Gamma-aminobutyric Acid Interneurons Research Articles

Mapping alterations in the local synchrony of the cerebral cortex in schizophrenia.

Observations from different fields of research coincide in indicating that a defective gamma-aminobutyric acid (GABA) interneuron system may be among the primary factors accounting for the varied clinical expression of schizophrenia. GABA interneuron deficiency is locally expressed in the form of neural activity desynchronization. We mapped the functional anatomy of local synchrony in the cerebral cortex in schizophrenia using functional connectivity MRI. Data from 86 patients with schizophrenia and 137 control subjects were obtained from publicly available repositories. Resting-state functional connectivity maps based on Iso-Distant Average Correlation measures across three distances were estimated detailing the local functional structure of the cerebral cortex. Patients with schizophrenia showed weaker local functional connectivity (i.e., lower MRI signal synchrony) in (i) prefrontal lobe areas, (ii) somatosensory, auditory, visual, and motor cortices, (iii) paralimbic system at the anterior insula and anterior cingulate cortex, and (iv) hippocampus. The distribution of the defect in cortical area synchrony largely coincided with the synchronization effect of the GABA agonist alprazolam previously observed using identical functional connectivity measures. There was also a notable resemblance between the anatomy of our findings and cortical areas showing higher density of parvalbumin (prefrontal lobe and sensory cortices) and somatostatin (anterior insula and anterior cingulate cortex) GABA interneurons in humans. Our results thus provide detail of the functional anatomy of synchrony changes in the cerebral cortex in schizophrenia and suggest which elements of the interneuron system are affected. Such information could ultimately be relevant in the search for specific treatments.

Depressive patient-derived GABA interneurons reveal abnormal neural activity associated with HTR2C.

Major depressive disorder with suicide behavior (sMDD) is a server mood disorder, bringing tremendous burden to family and society. Although reduced gamma amino butyric acid (GABA) level has been observed in postmortem tissues of sMDD patients, the molecular mechanism by which GABA levels are altered remains elusive. In this study, we generated induced pluripotent stem cells (iPSC) from five sMDD patients and differentiated the iPSCs to GABAergic interneurons (GINs) and ventral forebrain organoids. sMDD GINs exhibited altered neuronal morphology and increased neural firing, as well as weakened calcium signaling propagation, compared with controls. Transcriptomic sequencing revealed that a decreased expression of serotoninergic receptor 2C (5-HT2C) may cause the defected neuronal activity in sMDD. Furthermore, targeting 5-HT2C receptor, using a small molecule agonist or genetic approach, restored neuronal activity deficits in sMDD GINs. Our findings provide a human cellular model for studying the molecular mechanisms and drug discoveries for sMDD.

Motor Resonance During Action Observation and Its Relevance to Virtual Clinical Consultations: Observational Study Using Transcranial Magnetic Stimulation.

Virtual clinical interactions have increased tremendously since the onset of the COVID-19 pandemic. While they certainly have their advantages, there also exist potential limitations, for example, in establishing a therapeutic alliance, discussing complex clinical scenarios, etc. This may be due to possible disruptions in the accurate activation of the human mirror neuron system (MNS), a posited physiological template for effective social communication. This study aimed to compare motor resonance, a putative marker of MNS activity, estimated using transcranial magnetic stimulation (TMS) elicited while viewing virtual (video-based) and actual or real (enacted by a person) actions in healthy individuals. We hypothesized that motor resonance will be greater during real compared to virtual action observation. We compared motor resonance or motor-evoked potential (MEP) facilitation during the observation of virtual (presented via videos) and real (enacted in person) actions, relative to static image observation in healthy individuals using TMS. The MEP recordings were obtained by 2 single-pulse (neuronal membrane excitability-driven) TMS paradigms of different intensities and 2 paired-pulse (cortical gamma-aminobutyric acid-interneuron-driven) TMS paradigms. This study comprised 64 participants. Using the repeated measures ANOVA, we observed a significant time effect for MEP facilitation from static to virtual and real observation states when recorded using 3 of the 4 TMS paradigms. Post hoc pairwise comparisons with Benjamini-Hochberg false discovery rate correction revealed significant MEP facilitation in both virtual and real observation states relative to static image observation; however, we also observed a significant time effect between the 2 action observation states (real > virtual) with 2 of the 4 TMS paradigms. Our results indicate that visual cues expressed via both virtual (video) or real (in person) modes elicit physiological responses within the putative MNS, but this effect is more pronounced for actions presented in person. This has relevance to the appropriate implementation of digital health solutions, especially those pertaining to mental health.

