Excitatory amino acid-induced release of 3H-GABA from cultured mouse cerebral cortex interneurons

A newly developed continuous superfusion model was used for studies of 3H-GABA release from cultured mouse cerebral cortex neurons. It was found that a series of excitatory amino acids (EAAs) representing all receptor subtypes evoked Ca2+- dependent release of 3H-GABA from the neurons. Quisqualate was the most potent agonist tested, with an EC50 value of 75 nM. L-Glutamate, N-methyl-D-aspartate (NMDA), and kainate showed EC50 values of 12, 16 and 29 microM, respectively. The EAA-evoked 3H-GABA release could be blocked by a series of EAA antagonists. The highly selective NMDA antagonist D-2-amino-5-phosphonovaleric acid (D-APV) was found to block NMDA responses, whereas the nonselective antagonists cis-2,3-piperidine dicarboxylic acid (PDA) and gamma-D-glutamyl-aminomethyl sulphonic acid (GAMS) blocked responses to all agonists. NMDA responses were found to be sensitive to Mg+ blockade. EAA- as well as potassium-induced 3H-GABA release from the neurons could be detected as early as day 5 in culture. However, during the culture period up to 12 d, the responses to K+, quisqualate, and NMDA were increased. The ontogenetic development of binding sites for quisqualate, kainate, and NMDA in mouse cortex was studied using the radioligands 3H-alpha-amino-3-hydroxy-5-methyl-4-isoxasole propionate (3H-AMPA), 3H-kainate, and 3H-L-glutamate, respectively. The development of binding sites for the different EAA-receptor subtypes showed a good correlation with the development of neuronal 3H-GABA release evoked by the excitatory amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of Serotonin on the Glutamate-Induced Excitations of Secondary Vestibular Neurons in the Rat

The excitatory responses evoked by glutamate and its agonists in secondary vestibular neurons of the rat were studied during microiontophoretic application of 5-hydroxytryptamine (5-HT). Ejection of 5-HT modified neuronal responsiveness to glutamate in 86% of the studied units, the effect being a depression of the excitatory responses in two-thirds of cases and an enhancement in the remaining third. 5-HT was also effective in modifying 94% of the responses evoked by N-methyl-d-aspartate (NMDA), inducing a depressive effect in 76% of cases and an enhancement in the remaining ones. Quisqualate-evoked effects were depressed and enhanced by 5-HT in about the same number of cases; in contrast, kainate-evoked responses were enhanced. The depressive action of 5-HT was mimicked by application of alpha-methyl-5-hydroxytryptamine (alpha-Me-5-HT), a 5-HT(2) receptor agonist, whereas the enhancing effect could be evoked by application of 8-hydroxy-2(di-n-propylamino)tetralin (8-OH-DPAT), a selective 5-HT(1A) receptor agonist. The 5-HT(2) receptor antagonist ketanserin was able to reduce, but not to block totally, the depressive action of 5-HT on glutamate- or NMDA-evoked responses. No significant difference was detected between neuronal responses in the lateral and the superior vestibular nucleus. These results indicate that 5-HT is able to modulate the responsiveness of secondary vestibular neurons to excitatory amino acids. Its action is mostly depressive, involves 5-HT(2) receptors, and is exerted on NMDA receptors. A minor involvement of other 5-HT receptors (at least 5-HT(1A)) and other glutamate receptors (for quisqualate and kainate) in the modulatory action of 5-HT is plausible.     

Neurophysiology and neuropharmacology of projections from entorhinal cortex to striatum in the rat

We studied projections from the entorhinal cortex (Ent) to the striatum in anesthetized rats using extra- and intracellular recording and multibarrel iontophoresis. The majority of recordings were from the caudate-putamen (CPu) and core of the nucleus accumbens (AcbC). Electrical stimulation of the Ent evoked synaptic responses in 77% of tests with AcbC neurons and 48% of tests with CPu neurons. In the case of AcbC neurons, 61% of these tests proved to be excitatory and were often followed by inhibitory phases. In contrast to this, only 18% of tests from CPu neurons were excitatory. Intracellular HRP labeling showed that responsive cells were medium spiny neurons.

