Spontaneous Openings of NMDA Receptor Channels in Cultured Rat Hippocampal Neurons

Spontaneous and N-methyl-D-aspartate (NMDA)-evoked single-channel currents were studied in outside-out patches isolated from cultured rat hippocampal neurons. Both spontaneous and NMDA-evoked single-channel currents reversed at potentials close to 0 mV and exhibited multiple amplitude levels of similar amplitude. Both spontaneous and NMDA-evoked single-channel currents were inhibited by Mg²⁺ in a voltage-dependent manner and by 7-chlorokynurenic acid. The activity of spontaneous single-channel currents was reduced by the competitive NMDA receptor antagonists, but by one to three orders of magnitude less than expected assuming that the spontaneous activity is due to an ambient NMDA receptor agonist present in the extracellular solution. Our results suggest that, similar to other ligand-gated ion channels, NMDA receptor channels have a dual mode of activation - spontaneous and agonist induced.     

Copper Modulation of NMDA Responses in Mouse and Rat Cultured Hippocampal Neurons

The effect of Cu²⁺ on NMDA receptors was studied in cultured mouse and rat hippocampal neurons using whole-cell patch-clamp and a fast perfusion system. Analysis of the Cu²⁺ concentration-response curve for inhibition of NMDA-induced currents suggests that free Cu²⁺ directly inhibits NMDA receptors with an IC₅₀ of 0.27 μM. Cu²⁺ was ineffective in blocking NMDA receptor activity when complexed with NMDA or glycine; NMDA-Cu²⁺ and glycine-Cu²⁺ complexes acted as agonists of similar potency to the free amino acids. The inhibition by Cu²⁺ (10–100 μM) of responses to 10 μM NMDA was essentially voltage-independent. The onset of inhibition by 100 μM Cu²⁺ of responses to 2 FM glutamate acting at NMDA receptors was significantly faster than NMDA receptor deactivation evoked by a sudden decrease in the concentration of glycine or glutamate, or of both agonists. This suggests that CU²⁺ acts as a non-competitive antagonist, and does not directly interfere with the binding of glutamate or glycine to their recognition sites on the NMDA receptor complex. In the absence of NMDA the apparent association rate constant for binding of Cu²⁺ to NMDA receptors, calculated from the rate of onset of block by Cu²⁺ of test responses to NMDA, was 19 times slower than in the presence of 30 μM NMDA, suggesting that Cu^z+ interacts preferentially with agonist-bound receptors. Our results show that Cu²⁺ is a potent inhibitor of NMDA receptor-mediated responses.     

Single-channel Activity in Cultured Cortical Neurons of the Rat in the Presence of a Toxic Dose of Glutamate

Rat cortical neurons grown in cell culture were exposed to 500 μM glutamate for 5 min during continuous current recording from cell-attached patches. The Ca²⁺-dependence and ion selectivity of the membrane channels activated during and after glutamate application were studied in inside-out patches. Glutamate blocked spontaneous action potential firing. In 77% of the experiments glutamate activated several types of ion channels indirectly, i.e. via a change of cytoplasmic factors. Channel activity did not disappear after removing glutamate from the bath. A K⁺ channel requiring intracellular calcium ([Ca²⁺]_i) was activated in 44% of the experiments (conductance for inward currents in cell-attached patches 118 ± 6 pS;‘BK channel'). Another Ca²⁺-dependent channel permeable for Cl^- (conductance for outward currents in cell-attached patches 72±17 pS), acetate and methanesulphonate appeared in 26% of the patches. Other K⁺ channels of smaller conductance were infrequently observed. During and after glutamate application the activity of the BK channel showed an initial increase followed by a transient decay and a second rise to a plateau, probably reflecting a similar time course of changes in [Ca²⁺]_i. Both phases of increasing channel activity required the presence of extracellular Ca²⁺ suggesting that [Ca²⁺]_i was mainly increased by Ca²⁺ influx. The N-methyl-d -aspartate (NMDA) antagonists dizocilpine (MK-801, 10 μM) and dl -2-amino-5-phosphonovaleric acid (AP5; 100 μM), added within 5 min after glutamate application, stopped BK channel activity and restored the spontaneous action potential firing. We conclude that the influx of Ca²⁺ through NMDA receptor channels causes a strong activation of Ca²⁺-dependent K⁺ channels, which is likely to result in pronounced loss of intracellular K⁺. NMDA receptor channels seem to remain active for a long time (>10 min) after the end of glutamate application.     