Somatostatin Receptor 4 Agonism Normalizes Stress-Related Excessive Amygdala Glutamate Release and Pavlovian Aversion Learning and Memory in Rodents

BackgroundExcessive processing of aversive life events is a major pathology in stress-related anxiety and depressive disorders. Current pharmacological treatments have rather nonspecific mechanisms of action. Somatostatin is synthesized and released as an inhibitory co-neurotransmitter by specific GABA (gamma-aminobutyric acid) interneurons, and one of its receptors, SSTR4 (somatostatin receptor 4), is localized in brain regions involved in adaptive aversion processing and implicated in negative valence neuropathology, including the amygdala. MethodsRat and mouse experiments were conducted to investigate effects of specific SSTR4 agonism on neurobehavioral aversion processing, including any normalization of stress-related hyperresponsiveness. A mouse experiment to investigate stress and SSTR4 agonism effects on reward processing was also conducted. ResultsIn male rats (n = 5–10/group) fitted with glutamate biosensors in basolateral amygdala, SSTR4 agonism attenuated glutamate release to restraint stress in control rats and particularly in rats previously exposed to chronic corticosterone. In male mice (n = 10–18/group), SSTR4 agonism dose-dependently attenuated Pavlovian tone/footshock learning and memory measured as freezing behavior, in both control mice and mice exposed to chronic social stress, which induces excessive Pavlovian aversion learning and memory. Specificity of SSTR4 agonism effects to aversion learning/memory was demonstrated by absence of effects on discriminative reward (sucrose) learning/memory in both control mice and mice exposed to chronic social stress; SSTR4 agonism did increase reward-to-effort valuation in a dose-dependent manner and in both control mice and mice exposed to chronic social stress, which attenuates reward motivation. ConclusionsThese neuropsychopharmacological findings add substantially to the preclinical proof-of-concept evidence for SSTR4 agonism as a treatment in anxiety and depressive disorders.

Symptomatologic pathomechanism of N-methyl D-aspartate receptor encephalitis.

N-methyl D-aspartate receptor (NMDAR) encephalitis is a well-characterized clinical syndrome. The main molecular mechanism of NMDAR encephalitis is autoantibody-mediated NMDAR hypofunction in the neuronal synapse. Several pathomechanistic hypotheses might explain how NMDAR hypofunction causes the typical symptoms and prognosis of NMDAR encephalitis. Suppression of NMDAR-dependent gamma-aminobutyric acid interneurons provokes an accelerated activation of the positive feedback loops of the dorsolateral prefrontal cortex/subiculum-nucleus accumbens circuit in the striatum, the ventral tegmental area (VTA), and the nucleus reuniens in the thalamus-hippocampus-VTA loop. Dysregulated activation of the VTA and cortex via those positive feedback loops may explain the rapid clinical deterioration at acute stages of the disease and the well-characterized syndrome that includes limbic system dysfunction, intractable seizures, dyskinesia, coma, and the characteristic extreme delta brush. Progressive cerebellar atrophy is correlated with cumulative disease burden and is associated with worse long-term outcomes, which might be explained by the NMDAR-dependent pathways required to maintain neuronal survival. Those pathomechanistic hypotheses for NMDAR encephalitis support the rationale for the early introduction of combination immunotherapy and the use of adjuvant immunotherapy in patients with persisting symptoms in chronic disease phases.