During iontophoretic experiments, application of the glutamatergic AMPA antagonist DNQX could selectively decrease or block excitatory responses. The GABA_A antagonist bicuculline methiodide increased cellular firing rates and could reveal excitatory responses, suggesting block of a short-latency, short-duration inhibitory component. Ejection of the GABA_B antagonist CGP-35348 could attenuate a later, longer-duration component of inhibition. The results indicate that the Ent excites striatal neurons at least in part by glutamatergic receptors and suggest that this excitation is followed by secondary prolonged GABAergic inhibition.     

High concentrations of N-acetylaspartylglutamate (NAAG) selectively activate NMDA receptors on mouse spinal cord neurons in cell culture

We examined the membrane action of the endogenous dipeptide and putative neurotransmitter N-acetylaspartylglutamate (NAAG) on the excitatory amino acid receptors of cultured mouse spinal cord neurons using electrophysiological methods. Responses to NAAG (1 microM-5 mM) were compared to those elicited by N-methyl-D-aspartate (1 microM-1 mM) and L-glutamate (0.5-500 microM). Under voltage clamp, concentration-response curves of agonist-evoked currents demonstrated that NAAG was much less potent than either L-glutamate or N-methyl-D-aspartate (NMDA), so that inward currents could be evoked only at NAAG concentrations above 300 microM. Analysis of the dipeptide by high-pressure liquid chromatography showed no evidence of contamination by excitatory amino acids, suggesting that NAAG has an intrinsic, although weak, neuroexcitatory action on spinal neurons. Previous studies have shown that activation of NMDA receptors produces a voltage-dependent response. The current-voltage relationship of responses evoked by NAAG was also voltage-dependent. The peptide-activated conductance decreased with hyperpolarization in the presence of extracellular Mg2+, such that little inward current could be evoked at a membrane potential of -80 mV. In addition, responses to NAAG were completely antagonized by 250 microM DL-2-amino-5-phosphonovaleric acid, a specific NMDA-receptor antagonist. Application of NAAG in Mg2+-free medium resulted in an inward current with a large increase in membrane current noise. The spectral density function of this current noise could be fitted with a single Lorentzian with a decay time constant near 5 msec and a calculated single-channel conductance of 50-60 pS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrophysiological characteristic of corticoaccumbens synapses in rat mesolimbic system reconstructed using organotypic slice cultures

The nucleus accumbens (NAc) is a component of the mesolimbic system involved in drug dependence. Activity of nucleus accumbens neurons is modulated by glutamatergic afferents from the prefrontal cortex and by dopaminergic afferents from the ventral tegmental area (VTA). In the present study, we reconstructed the mesolimbic system using organotypic slice cultures and examined the effects of dopaminergic agents on synaptic activity in the prefrontal cortex-nucleus accumbens synapses. A slice of each of the prefrontal cortex, nucleus accumbens and ventral tegmental area in newborn rat, was arranged on a multi-electrode dish (MED) filled with culture medium so that they contacted each other, termed a 'triple culture'. Extracellular recording using microelectrodes on the multi-electrode dish showed that a single electrical stimulation of the prefrontal cortex slice evoked field excitatory postsynaptic potential, and that population spikes occurred spontaneously in the nucleus accumbens area of the triple culture. The amplitude of evoked field excitatory postsynaptic potentials and the frequency of spontaneous population spikes were decreased by glutamatergic antagonists, D(-)-2-amino-5-phosphonovaleric acid and 6-cyano-7-nitroquinoxaline-2,3-dione. The D1-like receptor agonist SKF38393, but not the D2-like receptor agonist quinpirole, reduced both the amplitude of field excitatory postsynaptic potential and frequency of spontaneous population spikes. Cocaine depressed field excitatory postsynaptic potential and this depression was reversed by D1-like receptor antagonist SCH23390, but not by D2-like receptor antagonist sulpiride. These results suggest that evoked field excitatory postsynaptic potentials and spontaneous population spikes were driven by glutamatergic neurons and were subject to exogenous and endogenous dopaminergic modulation in the triple culture that was similar to that shown in in vivo.     