Metabotropic Glutamate Receptors in the Rat Nucleus Accumbens

The effects of glutamate metabotropic receptors (mGluRs) on excitatory transmission in the nucleus accumbens were investigated using electrophysiological techniques in rat nucleus accumbens slices. The broad-spectrum mGluR agonist (1S,3 R )-1-aminocyclopentyl-1,3-dicarboxylate, the mGluR group 2 selective agonists (S)-4-carboxy-3-hydroxyphenylglycine, (1S,3S)-ACPD) and (2S,1'S,2'S)-2-(2'-carboxycyclopropyl)glycine (L-CCG1), and the mGluR group 3 specific agonist L-2-amino-4-phosphonobutyrate (L-AP4) all reversibly inhibited evoked excitatory synaptic responses. The specific group 1 mGluR agonist (R,S)-3,5-dihydroxyphenylglycine [(R,S)-DHPG] did not depress transmission. Dose-response curves showed that the rank order of agonist potencies was: L-CCGI > L-AP4 > (1 S,3S)-ACPD. Group 2 and 3 mGluRs inhibited transmission via a presynaptic mechanism, as they increased paired-pulse facilitation, decreased the frequency of miniature excitatory postsynaptic currents and had no effect on their amplitude. The mGluRs did not inhibit transmitter release by reducing voltage-dependent Ca²⁺ currents through N- or P-type Ca²⁺ channels, as inhibition persisted in the presence of a-conotoxin-GVIA or Aga-IVA. The depression induced by mGluRs was not affected by specific antagonists of dopamine D1, GABA-B or adenosine A1 receptors, indicating direct effects. Finally, (13,s)-DHPG specifically blocked the postsynaptic afterhyperpolarization current (I_ahp). Our results represent the first direct demonstration of functional mGluRs in the nucleus accumbens of the rat.     

Localization of glutamate receptors in developing cortical neurons in culture and relationship to susceptibility to excitotoxicity

Overactivation of glutamate receptors leading to excitotoxicity has been implicated in the neurodegenerative alterations of a range of central nervous system (CNS) disorders. We have investigated the cell-type-specific changes in glutamate receptor localization in developing cortical neurons in culture, as well as the relationship between glutamate receptor subunit distribution with synapse formation and susceptibility to excitotoxicity. Glutamate receptor subunit clustering was present prior to the formation of synapses. However, different receptor types showed distinctive temporal patterns of subunit clustering, localization to spines, and apposition to presynaptic terminals. N-methyl-D-aspartate (NMDA) receptor subunit immunolabelling was present in puncta along dendrites prior to the formation of synapses, with relatively little localization to spines. Vulnerability to NMDA receptor-mediated excitotoxicity occurred before receptor subunits became localized in apposition to presynaptic terminals. Clustering of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors occurred concurrently with development of vulnerability to excitotoxicity and was related to localization of AMPA receptors at synapses and in spines. Different AMPA receptor subunits demonstrated cell-type-specific localization as well as distribution to spines, dendrites, and extrasynaptic subunit clusters. A subclass of neurons demonstrated substantial perineuronal synaptic innervation, and these neurons expressed relatively high levels of GluR1 and/or GluR4 at receptor puncta, indicating the presence of calcium-permeable AMPA receptors and suggesting alternative synaptic signalling mechanisms and vulnerability to excitotoxicity. These data demonstrate the relationship between glutamate receptor subunit expression and localization with synaptogenesis and development of neuronal susceptibility to excitotoxicity. These data also suggest that excitotoxicity can be mediated through extrasynaptic receptor subunit complexes along dendrites.     