Behavioral Deficits Induced by Somatostatin-Positive GABA Neuron Silencing Are Rescued by Alpha 5 GABA-A Receptor Potentiation.

IntroductionDeficits in somatostatin-positive gamma-aminobutyric acid interneurons (SST+ GABA cells) are commonly reported in human studies of mood and anxiety disorder patients. A causal link between SST+ cell dysfunction and symptom-related behaviors has been proposed based on rodent studies showing that chronic stress, a major risk factor for mood and anxiety disorders, induces a low SST+ GABA cellular phenotype across corticolimbic brain regions; that lowering Sst, SST+ cell, or GABA functions induces depressive-/anxiety-like behaviors (a rodent behavioral construct collectively defined as “behavioral emotionality”); and that disinhibiting SST+ cells has antidepressant-like effects. Recent studies found that compounds preferentially potentiating receptors mediating SST+ cell functions, α5-GABAA receptor positive allosteric modulators (α5-PAMs), achieved antidepressant-like effects. Together, the evidence suggests that SST+ cells regulate mood and cognitive functions that are disrupted in mood disorders and that rescuing SST+ cell function via α5-PAM may represent a targeted therapeutic strategy.MethodsWe developed a mouse model allowing chemogenetic manipulation of brain-wide SST+ cells and employed behavioral characterization 30 minutes after repeated acute silencing to identify contributions to symptom-related behaviors. We then assessed whether an α5-PAM, GL-II-73, could rescue behavioral deficits.ResultsBrain-wide SST+ cell silencing induced features of stress-related illnesses, including elevated neuronal activity and plasma corticosterone levels, increased anxiety- and anhedonia-like behaviors, and impaired short-term memory. GL-II-73 led to antidepressant- and anxiolytic-like improvements among behavioral deficits induced by brain-wide SST+ cell silencing.ConclusionOur data validate SST+ cells as regulators of mood and cognitive functions and demonstrate that bypassing low SST+ cell function via α5-PAM represents a targeted therapeutic strategy.

In utero alcohol exposure exacerbates endothelial protease activity from pial microvessels and impairs GABA interneuron positioning

In utero alcohol exposure can induce severe neurodevelopmental disabilities leading to long-term behavioral deficits. Because alcohol induces brain defects, many studies have focused on nervous cells. However, recent reports have shown that alcohol markedly affects cortical angiogenesis in both animal models and infants with fetal alcohol spectrum disorder (FASD). In addition, the vascular system is known to contribute to controlling gamma-aminobutyric acid (GABA)ergic interneuron migration in the developing neocortex. Thus, alcohol-induced vascular dysfunction may contribute to the neurodevelopmental defects in FASD. The present study aimed at investigating the effects of alcohol on endothelial activity of pial microvessels. Ex vivo experiments on cortical slices from mouse neonates revealed that in endothelial cells from pial microvessels acute alcohol exposure inhibits both glutamate-induced calcium mobilization and activities of matrix metalloproteinase-9 (MMP-9) and tissue plasminogen activator (tPA). The inhibitory effect of alcohol on glutamate-induced MMP-9 activity was abrogated in tPA-knockout and Grin1flox/VeCadcre mice suggesting that alcohol interacts through the endothelial NMDAR/tPA/MMP-9 vascular pathway. Contrasting with the effects from acute alcohol exposure, in mouse neonates exposed to alcohol in utero during the last gestational week, glutamate exacerbated both calcium mobilization and endothelial protease activities from pial microvessels. This alcohol-induced vascular dysfunction was associated with strong overexpression of the N-methyl-d-aspartate receptor subunit GluN1 and mispositioning of the Gad67-GFP interneurons that normally populate the superficial cortical layers. By comparing several human control fetuses with a fetus chronically exposed to alcohol revealed that alcohol exposure led to mispositioning of the calretinin-positive interneurons, whose density was decreased in the superficial cortical layers II-III and increased in deepest layers. This study provides the first mechanistic and functional evidence that alcohol impairs glutamate-regulated activity of pial microvessels. Endothelial dysfunction is characterized by altered metalloproteinase activity and interneuron mispositioning, which was also observed in a fetus with fetal alcohol syndrome. These data suggest that alcohol-induced endothelial dysfunction may contribute in ectopic cortical GABAergic interneurons, that has previously been described in infants with FASD.

