compulsive disorder (OCD) etiology. The role of glutamate as an important neurotransmitter affecting OCD pathophysiology has been supported by neuroimaging, animal model, medication, and initial candidate gene studies. Genes involved in glutamatergic pathways, such as the glutamate receptor, ionotropic, kainate 2 (GRIK2), have been associated with OCD in previous studies. This study examines GRIK2 as a candidate gene for OCD susceptibility in a family-based approach. Probands had full DSM-IV diagnostic criteria for OCD. Forty-seven OCD probands and their parents were recruited from tertiary care OCD specialty clinics from France and USA. Genotypes of single nucleotide polymorphism (SNP) markers and related haplotypes were analyzed using Haploview and FBAT software. The polymorphism at rs1556995 (P = 0.0027; permuted P-value = 0.03) was significantly associated with the presence of OCD. Also, the two marker haplotype rs1556995/rs1417182, was significantly associated with OCD (P = 0.0019, permuted P-value = 0.01). This study supports previously reported findings of association between proximal GRIK2 SNPs and OCD in a comprehensive evaluation of the gene. Further study with independent samples and larger sample sizes is required.

5-HT(1A/1B), 5-HT(6), and 5-HT(7) serotonergic receptors recruitment in tonic-clonic seizure-induced antinociception: Role of dorsal raphe nucleus

Pharmacological studies have been focused on the involvement of different neural pathways in the organization of antinociception that follows tonic-clonic seizures, including 5-hydroxytryptamine (5-HT)-, norepinephrine-, acetylcholine- and endogenous opioid peptide-mediated mechanisms, giving rise to more in-depth comprehension of this interesting post-ictal antinociceptive phenomenon. The present work investigated the involvement of 5-HT(1A/1B), 5-HT(6), and 5-HT(7) serotonergic receptors through peripheral pretreatment with methiothepin at doses of 0.5, 1.0, 2.0 and 3.0 mg/kg in the organization of the post-ictal antinociception elicited by pharmacologically (with pentylenetetrazole at 64 mg/kg)-induced tonic-clonic seizures. Methiothepin at 1.0 mg/kg blocked the post-ictal antinociception recorded after the end of seizures, whereas doses of 2.0 and 3.0 mg/kg potentiated the post-ictal antinociception. The nociceptive thresholds were kept higher than those of the control group. However, when the same 5-hydroxytryptamine receptors antagonist was microinjected (at 1.0, 3.0 and 5.0 mu g/0.2 mu L) in the dorsal raphe nucleus, a mesencephalic structure rich in serotonergic neurons and 5-HT receptors, the post-ictal hypo-analgesia was consistently antagonized. The present findings suggest a dual effect of methiothepin, characterized by a disinhibitory effect on the post-ictal antinociception when peripherally administered (possibly due to an antagonism of pre-synaptic 5-HT(1A) serotonergic autoreceptors in the pain endogenous inhibitory system) and an inhibitory effect (possibly due to a DRN post-synaptic 5-HT(1B), 5-HT(6), and 5-HT(7) serotonergic receptors blockade) when centrally administered. The present data also Suggest that serotonin-mediated mechanisms of the dorsal raphe nucleus exert a key-role in the modulation of the post-ictal antinociception. (C) 2009 Elsevier Inc. All rights reserved.

Serotonergic neurotransmission in the dorsal raphe nucleus recruits in situ 5-HT2A/2C receptors to modulate the post-ictal antinociception

The post-ictal immobility syndrome is followed by a significant increase in the nociceptive thresholds in animals and humans. The aim of this study was to assess the involvement of the dorsal raphe nucleus (DRN) in the post-ictal antinociception. The second aim was to study the role of serotonergic intrinsic mechanisms of the DRN in this hypo-algesic phenomenon. Pentylenetetrazole (PTZ), an ionophore GABA-mediated Cl- influx antagonist, was peripherally used to induce tonic-clonic seizures in Wistar rats. The nociceptive threshold was measured by the tail-flick test. Neurochemical lesions of the DRN, performed with microinjection of ibotenic acid (1.0 mu g/0.2 mu L), caused a significant decrease of tonic-clonic seizure-induced antinociception, suggesting the involvement of this nucleus in this antinociceptive Process. Microinjections of methysergide (1.0 and 5.0 mu g/0.2 mu L), a non-selective serotonergic receptor antagonist, into DRN caused a significant decrease in the post-ictal antinociception in seizing animals, compared to controls, in all post-ictal periods Presently studied. These findings were corroborated by microinjections of ketanserin (at 1.0 and 5.0 mu g/0.2 mu L) into DRN. Ketanserin is an antagonist with large affinity for 5-HT2A/2C serotonergic receptors, which, in this Case, Caused a significant decrease in the tail-flick latencies in seizing animals, compared to controls after the first 20 min following tonic-clonic convulsive reactions. These results indicate that serotonergic neurotransmission of the DRN neuronal clusters is involved in the organization of the post-ictal hypo-algesia. The 5-HT2A/2C receptors of DRN neurons seem to be critically involved in the increase of nociceptive thresholds following tonic-clonic seizures. (c) 2008 Elsevier Inc, All rights reserved.