Brain-derived neurotrophic factor acutely depresses excitatory synaptic transmission to GABAergic neurons in visual cortical slices

Brain-derived neurotrophic factor (BDNF) acutely modulates synaptic transmission to excitatory neurons in hippocampus and neocortex. The question of whether BDNF acts similarly on excitatory synaptic transmission to GABAergic neurons was eluded in previous studies using cortical slices. To address this question, we used transgenic mice in which expression of green fluorescence protein (GFP) is regulated by glutamic acid decarboxylase 67 (GAD67) promoter. In cortical slices prepared from these GAD67-GFP knock-in mice, we could detect GABAergic neurons under a fluorescent microscope. An application of BDNF rapidly depressed excitatory postsynaptic currents (EPSCs) evoked by layer IV stimulation in most GFP-positive neurons in layer II/III of the cortex. This effect was seen at synapses activated during the BDNF application and blocked by anti-TrkB IgG, indicating that the acute inhibitory action of BDNF is activity-dependent and mediated through TrkB. Paired-pulse ratios of the amplitude of EPSCs to paired stimulation at intervals of 10-100 ms were not significantly changed after BDNF application, suggesting that the site of depression may be postsynaptic. Responses to directly applied glutamate were also depressed by BDNF in most of neurons, being consistent with the interpretation of postsynaptic action of BDNF. The depressive action of BDNF was blocked by an intracellular injection of a Ca(2+) chelator, suggesting that a rise in Ca(2+) is involved in the acute depression of EPSCs. This action of BDNF was seen in 67% of parvalbumin (PV)-positive neurons, but in only 19% of PV-negative neurons, indicating that the depressive action is biased to PV-positive GABAergic neurons.     

Cholinergic suppression of excitatory synaptic responses in layer II of the medial entorhinal cortex

Theta-frequency (4-12 Hz) electroencephalographic activity is thought to play a role in mechanisms mediating sensory and mnemonic processing in the entorhinal cortex and hippocampus, but the effects of acetylcholine on excitatory synaptic inputs to the entorhinal cortex are not well understood. Field excitatory postsynaptic potentials (fEPSPs) evoked by stimulation of the piriform (olfactory) cortex were recorded in the medial entorhinal cortex during behaviors associated with theta activity (active mobility) and were compared with those recorded during nontheta behaviors (awake immobility and slow wave sleep). Synaptic responses were smaller during behavioral activity than during awake immobility and sleep, and responses recorded during movement were largest during the negative phase of the theta rhythm. Systemic administration of cholinergic agonists reduced the amplitude of fEPSPs, and the muscarinic receptor blocker scopolamine strongly enhanced fEPSPs, suggesting that the theta-related suppression of fEPSPs is mediated in part by cholinergic inputs. The reduction in fEPSPs was investigated using in vitro intracellular recordings of EPSPs in Layer II neurons evoked by stimulation of Layer I afferents. Constant bath application of the muscarinic agonist carbachol depolarized membrane potential and suppressed EPSP amplitude in Layer II neurons. The suppression of EPSPs was not associated with a substantial change in input resistance, and could not be accounted for by a depolarization-induced reduction in driving force on the EPSP. The GABA(A) receptor-blocker bicuculline (50 microM) did not prevent the cholinergic suppression of EPSPs, suggesting that the suppression is not dependent on inhibitory mechanisms. Paired-pulse facilitation of field and intracellular EPSPs were enhanced by carbachol, indicating that the suppression is likely due to inhibition of presynaptic glutamate release. These results indicate that, in addition to well known effects on postsynaptic conductances that increase cellular excitability, cholinergic activation in the entorhinal cortex results in a strong reduction in strength of excitatory synaptic inputs from the piriform cortex.     

Inhibition of spontaneous and evoked unit activity in the rat medial prefrontal cortex by mesencephalic raphe nuclei

The rat medial prefrontal cortex (PFC) receives a serotoninergic (5-HT) innervation which originates from the mesencephalic raphe nuclei. In the present study we determined the influence of the 5-HT ascending systems on the spontaneous and evoked activity of PFC neurons in anesthetized rats. Stimulation of the dorsal (DRN) and of the median raphe (MRN) nuclei inhibited the spontaneous activity of 35.0% and 52.8% of the PFC cells tested (mean duration of the inhibition: 75.5 and 82.2 ms, respectively). These inhibitory responses are likely mediated by the 5-HT-containing neurons since they were decreased markedly following selective destruction of ascending 5-HT pathways induced by local injections of 5,7-dihydroxytryptamine. Moreover, the inhibitory effect of MRN stimulation could be blocked by systemic administration of the 5-HT2 receptor antagonists: ketanserin and ritanserin. The effects of MRN stimulation on two types of evoked responses were studied. The excitatory responses of PFC neurons induced by the stimulation of the mediodorsal nucleus of the thalamus (MD) were inhibited by MRN stimulation applied before that of MD. Similarly, the activation of PFC cells induced by a noxious tail pinch was suppressed by a concomitant stimulation of the MRN. These results indicate that 5-HT neurons exert an inhibitory control on spontaneous or evoked activity in the rat PFC.     