GABAergic Inhibitory Interneuron Deficits in Alzheimer's Disease: Implications for Treatment.

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized clinically by severe cognitive deficits and pathologically by amyloid plaques, neuronal loss, and neurofibrillary tangles. Abnormal amyloid β-protein (Aβ) deposition in the brain is often thought of as a major initiating factor in AD neuropathology. However, gamma-aminobutyric acid (GABA) inhibitory interneurons are resistant to Aβ deposition, and Aβ decreases synaptic glutamatergic transmission to decrease neural network activity. Furthermore, there is now evidence suggesting that neural network activity is aberrantly increased in AD patients and animal models due to functional deficits in and decreased activity of GABA inhibitory interneurons, contributing to cognitive deficits. Here we describe the roles played by excitatory neurons and GABA inhibitory interneurons in Aβ-induced cognitive deficits and how altered GABA interneurons regulate AD neuropathology. We also comprehensively review recent studies on how GABA interneurons and GABA receptors can be exploited for therapeutic benefit. GABA interneurons are an emerging therapeutic target in AD, with further clinical trials urgently warranted.

The Role of Dendritic Brain-Derived Neurotrophic Factor Transcripts on Altered Inhibitory Circuitry in Depression

BackgroundA parallel downregulation of brain-derived neurotrophic factor (BDNF) and somatostatin (SST), a marker of inhibitory gamma-aminobutyric acid interneurons that target pyramidal cell dendrites, has been reported in several brain areas of subjects with major depressive disorder (MDD). Rodent genetic studies suggest that they are linked and that both contribute to the illness. However, the mechanism by which they contribute to the pathophysiology of the illness has remained elusive. MethodsWith quantitative polymerase chain reaction, we determined the expression level of BDNF transcript variants and synaptic markers in the prefrontal cortex of patients with MDD and matched control subjects (n = 19/group) and of C57BL/6J mice exposed to chronic stress or control conditions (n = 12/group). We next suppressed Bdnf transcripts with long 3′ untranslated region (L-3′-UTR) using short hairpin RNA and investigated changes in cell morphology, gene expression, and behavior. ResultsL-3′-UTRs containing BDNF messenger RNAs, which migrate to distal dendrites of pyramidal neurons, are selectively reduced, and their expression was highly correlated with SST expression in the prefrontal cortex of subjects with MDD. A similar downregulation occurs in mice submitted to chronic stress. We next show that Bdnf L-3′-UTR knockdown is sufficient to induce 1) dendritic shrinkage in cortical neurons, 2) cell-specific MDD-like gene changes (including Sst downregulation), and 3) depressive- and anxiety-like behaviors. The translational validity of the Bdnf L-3′-UTR short hairpin RNA–treated mice was confirmed by significant cross-species correlation of changes in MDD-associated gene expression. ConclusionsThese findings provide evidence for a novel MDD-related pathological mechanism linking local neurotrophic support, pyramidal cell structure, dendritic inhibition, and mood regulation.

GABAergic inhibitory neurons as therapeutic targets for cognitive impairment in schizophrenia.