Opioidergic and GABAergic mechanisms in the rostral ventromedial medulla modulate the nociceptive response of vocalization in guinea pigs

Vocalization generated by the application of a noxious stimulus is an integrative response related to the affective-motivational component of pain. The rostral ventromedial medulla (RVM) plays an important role in descending pain modulation, and opiates play a major role in modulation of the antinociception mediated by the RVM. Further, it has been suggested that morphine mediates antinociception indirectly, by inhibition of tonically active GABAergic neurons. The current study evaluated the effects of the opioids and GABA agonists and antagonists in the RVM on an affective-motivational pain model. Additionally, we investigated the opioidergic-GABAergic interaction in the RVM in the vocalization response to noxious stimulation. Microinjection of either morphine (4.4 nmo1/0.2 mu l) or bicuculline (0.4 nmo1/0.2 mu l) into the RVM decreased the vocalization index, whereas application of the GABA(A) receptor agonist, musci-mol (0.5 nmo1/0.2 mu l) increased the vocalization index during noxious stimulation. Furthermore, prior microinjection of either the opioid antagonist naloxone (2.7 nmo1/0.2 mu l) or muscimol (0.25 nmo1/0.2 mu l) into the RVM blocked the reduction in vocalization index induced by morphine. These observations suggest an antinociceptive and pro-nociceptive role of the opioidergic and GABAergic neurotransmitters in the RVM, respectively. Our data show that opioids have an antinociceptive effect in the RVM, while GABAergic neurotransmission is related to the facilitation of nociceptive responses. Additionally, our results indicate that the antinociceptive effect of the opioids in the RVM could be mediated by a disinhibition of tonically active GABAergic interneurons in the downstream projection neurons of the descending pain control system; indicating an interaction between the opioidergic and GABAergic pathways of pain modulation. (C) 2010 Elsevier Inc. All rights reserved.

The GABAergic system contributes to the anxiolytic-like effect of essential oil from Cymbopogon citratus (lemongrass)

Ethnopharmacological relevance: The essential oil (EO) from Cymbopogon citratus (DC) Stapf is reported to have a wide range of biological activities and is widely used in traditional medicine as an infusion or decoction. However, despite this widely use, there are few controlled studies confirming its biological activity in central nervous system. Materials and methods: The anxiolytic-like activity of the EO was investigated in light/dark box (LDB) and marble-burying test (MBT) and the antidepressant activity was investigated in forced-swimming test (FST) in mice. Flumazenil, a competitive antagonist of benzodiazepine binding and the selective 5-HT(1A) receptor antagonist WAY100635 was used in experimental procedures to determine the action mechanism of EO. To exclude any false positive results in experimental procedures, mice were submitted to the rota-rod test. We also quantified some neurotransmitters at specific brain regions after EO oral acute treatment. Results: The present work found anxiolytic-like activity of the EO at the dose of 10 mg/kg in a LDB. Flumazenil, but not WAY100635, was able to reverse the effect of the EO in the LOB, indicating that the EO activity occurs via the GABA(A) receptor-benzodiazepine complex. Only at higher doses did the EO potentiate diethyl-ether-induced sleeping time in mice. In the FST and MBT, EO showed no effect. Finally, the increase in time spent in the light chamber, demonstrated by concomitant treatment with ineffective doses of diazepam (DZP) and the EO, revealed a synergistic effect of the two compounds. The lack of activity after long-term treatment in the LDB test might be related to tolerance induction, even in the DZP-treated group. Furthermore, there were no significant differences between groups after either acute or repeated treatments with the EO in the rota-rod test. Neurochemical evaluation showed no amendments in neurotransmitter levels evaluated in cortex, striatum, pons, and hypothalamus. Conclusions: The results corroborate the use of Cymbopogon citratus in folk medicine and suggest that the anxiolytic-like effect of its EO is mediated by the GABA(A) receptor-benzodiazepine complex. (C) 2011 Elsevier Ireland Ltd. All rights reserved.