The pharmacology of synapses formed by identified corticocollicular neurons in primary cultures of rat visual cortex

Primary cultures of neurons from the visual cortex of 7-10-d-old Long Evans rats were used to study the pharmacology of synaptic transmission. Dissociated cells were grown either in mass cultures, which contained 8000-10,000 neurons, or in miniature island cultures of 50-100 cells. Prior to dissociation, cells in layer V of visual cortex that project to the superior colliculus were labeled in vivo by retrograde transport of fluorescent latex microspheres-a permanent fluorescent marker. After 2 d to 8 weeks in culture, labeled neurons were identified by epifluorescent illumination, and electrophysiological recordings were obtained from a labeled cell and, simultaneously, from a nearby unlabeled neuron in the same field of view. The 2 neurons were stimulated sequentially by current injection and the pharmacology of evoked postsynaptic potentials (PSPs) was investigated. In mass cultures, relatively few pairs of neurons from which we recorded were synaptically connected, although nearly every cell exhibited abundant spontaneous EPSPs and IPSPs. Neurons grown on island cultures generally did not exhibit spontaneous synaptic activity; however, stimulation of one of the cells in a pair frequently elicited a short-latency PSP in the follower neuron. Retrogradely labeled corticocollicular neurons produced only excitatory PSPs in follower cells, while unlabeled neurons were either excitatory or inhibitory. Three antagonists of excitatory amino acid receptors, kynurenic acid, piperidine dicarboxylic acid, and gamma-D-glutamylglycine, completely blocked EPSPs produced by labeled corticocollicular neurons, as well as EPSPs produced by nearly all of the unlabeled excitatory cells. We have previously shown that these compounds block both N-methyl-D-aspartate (NMDA)-type and non-NMDA receptors on cultured cortical neurons (Huettner and Baughman, 1986). The specific NMDA receptor antagonist 2-amino-5-phosphonovaleric acid (APV) did not alter short-latency EPSPs recorded in 1 mM Mg2+, but did reduce longer-latency EPSPs polysynaptic activity. Since responses mediated by the NMDA receptor are known to be antagonized by Mg2+ (Mayer and Westbrook, 1985), we perfused cultures with Mg2+-free medium and found that the falling phase of some monosynaptic EPSPs was prolonged. Addition of APV to Mg2+-free medium reduced the duration of the falling phase of EPSPs such that they returned to the time course obtained in 1 mM Mg2+.(ABSTRACT TRUNCATED AT 400 WORDS)     

Plasticity of responses to synaptic inputs in rat ventral striatal neurons after repeated administration of the dopamine D2 antagonist raclopride

The effects of acute and repeated systemic administration of the dopaminergic D2 antagonist raclopride on responses of ventral striatal neurons were examined in chloral hydrate-anesthetized rats. Stimulating electrodes were placed in the entorhinal and perirhinal cortices, medial thalamus, and prelimbic cortex. Stimulation in water-injected control rats evoked one or occasionally two action potentials. Results were similar in rats injected acutely with raclopride (4 or 8 mg/kg) except that a small proportion of cells (5%) produced burst responses (defined as three or more evoked action potentials). In rats injected with raclopride daily for 7-14 days, burst responses were seen in a larger proportion of cells (17%) and bursts of up to nine action potentials could be evoked. The results suggest that repeated administration of dopaminergic agents can induce striking plastic changes of excitatory responses in a subset of ventral striatal neurons.     

Participation of low-threshold calcium spikes in excitatory synaptic transmission in guinea pig medial frontal cortex

