Tetanic stimulation of schaffer collaterals induces rhythmic bursts via NMDA receptor activation in rat CA1 pyramidal neurons

Exploring the principles that regulate rhythmic membrane potential (Vm) oscillations and bursts in hippocampal CA1 pyramidal neurons is essential to understanding the theta rhythm (theta). Recordings were performed in vitro in hippocampal slices from young rats, and a group of the recorded CA1 pyramidal cells were dye-filled with carboxifluorescein and immunolabeled for the R1 subunit of the NMDA receptor. Tetanic stimulation of Schaffer collaterals (SCs) and iontophoresis of glutamate evoked rhythmic Vm oscillations and bursts (approximately 10 mV, approximately 7 Hz, 2-5 spikes per burst) in cells (31%) placed close to the midline ("medial cells"). Rhythmic bursts remained under picrotoxin (10 microM) and Vm oscillations persisted with tetrodotoxin (1.5 microM), but bursts were blocked by AP5 (25 microM) and Mg2+-free solutions. Depolarization and AMPA never induced rhythmic bursts. The rest of the neurons (69%), recorded closer to the CA3 region ("lateral cells"), discharged rhythmically single repetitive spikes under SC stimulation and glutamate in control conditions, but fired rhythmic bursts under similar stimulation, both when NMDA was applied and when non-NMDA receptors were blocked with CNQX (20 microM). Medial cells exhibited a larger NMDA current component and a higher NMDAR1 density at the apical dendritic shafts than lateral cells, suggesting that these differences underlie the dissimilar responses of both cell groups. We conclude that the "theta-like" rhythmic oscillations and bursts induced by glutamate and SC stimulation relied on the activation of NMDA receptors at the apical dendrites of medial cells. These results suggest a role of CA3 pyramidal neurons in the generation of CA1 theta via the activation of NMDA receptors of CA1 pyramidal neurons.     

Activation of N-methyl-d-aspartate Receptors Induces Endogenous Rhythmic Bursting Activities in Nucleus Tractus Solitarii Neurons: An Intracellular Study on Adult Rat Brainstem Slices

A brainstem slice preparation and intracellular recording techniques were used to examine the effects of N-methyl-d-aspartate (NMDA) application on neurons within the swallowing area of the nucleus tractus solitarii (NTS). According to their cellular properties, NTS neurons were classified into type I and type II neurons. The most striking difference was the occurrence of delayed excitation in type I but not in type II neurons, when they were depolarized from membrane potentials more negative than -60 mV. Bath application of NMDA (30 - 60 microM) elicited depolarization and triggered stable repetitive firing in all the NTS neurons but one. During the NMDA-induced depolarization, hyperpolarization below -60 mV elicited, in some type I neurons, a rhythmic bursting pattern. The duration of the bursts (300 - 1000 ms) and their frequency (0.5 - 2 Hz) depended on the membrane potential. With hyperpolarizations below -75 mV, rhythmic bursting was converted into rhythmic single discharges, a pattern elicited directly in the other type I neurons. In all cases, rhythmic patterns were superimposed on cyclic depolarizations of the membrane potential characterized by an initial ramp-shaped phase. In type II neurons, rhythmic bursting discharges, superimposed on rhythmic oscillations of the membrane potential, were also obtained upon hyperpolarization during the NMDA-induced depolarization. In all type I neurons tested, NMDA-induced cyclic ramp-shaped depolarizations continued after addition of tetrodotoxin to the medium. Rhythmic bursting was not elicited by bath application of kainate (10 - 20 microM). Application of d-2-amino-5-phosphonovalerate (50 microM) blocked NMDA-induced depolarizations without modifying those elicited by kainate, which were selectively depressed by 6-cyano-7-nitroquinoxaline-2,3-dione (10 microM). Moreover, removal of Mg2+ from the medium suppressed NMDA-induced cyclic depolarizations. Results demonstrate that both NMDA and non-NMDA receptors are present in NTS neurons and that selective activation of NMDA receptors induced rhythmic bursting and/or rhythmic single discharges. Rhythmic patterns were not driven by synaptic mechanisms but originated from endogenous properties of NTS neurons activated by NMDA. Thus, NTS neurons can be considered as conditional pacemakers. According to the location of the neurons, the conditional properties shown in these in vitro experiments might be involved in vivo in the generation of rhythmic motor activities set up at the NTS level, such as swallowing.     

The effects of selective glutamate receptor antagonists on synchronized firing bursts in layer III of rat visual cortex.