Critical Role of S465 in Protein Kinase C-Increased Rat Glutamate Transporter Type 3 Activity

Glutamate transporters, also called excitatory amino acid transporters (EAATs), uptake extracellular glutamate and regulate neurotransmission. Activation of protein kinase C (PKC) increases the activity of EAAT type 3 (EAAT3), the major neuronal EAAT. We designed this study to determine which amino acid residue(s) in EAAT3 may be involved in this PKC effect. Selective potential PKC phosphorylation sites were mutated. These EAAT3 mutants were expressed in the Xenopus oocytes. Phorbol 12-myristate 13-acetate, a PKC activator, significantly increased wild-type EAAT3 activity. Mutation of serine 465 to alanine or aspartic acid, but not the mutation of threonine 5 to alanine, abolished PKC-increased EAAT3 activity. Our results suggest a critical role of serine 465 in the increased EAAT3 activity by PKC activation.     

Cholinergic Modulation of the Action Potential in Rat Hippocampal Neurons

The cholinergic input to the hippocampus from the medial septum is important for modulating hippocampal activity and functions, including theta rhythm and spatial learning. Neuromodulation by transmitters in central nervous system neurons usually affects cell excitability by modifying the membrane potential, discharge pattern and spike frequency. Here we describe another type of neuromodulation: changing the action potential waveform. During intracellular recordings from CA1 pyramidal cells in hippocampal slices from rats, the cholinergic agonist carbachol caused several reversible changes in the action potential: low doses (2 μM) caused an increase in spike duration; high doses (10–40 μM) or long-lasting applications also reduced the spike amplitude and rate of rise, and raised the spike threshold. These effects are similar to those of metabotropic glutamate receptor agonists or phorbol esters, both of which activate protein kinase C. The effects were blocked by the muscarinic antagonist atropine, and were prevented by Ca²⁺-free medium and by Ca²⁺-channel blockers. However, the cholinergic spike modulation was not occluded or mimicked by blocking the Ca²⁺-dependent K⁺ currents I_c or I_AHP, suggesting that these K⁺ currents are not involved in the modulation.     

Survival-promoting and Protein Kinase C-regulating Roles of Basic FGF for Hippocampal Neurons Exposed to Phorbol Ester, Glutamate and Ischaemia-like Conditions

Recent evidence suggests that protein kinase C (PKC) is involved in the pathophysiology of neurodegenerative diseases. We examined the effect of basic fibroblast growth factor (bFGF) on the survival of cultured rat hippocampal neurons exposed to conditions in which PKC is likely to play a role. bFGF reduced neuron damage caused by the PKC-activating phorbol ester 12- O -tetradecanoylphorbol 13-acetate (TPA), glutamate and ischaemia-like culture conditions. bFGF was able to counteract the excessive activation of PKC caused by these treatments. Moreover, bFGF prevented the loss of PKC occurring after prolonged exposure to TPA or ischaemia-like conditions. These results indicate that both the overactivation and the abnormal degradation of PKC can lead to neuron degeneration, and that the neurotrophic competence of bFGF may reside in its ability to regulate and normalize the PKC phosphorylating system.     

Effect of Acute Soman Exposure on GABAA Receptors in Rat Hippocampal Slices and Cultured Hippocampal Neurons

Exposure of the central nervous system to organophosphorus (OP) nerve agents causes seizures and neuronal cell death. Benzodiazepines are commonly used to treat seizures induced by OPs. However, it is known that soman-induced seizures are particularly resistant to benzodiazepine treatment, as compared with other OPs. This study investigated the effect of soman on γ-aminobutyric acid (GABA) neurotransmission in acute rat hippocampal slices and the surface expression of GABA(A) receptors in cultured rat hippocampal neurons. Results showed that GABA-mediated inhibitory post synaptic currents (IPSCs) are significantly reduced by soman in a concentration-dependent manner in acute rat hippocampal slices. Furthermore, confocal microscopic and cell-based ELISA assays revealed that soman caused rapid internalization of GABA(A) receptors in cultured rat hippocampal neurons. The effect of soman on GABA(A)R endocytosis was not due to inhibition of acetylcholinesterase (AChE) because (1) the acetylcholine muscarinic receptor antagonist atropine did not block soman-induced GABA(A)R endocytosis; and (2) physostigmine, at concentrations that completely inhibit AChE activity, did not cause GABA(A)R endocytosis. Moreover, blocking of the N-methyl-D-aspartate (NMDA) receptors by 2-amino-5-phosphonovalerate (APV) had no effect on soman-induced GABA(A)R endocytosis, suggesting that the soman effect was not secondary to glutamate receptor over activation. Regardless of the exact mechanism, the observation that soman induces rapid GABA(A)R endocytosis may have significant implications in the development of effective countermeasures against soman-induced seizures.     