We studied the activation of low-threshold calcium spikes (LTS) by excitatory postsynaptic potentials in pyramidal neurons from guinea pig medial frontal cortex with intracellular recording. We used extracellular bicuculline and phaclofen and intracellular QX-314 to block inhibitory synaptic potentials and sodium currents. Postsynaptic potentials were evoked by stimulation of layer I. We found that large (> 10-15 mV) excitatory synaptic potentials evoked from membrane potentials more negative than -75 mV were able to trigger LTS. The activation of LTS resulted in an increase of the rising slope or amplitude of the synaptic potentials depending on the size of the excitatory postsynaptic potential (EPSP). We used 100 microM NiCl2 to confirm the presence of LTS as part of the EPSPs. The N-methyl-D-aspartate (NMDA) and non-NMDA components of the excitatory synaptic potentials were isolated using (+/-)2-amino-5-phosphonovaleric acid (APV; 50 microM) or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 20 microM); both components could, independently, trigger an LTS. With recordings made with K+ acetate-filled electrodes, we show that the activation of LTS was critical to allow excitatory synaptic potentials to reach the threshold of action potential firing; also, this amplification of synaptic responses produced the firing of more than a single action potential by the postsynaptic cell. These results demonstrate that in cortical pyramidal neurons the activation of low-threshold calcium spikes results in the amplification of synaptic responses.     

Excitatory amino acid-evoked membrane currents and excitatory synaptic transmission in lamprey reticulospinal neurons

The characteristics of excitatory amino acid-evoked currents and of excitatory synaptic events have been examined in lamprey Müller neurons using voltage clamp and current clamp recording techniques. Application of glutamate evoked depolarizations associated with a decrease in input resistance. The reversal potential of the responses was -35 mV. Under voltage clamp conditions, a series of excitatory amino acid agonists evoked inward currents associated with little or no increase in baseline current noise. The order of potency of the excitatory amino acid agonists was quisqualate greater than kainate greater than glutamate greater than aspartate, while N-methyl-D-aspartic acid (NMDA) was inactive. Inward currents evoked by glutamate, as well as by kainate and quisqualate were attenuated reversibly by 1 mM kynurenic acid (KYN). In contrast, glutamate-evoked currents were not affected by 100 microM D(-)-2-amino-5-phosphonovaleric acid (APV), a selective NMDA antagonist. Spontaneously occurring and stimulus-evoked excitatory postsynaptic events were antagonized reversibly by 1 mM KYN. At this concentration, KYN had no effect on membrane potential, input resistance, or excitability of the cells. In contrast, excitatory postsynaptic currents were unaffected by APV. It is concluded that both glutamate responses and excitatory synaptic transmission in lamprey Müller neurons are mediated by non-NMDA-type receptors and that these receptors are associated with ionic channels with a low elementary conductance. The combined pharmacological and biophysical characteristics of these responses are therefore different from those previously reported in other preparations. Spontaneous (but not stimulus-evoked) inhibitory synaptic events in Müller neurons were blocked reversibly by 1 mM KYN but not by 100 microM APV, suggesting that excitation of interneurons inhibitory to Müller cells was also mediated by non-NMDA receptors.     

Evoked epileptiform discharges in the rat anterior piriform cortex: generation and local propagation

The purpose of this study was to identify cellular and synaptic properties of neurons in a small region within the anterior piriform cortex (aPC), termed the area tempestas (AT), responsible for triggering forebrain seizures in rats. Using a brain slice preparation, we performed whole-cell patch recordings from neurons in the regions overlapping the functionally defined AT. Local electrical stimulation activated synaptic inputs to neurons in these regions, collectively termed the deep aPC (daPC). Synaptic inputs were blocked by selective ionotropic glutamate receptor antagonists. Excitatory bursts were evoked from 59% of daPC neurons as the stimulus intensity was raised above a precise threshold. Secondary bursts (6-15 Hz) occurred in 34% of daPC neurons. Evoked bursts were synaptically driven, as they were blocked by TTX (1 microM) or 2, 3-dihydroxy-6-nitro-7-sulfamoyl-benzo(f)quinoxaline (NBQX, 1 microM), but not by inclusion of cesium and N-(2, 6-dimethylphenylcarbamoylmethyl) triethylammonium (QX-314) in the internal patch solution. Neither augmentation of excitatory nor suppression of inhibitory transmission were required to evoke bursts from daPC neurons. However, bicuculline (20 microM) lowered the threshold intensity for evoking discharges and increased the incidence and duration of evoked bursts, indicating active inhibitory control of daPC neurons. Stimulation in the daPC evoked epileptiform field potentials from layer II of the adjacent PC and bursts from layer II pyramidal neurons. This work demonstrates that synaptically dependent excitatory burst discharges can be evoked from daPC neurons without altering the balance between synaptic excitation and inhibition. Stimuli that trigger bursts in daPC neurons also generate epileptiform activity in layer II pyramidal cells, indicating that propagation of excitatory activity triggered from the daPC to the pyramidal neurons of the aPC can contribute to the initiation of seizures induced by disinhibition of the AT in vivo.     