Excited to death: different ways to lose your neurones

The selective loss of neurones in a range of neurodegenerative diseases is widely thought to involve the process of excitotoxicity, in which glutamate-mediated neuronal killing is elaborated through the excessive stimulation of cell-surface receptors. Every such disease exhibits a distinct regional and subregional pattern of neuronal loss. so processes must be locally triggered to different extents to account for this. We have studied several mechanisms which could lead to excitotoxic glutamate pathophysiology and compared them in different diseases. Our data suggest that glutamate can reach toxic extracellular levels in Alzheimer disease by malfunctions in cellular transporters, and that the toxicity may be exacerbated by continued glutamate release from presynaptic neurones acting on hypersensitive postsynaptic receptors. Thus the excitotoxicity is direct. In contrast, alcoholic brain damage arises in regions where GABA-mediated inhibition is deficient, and fails properly to dampen trans-synaptic excitation, Thus the excitotoxicity is indirect. A variety of such mechanisms is possible, which may combine in different ways.

Untersuchungen zur Quartärstruktur neuronaler nikotinischer Acetylcholinrezeptoren von Drosophila melanogaster

Fruitfly, Drosophila melanogaster, neuronal nicotinic acetylcholine receptor, receptor subunit, quarternary structure

Muscimol-induced death of GABAergic neurons in rat brain aggregating cell cultures.

During brain development, spontaneous neuronal activity has been shown to play a crucial role in the maturation of neuronal circuitries. Activity-related signals may cause selective neuronal cell death and/or rearrangement of neuronal connectivity. To study the effects of sustained inhibitory activity on developing inhibitory (GABAergic) neurons, three-dimensional primary cell cultures of fetal rat telencephalon were used. In relatively immature cultures, muscimol (10 microns), a GABAA receptor agonist, induced a transient increase in apoptotic cell death, as evidenced by a cycloheximide-sensitive increase of free nucleosomes and an increased frequency of DNA double strand breaks (TUNEL labeling). Furthermore, muscimol caused an irreversible reduction of glutamic acid decarboxylase activity, indicating a loss of GABAergic neurons. The muscimol-induced death of GABAergic neurons was attenuated by the GABAA receptor blockers bicuculline (100 microns) and picrotoxin (100 microns), by depolarizing potassium concentrations (30 mM KCl) and by the L-type calcium channel activator BAY K8644 (2 microns). As compared to the cholinergic marker (choline acetyltransferase activity), glutamic acid decarboxylase activity was significantly more affected by various agents known to inhibit neuronal activity, including tetrodotoxin (1 micron), flunarizine (5 microns), MK 801 (50 microns) and propofol (40 microns). The present results suggest that the survival of a subpopulation of immature GABAergic neurons is dependent on sustained neuronal activity and that these neurons may undergo apoptotic cell death in response to GABAA autoreceptor activation.

Neuronal in vitro models for the estimation of acute systemic toxicity.

The objective of the EU funded integrated project "ACuteTox" is to develop a strategy in which general cytotoxicity, together with organ-specific endpoints and biokinetic features, are taken into consideration in the in vitro prediction of oral acute systemic toxicity. With regard to the nervous system, the effects of 23 reference chemicals were tested with approximately 50 endpoints, using a neuronal cell line, primary neuronal cell cultures, brain slices and aggregated brain cell cultures. Comparison of the in vitro neurotoxicity data with general cytotoxicity data generated in a non-neuronal cell line and with in vivo data such as acute human lethal blood concentration, revealed that GABA(A) receptor function, acetylcholine esterase activity, cell membrane potential, glucose uptake, total RNA expression and altered gene expression of NF-H, GFAP, MBP, HSP32 and caspase-3 were the best endpoints to use for further testing with 36 additional chemicals. The results of the second analysis showed that no single neuronal endpoint could give a perfect improvement in the in vitro-in vivo correlation, indicating that several specific endpoints need to be analysed and combined with biokinetic data to obtain the best correlation with in vivo acute toxicity.

Oxytocin selectively gates fear responses through distinct outputs from the central amygdala.

Central amygdala (CeA) projections to hypothalamic and brain stem nuclei regulate the behavioral and physiological expression of fear, but it is unknown whether these different aspects of the fear response can be separately regulated by the CeA. We combined fluorescent retrograde tracing of CeA projections to nuclei that modulate fear-related freezing or cardiovascular responses with in vitro electrophysiological recordings and with in vivo monitoring of related behavioral and physiological parameters. CeA projections emerged from separate neuronal populations with different electrophysiological characteristics and different response properties to oxytocin. In vivo, oxytocin decreased freezing responses in fear-conditioned rats without affecting the cardiovascular response. Thus, neuropeptidergic signaling can modulate the CeA outputs through separate neuronal circuits and thereby individually steer the various aspects of the fear response.