G-Protein Modulation of Glycine-resistant NMDA Receptor Desensitization in Rat Cultured Hippocampal Neurons

Activation of N-methyl-D-aspartate (NMDA) subtype glutamate receptors increases the excitability of most neurons within the CNS. A common feature of ionotropic glutamate receptors is their ability to undergo desensitization. In the present experiments we have examined the role of guanine nucleotide-binding proteins(G-proteins) in the regulation of NMDA receptor desensitization. Repeated NMDA receptor activation with 2 mM extracellular Ca²+ increased the degree of glycine-resistant NMDA receptor desensitization of subsequent responses to NMDA recorded in the presence of 0.2 mM Ca²+. The recovery of glycine-resistant NMDA receptor desensitization after repeated NMDA receptor activation in the presence of 2 mM Ca²+ was significantly reduced in neurons intracellularly dialysed with guanosine-5'-0-(3-thiotriphosphate), guanosine-5'-triphosphate or AIC1₃ and CsF, compounds known to activate G-proteins. lntracellular dialysis with guanosine-5'-0(2thiodiphosphate), adenosine triphosphate, or adenosine-5'-G(3-thiotriphosphate) was ineffective. The calcium permeability of NMDA receptor-channels was not altered by intracellular dialysis with GTPyS. This suggests that modulation of NMDA receptor desensitization by G-proteins represents a novel mechanism forregulation of glutamate-gated ion channel activity.     

Glutamate dependent dendritic outgrowth in developing neuronal networks of rat hippocampal cells in vitro

The present study was undertaken to determine whether release of glutamate is capable of influencing dendritic morphology in a developing network of rat hippocampal neurons in vitro. Control cultures developed a dense network of fibers and evinced spontaneous electrical activity from the third day in vitro. Dendrites were examined in cultures maintained for 2 weeks in vitro: the experimental group grown in medium containing the glutamate receptor antagonists AP-5 and DNQX. Dendritic extensions were analyzed as a function of time (days in vitro) using a number of morphometric parameters, viz. the number of processes, the number and length of intermediate and terminal segments, as well as the total length of all segments. We found that the effect of age and treatment was most prominently reflected in the length of the terminal segments. Chronic addition of ionotropic glutamate receptor antagonists from day 2 in culture arrested all dendrite parameters at the prefunctional level. The results suggest that glutamate release is crucial for the onset of dendritic morphological development in hippocampal neurons.     

Hippocampal dendritic spines remodeling and fear memory are modulated by GABAergic signaling within the basolateral amygdala complex

GABAergic signaling in the basolateral amygdala complex (BLA) plays a crucial role on the modulation of the stress influence on fear memory. Moreover, accumulating evidence suggests that the dorsal hippocampus (DH) is a downstream target of BLA neurons in contextual fear. Given that hippocampal structural plasticity is proposed to provide a substrate for the storage of long‐term memories, the main aim of this study is to evaluate the modulation of GABA neurotransmission in the BLA on spine density in the DH following stress on contextual fear learning. The present findings show that prior stressful experience promoted contextual fear memory and enhanced spine density in the DH. Intra‐BLA infusion of midazolam, a positive modulator of GABAa sites, prevented the facilitating influence of stress on both fear retention and hippocampal dendritic spine remodeling. Similarly to the stress‐induced effects, the blockade of GABAa sites within the BLA ameliorated fear memory emergence and induced structural remodeling in the DH. These findings suggest that GABAergic transmission in BLA modulates the structural changes in DH associated to the influence of stress on fear memory. © 2015 Wiley Periodicals, Inc.     