Localization of glutaminase-like and aspartate aminotransferase-like immunoreactivity in neurons of cerebral neocortex

The distribution of glutaminase (GLNase)- and aspartate aminotransferase (AATase)-immunoreactive cells was examined in the cerebral neocortex of rat and guinea pig and in the somatic sensorimotor and primary visual cortex of the Macaca fascicularis monkey. These enzymes are involved in the metabolism of glutamate and aspartate, two amino acids thought to be excitatory amino acid transmitters for cortical neurons. In each of the species examined a large percentage of layer V and VI pyramidal neurons have pronounced glutaminase-like immunoreactivity (GLNase IR). In contrast, neurons in layers I, II, and IV show little GLNase IR. Layer III in the rat and guinea pig contains only a few, densely labeled GLNase-like-immunoreactive (GLNase-Ir) pyramidal neurons, whereas in the monkey the number of GLNase-Ir cells in layer III varies between cytoarchitectonic fields. Area 3b of the primary somatic sensory cortex and area 17 (primary visual cortex) contain few GLNase-Ir cells in layer III. However, layer III contains moderate numbers of GLNase IR in cells in areas 3a, 1, 2, 5, and in the primary motor cortex. Within the motor cortex the largest pyramidal ("Betz") cells are not labeled. In marked contrast to the results with antibody to GLNase, antibody to AATase labels cells that appear nonpyramidal in form, and these cells are in all cortical layers in each of the species examined. This distribution is roughly similar throughout all areas of rodent neocortex, but in monkey visual cortex AATase-immunoreactive neurons are more numerous in layers II-III, IVc, and VI. When combined with the findings of other studies, our results suggest that GLNase IR marks pyramidal neurons that use an excitatory amino acid transmitter. Antibody to AATase appears to mark intrinsic cortical neurons. The AATase immunoreactivity of these cells could indicate that they use an excitatory amino acid transmitter. However, their form and distribution in cortex suggest that this antibody labels GABAergic neurons.     

Unmyelinated cutaneous afferent neurons activate two types of excitatory amino acid receptor in the spinal cord of Xenopus laevis embryos

The trunk and tail skin of Xenopus laevis embryos near the time of hatching is innervated by the mechanoreceptive free nerve endings of Rohon-Beard neurons, a homogeneous class of cutaneous primary afferent fibers. Rohon-Beard neurons have cell bodies and axons in the dorsal spinal cord, where they monosynaptically excite a population of dorsolaterally situated interneurons (Clarke and Roberts, 1984). EPSPs can be recorded in these dorsolateral interneurons following electrical stimulation of the unmyelinated neurites of Rohon-Beard neurons in the skin. The EPSPs are dual component, consisting of separate fast and slow potentials that are usually evoked synchronously and that closely resemble those described previously in Xenopus and lamprey motoneurons (Dale and Roberts, 1985; Dale and Grillner, 1986). The excitation of dorsolateral interneurons by Rohon-Beard neurons is reduced by the bath application of excitatory amino acid antagonists. Kynurenic acid suppresses both the fast and slow components of the EPSPs, while both (+/-)-2-amino-5-phosphonovaleric acid (APV) and 1 mM magnesium reduce the slow component but have little or no effect on the peak amplitude of the EPSPs. These data suggest that Rohon-Beard neurons release an excitatory amino acid neurotransmitter, which acts simultaneously at both N-methyl-D-aspartate (NMDA) and non-NMDA receptor types. This is the first direct demonstration of dual-component excitatory amino acid-mediated synaptic transmission from cutaneous primary afferent neurons in the vertebrate spinal cord. The bath application of the agonists NMDA, kainate, or quisqualate in salines containing 1 microM TTX depolarized the interneurons and reduced their input resistance, which suggests that the interneurons possess all 3 types of excitatory amino acid receptor. Kynurenic acid strongly inhibits responses to NMDA and kainate, but is relatively less effective against the larger responses of quisqualate in this system.     