Schizophrenia is considered primarily as a cognitive disorder. However, functional outcomes in schizophrenia are limited by the lack of effective pharmacological and psychosocial interventions for cognitive impairment. GABA (gamma-aminobutyric acid) interneurons are the main inhibitory neurons in the central nervous system (CNS), and they play a critical role in a variety of pathophysiological processes including modulation of cortical and hippocampal neural circuitry and activity, cognitive function-related neural oscillations (eg, gamma oscillations) and information integration and processing. Dysfunctional GABA interneuron activity can disrupt the excitatory/inhibitory (E/I) balance in the cortex, which could represent a core pathophysiological mechanism underlying cognitive dysfunction in schizophrenia. Recent research suggests that selective modulation of the GABAergic system is a promising intervention for the treatment of schizophrenia-associated cognitive defects. In this review, we summarized evidence from postmortem and animal studies for abnormal GABAergic neurotransmission in schizophrenia, and how altered GABA interneurons could disrupt neuronal oscillations. Next, we systemically reviewed a variety of up-to-date subtype-selective agonists, antagonists, positive and negative allosteric modulators (including dual allosteric modulators) for α5/α3/α2 GABAA and GABAB receptors, and summarized their pro-cognitive effects in animal behavioral tests and clinical trials. Finally, we also discuss various representative histone deacetylases (HDAC) inhibitors that target GABA system through epigenetic modulations, GABA prodrug and presynaptic GABA transporter inhibitors. This review provides important information on current potential GABA-associated therapies and future insights for development of more effective treatments.

Nicotine self-administration remodels perineuronal nets in ventral tegmental area and orbitofrontal cortex in adult male rats.

Nicotine, a major psychoactive component of tobacco smoke, alters gamma-aminobutyric acid (GABA) modulation of dopamine neurons in the ventral tegmental area (VTA). Changes in structural neuroplasticity can occur in GABAergic parvalbumin (PRV) positive neurons, which are enveloped by structures of the extracellular matrix called perineuronal nets (PNNs). In the current study, rats were trained to self-administer intravenous nicotine (0.03 mg/kg/infusion) for 21 days in 1-hour daily sessions with an incrementing fixed ratio requirement; a control group received saline infusions. At either 45 minutes or 72 hours after the last session, immunofluorescence measurements for PNNs, PRV and c-Fos were conducted. In VTA, nicotine self-administration reduced the number of PRV+ cells surrounded by PNNs at 45 minutes, as well as reducing the intensity of PNNs, suggesting a remodeling of GABA interneurons in this region; the number of PRV+ cells surrounded by PNNs was also reduced at 72 hours. A similar reduction of PNNs occurred in orbitofrontal cortex (OFC) but not in medial prefrontal cortex (prelimbic or infralimbic), 45 minutes after the last session; PNNs were not detected in nucleus accumbens (shell or core). The reduction of PNNs in VTA and OFC was unrelated to c-Fos + cells, as the percent of wisteria floribunda agglutinin + cells co-expressing c-Fos was decreased in OFC but not in VTA. Thus, nicotine self-administration remodeled PNNs surrounding GABA interneurons in VTA and its indirect connections to OFC, suggesting a new possible molecular target where nicotine-induced neuroplasticity takes place. PNN manipulations may prevent or reverse the different stages of tobacco addiction.

Effects of context-drug learning on synaptic connectivity in the basolateral nucleus of the amygdala in rats.

Context-drug learning produces structural and functional synaptic changes in the circuitry of the basolateral nucleus of the amygdala (BLA). However, how the synaptic changes translated to the neuronal targets was not established. Thus, in the present study, immunohistochemistry with a cell-specific marker and the stereological quantification of synapses was used to determine if context-drug learning increases the number of excitatory and inhibitory/modulatory synapses contacting the gamma-aminobutyric acid (GABA) interneurons and/or the pyramidal neurons in the BLA circuitry. Amphetamine-conditioned place preference increased the number of asymmetric (excitatory) synapses contacting the spines and dendrites of pyramidal neurons and the number of multisynaptic boutons contacting pyramidal neurons and GABA interneurons. Context-drug learning increased asymmetric (excitatory) synapses onto dendrites of GABA interneurons and increased symmetric (inhibitory or modulatory) synapses onto dendrites but not perikarya of these same interneurons. The formation of context-drug associations alters the synaptic connectivity in the BLA circuitry, findings that have important implications for drug-seeking behavior.