Galanin and Glibenclamide Modulate the Anoxic Release of Glutamate in Rat CA3 Hippocampal Neurons

The effects of brief anoxic episodes on intracellularly recorded CA3 pyramidal neurons have been studied in the hippocampal slice preparation. Anoxia induced a depolarization occasionally preceded by a transient hyperpolarization associated with a fall in input resistance. The anoxic depolarization was due to the release of glutamate from presynaptic terminals since it was blocked by tetrodotoxin (TTX) (1 microM) or by the broad spectrum excitatory amino acid antagonist kynurenate (1 mM). In the presence of TTX (1 microM) or kynurenate (1 mM), anoxia only induced a hyperpolarization which was due to activation of a K+ conductance. The anoxic depolarization was blocked by galanin, a hormone which activates ATP sensitive K+ (K+ATP) channels. Anoxic depolarization was increased by the potent sulfonylurea agent glibenclamide (GLIB) which blocks K+ATP channels. Bath applications of these agents had little effect when applied in oxygenated Krebs solution suggesting that their action may be mediated by K+ATP channels. Since excessive release of glutamate during anoxia is neurotoxic, agents such as galanin which activate K+ATP channels may provide tissue specific protection against anoxic damage.     

Hippocampal α2A-adrenergic receptors are located predominantly presynaptically but are also found postsynaptically and in selective astrocytes

Alpha-adrenergic receptor, subtype 2A (α_2A-AR), activation is one of the primary modes of action for norepinephrine (NE) in the rat hippocampal formation. In this study, α_2A-AR immunoreactivity (α_2A-AR-I) was localized by light and electron microscopy in the rat hippocampus and dentate gyrus by using a previously characterized antibody to the rat α_2A-AR. By light microscopy, dense α_2A-AR-I was observed in the pyramidal and granule cell layers. Diffuse and slightly granular α_2A-AR-I was found in the neuropil in all other laminae, notably stratum lacunosum-moleculare. Ultrastructurally, α_2A-AR-I was found in neuronal cytoplasm associated with large multivesicular-like organelles and with clusters adjacent to endoplasmic reticula and/or plasmalemma. The distribution of α_2A-AR-I in the strata oriens, radiatum, and lacunosum-moleculare of hippocampal CA1 and CA3 regions and in the molecular layer of the dentate gyrus was remarkably similar (n > 2,000 profiles examined): α_2A-AR-I was found in axons and terminals (∼40%), glia (∼30%), dendritic spines (∼25%), and dendritic shafts (∼5%). This mixed pre- and postsynaptic distribution was not seen in the stratum lucidum of the CA3 region and the dentate hilar region, where most α_2A-AR-I was found in axons (∼60%) and glia (∼30%). Alpha-2A—AR-labeled axons were small and unmyelinated; labeled terminals usually formed asymmetric synapses on unlabeled spines; and labeled dendritic spines were morphologically similar to pyramidal or granule cells. Dual labeling studies demonstrated that some axons contained α_2A-AR-I and tyrosine hydroxylase (TH), the catecholaminergic synthesizing enzyme, and that some TH-labeled terminals were in close proximity to α_2A-AR-labeled spines and glia. These studies demonstrate that hippocampal α_2A-AR-I is localized (1) presynaptically in both noncatecholaminergic and catecholaminergic terminals, (2) postsynaptically in the dendritic spines of pyramidal and granule cells near catecholaminergic terminals, and (3) in some glial processes. These results suggest several sites for NE to exert its effects on hippocampal α_2A-ARs. J. Comp. Neurol. 395:310–327. 1998. © 1998 Wiley-Liss, Inc.     

Differential Regulation of Preprotachykinin-A mRNA Expression in Striatum by Excitation of Hippocampal Neurons