Development of binding sites for excitatory amino acids in cultured cerebral cortex neurons

Binding of [3H]glutamate, [3H]AMPA (RS-alpha-amino-3-hydroxy-5-methyl-4-isoxazolo-propionate) and [3H]kainate was investigated in membranes prepared from cerebral cortex of 4-day-old and adult mice and from cerebral cortex neurons cultured for different periods of time (2, 4, 8 and 14 days). For all ligands, the number of binding sites increased as a function of development both in vivo and in culture. A significant number of binding sites for the ligands could be demonstrated on the cultured neurons already after 2 days in culture. Scatchard analysis of the binding data showed a single population of binding sites for glutamate (KD approximately 200 nM) and kainate (approximately 6 nM) regardless of the developmental stage in vivo or in culture. In case of [3H] AMPA binding two binding sites with KD values of approximately 6 nM and 100-200 nM could be demonstrated both in vivo and in culture. Binding of [3H]glutamate to cultured neurons could be displaced by N-methyl-D-aspartate (100 microM) and quisqualate (3 microM) in an additive manner but D,L-4-aminophosphonobutyrate (100 microM) had no effect. AMPA binding to cultured neurons was much more (40-fold) sensitive than kainate binding to the newly developed AMPA selective antagonist NBQX (2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline) indicating that kainate and AMPA bind to independent binding sites. Monitoring membrane potentials in the cultured neurons using the lipophilic cation TPP+ (tetraphenylphosphonium) it was demonstrated that potassium (55 mM) as well as glutamate, AMPA and kainate (100 microM) could depolarize the neurons both at early (2 days) and late (9 days) developmental stages in culture. The demonstration of functionally active receptors for the 3 excitatory amino acids in both immature (2 days in culture) and mature (8-9 days in culture) neurons is discussed in the light of previous studies of the development as a function of the culture period of effects of excitatory amino acids in neurons. It is concluded that no simple correlation exists between expression of binding sites for the excitatory amino acids and their ability to induce cytotoxicity and neurotransmitter release.     

The perforant path projection from the medial entorhinal cortex layer III to the subiculum in the rat combined hippocampal–entorhinal cortex slice

Intracellular recordings were performed to examine the perforant path projection from layer III of the entorhinal cortex to the subiculum in rat combined hippocampal–entorhinal cortex slices. Electrical stimulation in the medial entorhinal cortex layer III caused short latency combined excitatory and inhibitory synaptic responses in subicular cells. In the presence of the GABA_A antagonist bicuculline and the GABA_B antagonist CGP-55845 A inhibition was blocked and isolated AMPA- or NMDA receptor-mediated EPSPs could be elicited. After application of the non-NMDA antagonist NBQX and the NMDA antagonist APV excitatory responses were completely blocked indicating a glutamatergic input from the neurons of the medial entorhinal cortex layer III. By stimulation from a close (< 0.2 mm) position in the presence of NBQX and APV and either CGP-55845 A or bicuculline we could record monosynaptic fast GABA_A or slow GABA_B receptor-mediated IPSPs, respectively. We compared synaptic responses in subicular cells induced by stimulation in the medial entorhinal cortex layer III with responses elicited by stimulation of afferent fibres in the alveus. The EPSPs of subicular cells induced by stimulation of alvear fibres could be significantly augmented by simultaneous activation of perforant path fibres originating in the medial entorhinal cortex layer III, while delayed activation of alvear fibres after stimulation of the perforant path resulted in a weak inhibition of the alveus evoked EPSPs. Thus, the perforant path projection activates monosynaptic excitation of subicular neurons. Therefore the entorhinal cortex does not only function as an important input structure of the hippocampal formation but is also able to modulate the hippocampal output via the entorhinal–subicular circuit.     

Intracellular neurophysiological analysis reveals alterations in excitation in striatal neurons in aged rats

Intracellular recordings were used to characterize the physiological changes underlying decreases in excitation observed in striatal neurons during the aging process. Rats were divided into 3 age groups: young (3-5 months), middle-aged (10-12 months) and aged (greater than 24 months). All experiments were performed in urethane-anesthetized rats. Recordings were obtained from 33 neurons in young, 17 in middle-aged and 20 in aged rats. When identified by intracellular injections of Lucifer yellow all recorded neurons were medium-sized spiny cells. Resting membrane potentials were at least -40 mV and action potentials greater than 35 mV. Postsynaptic responses were evoked by stimulation of frontal cortex. In all recorded neurons, regardless of age, excitatory postsynaptic potentials (EPSPs) could be evoked. However, the threshold currents for eliciting both EPSPs and synaptically driven action potentials were significantly higher in neurons obtained from aged rats than those recorded in the other two groups. Other changes in excitation in aged striatal neurons consisted of absence of spontaneously occurring EPSPs, higher current to induce firing by intracellular injections of depolarizing current and an inability of orthodromically induced action potentials to follow paired stimulation pulses to the cortex at short interpulse intervals. These data were interpreted to indicate that a combination of changes in synaptic connectivity and in membrane properties underlie the decreases in excitation. Together with our previous findings obtained from aged cats these results indicate that decreased neuronal excitability is a major effect of aging in the striatum.     