Process of cortical network formation and impact of early brain damage

The aim is to review mechanisms that are central to the formation of proper cortical circuitry and relevant to perinatal brain injury and premature birth. Clinical investigations using noninvasive imaging techniques suggest that impaired connectivity of cortical circuitry is associated with perinatal adverse conditions. Recent experimental and translational studies revealed developmental mechanisms that are critical for circuit formation and potentially at risk in the perinatal period. These include existence of last wave genesis, migration and integration of gamma-aminobutyric acid (GABA) interneurons in the perinatal period; maturation of GABA interneuron networks that are central to critical period plasticity; transient connections by subplate neurons that guide thalamocortical connectivity, and a perineuronal microglia network that maintains axonal growth and neuronal survival as well as executing synaptic pruning. In addition, recent work has demonstrated that birth plays a key role in triggering the maturation cascade of cortical circuits. Altered maturation of cortical circuits is an increasingly recognized aspect of perinatal injury and premature birth. Potential mechanisms are revealed but further translational studies are required to associate fine changes at the cellular and molecular level with imaging data in experimental models.

Enduring Effects of Adolescent Drug Exposure

Enduring Effects of Adolescent Drug Exposure

Gamma-aminobutyric acid interneuron hypoplasia in the hypothyroidal growth-retarded mouse brain is improved by triiodothyronine treatment at early stage of the postnatal life

Gamma-aminobutyric acid interneuron hypoplasia in the hypothyroidal growth-retarded mouse brain is improved by triiodothyronine treatment at early stage of the postnatal life

The immunhistochemical examination of GABAergic interneuron markers in the dorsolateral prefrontal cortex of patients with late-life depression

The "vascular depression" hypothesis has sought to explain differences in etiology between early and late life depression, and has been reinforced by recent imaging and morphometric studies. Gamma-aminobutyric acid (GABA) is thought to play a major role in the neurobiology of depression. However, it is unclear whether there is an effect on GABA neuronal subpopulations in an elderly depressed cohort. This study therefore examined immunohistochemically two calcium-binding proteins, calretinin and parvalbumin, which have been demonstrated to bind to two distinct GABAergic interneuron subpopulations, within the dorsolateral prefrontal cortex (DLPFC) of elderly depressed patients, against age-matched controls. Post-mortem tissue was obtained from nine controls and 11 depressed patients for the parvalbumin study and seven controls and 14 depressed patients in the calretinin study, and the mean percentage per area of immunohistochemical staining of the two antibodies was measured in individual layers and across the whole of the DLPFC. The study found a reduction in parvalbumin immunostaining in layer 6 (p = 0.05) of the DLFPC in elderly depressed patients. However, no significant changes were found in parvalbumin or calretinin immunostaining in the any other layer of the DLPFC in elderly depressed patients. The study does not suggest any change in GABA interneuron subpopulations, though significant reductions in layer 6 may represent subtle disturbance in GABA parvalbumin-expressing interneuron and glumatatergic pyramidal projection neuron regulation in late-life depression.

Individual Differences in Subconscious Motor Control Predicted by GABA Concentration in SMA

Subliminal visual stimuli affect motor planning, but the size of such effects differs greatly between individuals. Here, we investigated whether such variation may be related to neurochemical differences between people. Cortical responsiveness is expected to be lower under the influence of more of the main inhibitory neurotransmitter, GABA. Thus, we hypothesized that, if an individual has more GABA in the supplementary motor area (SMA)--a region previously associated with automatic motor control--this would result in smaller subliminal effects. We measured the reversed masked prime--or negative compatibility--effect, and found that it correlated strongly with GABA concentration, measured with magnetic resonance spectroscopy. This occurred specifically in the SMA region, and not in other regions from which spectroscopy measurements were taken. We replicated these results in an independent cohort: more GABA in the SMA region is reliably associated with smaller effect size. These findings suggest that, across individuals, the responsiveness of subconscious motor mechanisms is related to GABA concentration in the SMA.