In this report we have studied the influence of hippocampal neurons on neuropeptide mRNA expression in both dorsal and ventral striatum in the rat. Intrahippocampal unilateral kainic acid injections were performed in control animals and in animals with a unilateral 6-hydroxydopamine-induced dopamine deafferentation of the striatum. In situ hybridization combined with quantitative image analysis was used to study the expression of preprotachykinin A mRNA encoding the neuropeptides substance P and neurokinin A. The 6-hydroxydopamine-induced lesion caused a decrease of preprotachykinin A mRNA levels in the ipsilateral dorsal striatum and in both sides of the ventral striatum. In normal rats, the intrahippocampal kainic acid injection caused a twofold increase in preprotachykinin A mRNA in the limbic parts of the striatum, which are innervated by the hippocampus. No effect of the kainic acid injection was seen in the lateral parts of the dorsal striatum, a region which does not appear to be innvervated by the hippocampus. Animals with a 6-hydroxydopamine lesion showed a similar kainic acid-mediated increase in preprotachykinin A mRNA in parts of the ventral striatum. In the dopamine-lesioned dorsal striatum and ventral striatum the decreased preprotachykinin A mRNA levels were normalized by the intrahippocampal kainic acid injection. These results show that kainic acid-mediated excitation of hippocampal neurons causes a dopamine-independent induction of preprotachykinin A mRNA expression in parts of the ventral striatum, and reverses the dopamine deafferentation-induced decrease of preprotachykinin A mRNA in both dorsal and ventral striatum. Combined, our results suggest that hippocampal neurons can regulate preprotachykinin A mRNA expression in both the ventral and the dorsal striatum.     

Polymorphism of the Glutamate Transporter Protein EAAT2 and Migraine Transformation into Chronic Daily Headache

Background and Purpose

The progression of migraine into chronic daily headache involves multiple risk factors, but the main contributor is not known. Glutamate is the major excitatory neurotransmitter in central sensitization, which is an important process in the pathogenesis of migraine transformation. The glutamate transporter protein excitatory amino acid transporter 2 (EAAT2) is the primary modulator of glutamatergic neurotransmission, and genetic polymorphisms of its gene, EEAT2, have been identified. The aim of this study was to determine the effect of EAAT2 polymorphisms on migraine transformation into chronic daily headache.

Methods

We included 74 migraine patients with episodic attack (M-E) and 59 migraine patients with chronic daily headache (M-CDH). After amplifying EAAT2 by polymerase chain reaction, we assessed its genotype frequencies based on restriction fragment length polymorphisms. We reclassified all migraine patients into two groups according to their EAAT2 genotype, either with the A allele (n=62) or without it (n=71), and compared the clinical variables between the two groups.

Results

The genotype frequencies of EAAT2 polymorphisms did not differ between the M-E and M-CDH groups. Comparison between EEAT2 genotypes revealed that the frequency of analgesic usage was significantly higher among migraine patients with the A allele (12.9±1.6 days/month) than in those without the A allele (8.1±1.2 days/month; p=0.019). The other clinical variables of migraine did not differ between the two groups.

Conclusions

The results suggest that EEAT2 polymorphism contributes to the tendency toward frequent analgesic usage in migraine patients. This implies a potential genetic influence on the progression of migraine into chronic daily headache through the development of medication-overuse headache.     

β-淀粉样蛋白对海马神经元钙离子浓度的影响及其机制研究

目的　观察β-淀粉样蛋白 (Aβ1 -40 )对海马神经元内钙离子浓度的影响及尼莫地平的拮抗作用。方法　采用大鼠海马神经元的原代培养技术 ,分别用 MTT法和激光扫描共聚焦显微镜结合 Fluo-3 /AM标记观察神经元存活率和细胞内游离钙离子浓度变化。结果　 Aβ1 -40在较高浓度 (1μmol/L和 1 0μmol/L )下 ,对神经元存活率和 [Ca2 + ]i 的影响与对照组相比 ,差异均有显著性 (P <0 .0 5 ,P <0 .0 1 ) ;5 μmol/L尼莫地平可部分降低Aβ1 -40引起的 [Ca2 + ]i 升高。结论　 Aβ1 -40可引起培养海马神经元存活率下降及胞内钙离子浓度升高 ,尼莫地平有部分拮抗作用。     

Neurochemical Signaling of Reward and Aversion to Ventral Tegmental Area Glutamate Neurons