Synaptic transmission in human neocortex removed for treatment of intractable epilepsy in children

Synaptic transmission to pyramidal cells was studied in slices of neocortex resected from infants and children (n = 10, age 8 months to 13 years) undergoing surgical treatment for intractable epilepsy. Most specimens were from the least abnormal area of the resection. Stable intracellular recordings could be obtained for up to 8 hours. Most of the recorded neurons had electrophysiological characteristics similar to those of regular-firing pyramidal cells and were in layers III to V, which was confirmed by intracellular staining with Lucifer yellow. Local extracellular stimulation evoked a sequence of excitatory and inhibitory postsynaptic potentials. After application of the gamma-aminobutyric acid antagonist, bicuculline (10-30 microM), extracellular stimulation induced large excitatory postsynaptic potentials and epileptiform bursts. Spontaneous bursts occasionally occurred in bicuculline. This effect of bicuculline was observed in all the tissue samples, even those from infant patients (n = 4, age 8-16 months). Kynurenic acid depressed or abolished both spontaneous and stimulation-induced bursts. The competitive antagonist for N-methyl-D-aspartate receptors, DL-2-amino-5-phosphonopentanoic acid decreased the duration of bicuculline-induced bursts. These data provide evidence that, similar to rat and cat neocortex, excitatory and inhibitory amino acids are important transmitters to pyramidal cells in immature human neocortex.     

Activation of locus coeruleus neurons by nucleus paragigantocellularis or noxious sensory stimulation is mediated by intracoerulear excitatory amino acid neurotransmission

The nucleus paragigantocellularis (PGi), located in the rostral ventrolateral medulla, is one of two major afferents to the nucleus locus coeruleus (LC). Electrical stimulation of PGi exerts a robust, predominantly excitatory influence on LC neurons that is blocked by intracerebroventricular (i.c.v.) administration of the broad spectrum excitatory amino acid (EAA) antagonists kynurenic acid (KYN) or gamma-D-glutamylglycine (DGG), but not by the selective N-methyl-D-aspartate (NMDA) receptor antagonist 2-amino-7-phosphonoheptanoate (AP7). I.c.v. injection of KYN or DGG also blocked activation of LC neurons evoked by noxious somatosensory stimuli. These results indicate that activation of LC neurons by PGi and noxious stimuli may be mediated by an EAA acting at a non-NMDA receptor in LC. In the present study, microiontophoretic techniques were used to determine the sensitivity of LC neurons in vivo to the selective EAA receptor agonists kainate (KA), NMDA and quisqualate (QUIS). Microinfusion and microiontophoresis were also used to determine whether direct application of KYN, the preferential non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3 dione (CNQX) or the selective NMDA receptor antagonist 2-amino-5-phosphonovalerate (AP5) onto LC neurons blocked excitation elicited by stimulation of PGi or the sciatic nerve. The results demonstrated that individual LC neurons were robustly activated by direct application of KA, NMDA and QUIS. Iontophoretically applied KYN reduced or completely antagonized responses evoked by all 3 agonists. In contrast, iontophoretically applied AP5 strongly attenuated NMDA-evoked excitation, while KA-and QUIS-evoked responses were not affected by this agent. Furthermore, direct application of KYN or the specific non-NMDA receptor antagonist, CNQX, onto LC neurons substantially attenuated or completely blocked synaptic activation produced by PGi or sciatic nerve stimulation in nearly every LC neuron tested. Microinfusion of the selective NMDA receptor antagonist AP5 had no effect on sciatic nerve-evoked responses. These results confirm our hypothesis that activation of LC neurons from PGi is mediated by an EAA operating primarily at a non-NMDA receptor subtype on LC neurons. Furthermore, these findings provide additional support for the hypothesis that this pathway mediates at least some sensory-evoked responses of LC neurons.