Abnormal neural oscillations and synchrony in schizophrenia

Converging evidence from electrophysiological, physiological and anatomical studies suggests that abnormalities in the synchronized oscillatory activity of neurons may have a central role in the pathophysiology of schizophrenia. Neural oscillations are a fundamental mechanism for the establishment of precise temporal relationships between neuronal responses that are in turn relevant for memory, perception and consciousness. In patients with schizophrenia, the synchronization of beta- and gamma-band activity is abnormal, suggesting a crucial role for dysfunctional oscillations in the generation of the cognitive deficits and other symptoms of the disorder. Dysfunctional oscillations may arise owing to anomalies in the brain's rhythm-generating networks of GABA (gamma-aminobutyric acid) interneurons and in cortico-cortical connections.

Actions of Midazolam on Excitatory Transmission in Dorsal Horn Neurons of Adult Rat Spinal Cord

Although intrathecal administration of midazolam, a water-soluble imidazobenzodiazepine derivative, has been found to produce analgesia, how it exerts this effect at the neuronal level in the spinal cord is not fully understood. The effects of midazolam on electrically evoked and spontaneous excitatory transmission were examined in lamina II neurons of adult rat spinal cord slices using the whole cell patch clamp technique. Bath-applied midazolam (1 microm) diminished Adelta- and C-fiber evoked polysynaptic excitatory postsynaptic currents in both amplitude and integrated area. However, it affected neither Adelta- and C-fiber evoked monosynaptic excitatory postsynaptic currents in amplitude nor miniature excitatory postsynaptic currents in amplitude, frequency, and decay time constant. In the presence of a benzodiazepine receptor antagonist, flumazenil (5 microm), midazolam (1 microm) did not diminish Adelta-fiber evoked polysynaptic excitatory postsynaptic currents, suggesting that midazolam modulate the gamma-aminobutyric acid interneurons in the dorsal horn. Midazolam reduced excitatory synaptic transmission by acting on the gamma-aminobutyric acid type A/benzodiazepine receptor in interneurons, leading to a decrease in the excitability of spinal dorsal horn neurons. This may be a possible mechanism for the antinociception by midazolam in the spinal cord.

Glutamatergic Regulation of the p70S6 Kinase in Primary Mouse Neurons

Brief glutamatergic stimulation of neurons from fetal mice, cultured in vitro for 6 days, activates the mTOR-S6 kinase, ERK1/2 and Akt pathways, to an extent approaching that elicited by brain-derived neurotrophic factor. In contrast, sustained glutamatergic stimulation inhibits ERK, Akt, and S6K. Glutamatergic activation of S6K is calcium/calmodulin-dependent and is prevented by inhibitors of calcium/calmodulin-dependent protein kinase 2, phosphatidylinositol 3-OH-kinase and by rapamycin. 2-Amino-5-phosphonovaleric acid, an inhibitor of N'-methyl-D-aspartate receptors, abolishes glutamatergic activation of ERK1/2 but not the activation of mTOR-S6K; the latter is completely abolished by inhibitors of voltage-dependent calcium channels. Added singly, dopamine gives slight, and norepinephrine a more significant, activation of ERK and S6K; both catecholeamines, however, enhance glutamatergic activation of S6K but not ERK. After 12 days in culture, the response to direct glutamatergic activation is attenuated but can be uncovered by suppression of gamma-aminobutyric acid interneurons with bicuculline in the presence of the weak K(+) channel blocker 4-aminopyridine (4-AP). This selective synaptic activation of mTOR-S6K is also resistant to APV and inhibited by Ca(2+) channel blockers and higher concentrations of glutamate. Elongation factor 2 (EF2) is phosphorylated and inhibited by the eEF2 kinase (CaM kinase III); the latter is inhibited by the S6K or Rsk. Bicuculline/4-AP or KCl-induced depolarization reduces, whereas higher concentrations of glutamate increases, EF2 phosphorylation. Thus the mTOR-S6K pathway in neurons, a critical component of the late phase of LTP, is activated by glutamatergic stimulation in a calcium/calmodulin-dependent fashion through a calcium pool controlled by postsynaptic voltage-dependent calcium channels, whereas sustained stimulation of extrasynaptic glutamate receptors is inhibitory.