Ventral tegmental area (VTA) glutamate neurons signal and participate in reward and aversion-based behaviors. However, the neurochemical mechanisms that underlie how these neurons contribute to motivated behaviors is unknown. We used a combination of optical sensors to identify how distinct neurochemical inputs to VTA glutamate neurons participate in motivated behavior within female and male transgenic mice. Activity of glutamate inputs to VTA glutamate neurons increased for both reward-predicting and aversion-predicting cues and aversive outcomes, but subpopulations of glutamate inputs were increased or decreased by reward. For both reward and aversion-based cues and outcomes, activity of GABA inputs to VTA glutamate neurons mostly decreased. GCaMP recordings showed overall population increases in VTA glutamate neuron intracellular calcium during reward and aversion-based cues and outcomes. Electrophysiological recordings of VTA VGluT2 neurons showed that glutamate receptor activation increases firing while loss of excitation via glutamate receptor blockade decreases firing. GABA-A receptor activation decreased VTA glutamate neuron firing but GABA-A receptor blockade did not significantly change VTA glutamate neuron firing. Electrophysiological recordings in coordination with our sensor data suggest that glutamate inputs strongly regulate VTA glutamate neuron participation in diverse motivated behaviors.SIGNIFICANCE STATEMENT Glutamate and GABA are the primary excitatory and inhibitory neurotransmitters of the nervous system. However, identifying how these neurotransmitters regulate motivated behavior has remained challenging because of a lack of tools (1) capable of measuring neurotransmission at the temporal scale of motivated behaviors and (2) capable of capturing chemical signaling onto genetically-distinct neuronal populations. We have overcome these obstacles by implementing genetically-encoded fluorescent indicators to monitor both glutamate and GABA input dynamics exclusively to ventral tegmental area (VTA) glutamate neurons during reward and aversion-based behaviors. We identify that glutamate and GABA inputs to VTA glutamate neurons differentially and dynamically signal reward and aversion-based cues and outcomes. This research provides foundational evidence that links distinct neurotransmitters to motivated behaviors regulated by VTA glutamate neurons.     

Ion Dependence and Receptor Mediation of Glutamate Toxicity in the Immature Rat Hippocampal Slice

Glutamate (glu) is a major excitatory transmitter and a toxin in the brain. In the present study, the immature rat hippocampal slice was used to determine the morphology, topography, ionic mediation and receptor specificity of glu toxicity. Slices were exposed to glu for 30 min, and the damage was evaluated after 3 h of recovery in regular medium. The effects on glu toxicity of changes of [Ca2+], [Cl-] and [Na+] were determined. The receptor preference of glu was assessed by using the N-methyl-D-aspartate (NMDA) antagonist MK-801 and the kainate (KA)/quisqualate (QA) antagonist DNQX, alone or in combination. Further, to see whether glu produces cytotoxicity via osmolysis, the effects of hyperosmolal sucrose on glu toxicity were studied. Glu toxicity was similar to the previously described NMDA toxicity with regard to cytopathology, but differed in some aspects from that caused by KA and QA. The severity of the lesion was determined by the proximity of neurons to the incubation fluid, probably as a consequence of cellular accumulation of the amino acid. Omission of Ca2+ abolished glu toxicity in all neurons except the granule cells of the outer blade. This population was completely protected when Ca2+ was omitted and [Cl-] was reduced. Elevation of [Ca2+] markedly aggravated the lesion caused by glu. Substitution of isethionate for Cl- worsened the glu-induced damage, whilst the amino acid produced qualitatively different neuropathology when choline substituted for Na+. Apparently glu did not damage hippocampal nerve cells through an osmolytic mechanism as medium supplemented with 100 mM sucrose increased the toxicity of glu. Since the lesion produced by glu was more widespread in the presence of high [Ca2+], the effects of receptor antagonists were studied under this condition. MK-801 inhibited glu toxicity whereas DNQX had no effect. Combination of MK-801 and DNQX did not offer better protection than did MK-801 alone. The results suggest that Ca2+ is the main (but not single) determinant of glu toxicity in the immature hippocampal slice. The ionic requirements of glu neurotoxicity are identical to those of NMDA, but differ from those of KA and QA. The notion that glu is a selective NMDA agonist in the present model was confirmed by the protection of MK-801, and by the lack of an effect of DNQX. This is the first report demonstrating that the toxicity of glu is mediated by NMDA receptors in brain tissue which has developed normally. The findings indicate that specific blockade of NMDA receptors may be the most rational strategy in the prevention of glu-related neuronal death occurring in certain neurological anomalies.