In the rat visual cortex in vitro, single-shock stimulations applied to the border between layer VI and the white matter evoke synchronized burst-firing by units in layer III. We have examined the effects of glutamate receptor antagonists on this activity, with antagonists applied via the bath to allow correlation of effects with concentrations. All synaptically driven components (recorded extracellularly as field potential 'S2' spikes, dipoles 'W1' and 'W2', and coinciding single-unit spikes) were inhibited by greater than 90% in 1.0 mM kynurenic acid and in 3 or 10 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, which selectively blocks AMPA/kainate receptors). S2 spike amplitudes were reduced by half in 0.7 microM CNQX. 2-Amino-5-phosphonovalerate (APV), a specific blocker of NMDA receptors, did not prevent S2 spike burst or horizontal spread of bursting within layer III. However, APV reduced the duration of synchronized bursts and the slower potentials which followed. In Mg(2+)-free medium, new components appeared which were APV-sensitive: (1) low amplitude spikes, distributed spatially like S2 spike, but recurring more slowly, and (2) slow potentials, distributed spatially like W1 and W2 potentials, but lasting for hundreds of milliseconds. The amplitudes of these spikes were reduced by half in 3 microM D-APV. Our data imply that: (1) glutamate receptors play a major role in mediating local, excitatory neurotransmission in the supragranular layers of neocortex, with NMDA and AMPA/kainate subtypes each subserving somewhat different functions; (2) AMPA/kainate receptors mediate rapid excitatory transmission between layer III neurons, responsible for driving the first 15 ms of synchronized bursts; (3) currents gated by NMDA receptors determine the duration of coherent firing bursts, and drive asynchronous neuronal firing following bursts; and (4) under conditions which circumvent block by extracellular Mg2+, activation of NMDA receptors greatly enhances and prolongs the response to single-shock stimulations. In vivo, activation of layer III neurons is likely to depend significantly upon currents gated by NMDA receptors whenever repetitively firing excitatory inputs summed over several tens of milliseconds provide enough depolarization to lift block by extracellular Mg2+.     

Different changes in spontaneous field potential oscillations precede epileptiform bursting in hippocampal slices perfused with penicillin or reduced magnesium

Power spectra were used to analyse spontaneous field potentials (SFPs) recorded in the CA3 distal apical dendritic region of guinea pig hippocampal slices perfused with either penicillin or reduced Mg2+. High concentrations of penicillin (2000 IU/ml) progressively converted the low amplitude, irregular oscillations observed in control medium to higher amplitude, low frequency, rhythmic oscillations at approximately 2-3 Hz just prior to the onset of spontaneous, synchronized bursting. Low concentrations (50-300 IU/ml) increased the power of frequencies below 10 Hz and suppressed higher frequencies in a dose-dependent fashion. Although Mg2(+)-free medium also increased the magnitude of the SFPs prior to the onset of synchronous bursting, the changes were smaller than with penicillin and the frequency distribution was completely different. Low concentrations of Mg2+ (0.0-0.5 mM) increased the power across all frequencies, however, the maximal effect was on frequencies between 5 and 25 Hz. The transition from normal to epileptiform activity may proceed through at least 2 distinct intermediate states. When recurrent inhibition is blocked (penicillin), synchronous synaptic activity precedes the onset of bursting, whereas non-specific increases in excitability and activation of NMDA receptors (reduced Mg2+) produce an asynchronous transition state.     

The induction and modification of voltage-sensitive responses in cat neocortical nuerons by N-methyl-d-aspartate

The actions of the excitatory amino acid, N-methyl-D-aspartate (NMDA), on layer V neurons of cat sensorimotor cortex were examined in an in vitro slice preparation using current clamp, single electrode voltage clamp (SEVC), and ionic substitution techniques. Low doses of NMDA evoked a slow depolarization with a net decrease of input conductance. Larger doses additionally evoked repetitive firing, rhythmic depolarization shifts (DSs), low-threshold calcium spikes (in the presence of TEA+) and bistable membrane potential behavior. Ionic substitution experiments suggested that entry of both Ca2+ and Na+ ions contributed to the NMDA responses. Attention was focused on the NMDA response with Ca2+ entry blocked. Examination by SEVC revealed that, in both normal cells and in the presence of several blocking agents, NMDA induced a highly voltage-dependent inward ionic current which could result in a region of negative slope conductance on the cell's current-voltage relation. The development of this current seems capable of accounting for all aspects of the observed response, including the DSs and low-threshold Ca2+ spikes. Substitution of TEA+ for most external Na+ (with Ca2+ entry blocked) largely eliminated the NMDA responses and corresponding ionic current. Our results in neocortical neurons are compared to those recently obtained in cultured murine neurons.     

Cellular mechanisms underlying the rhythmic bursts induced by NMDA microiontophoresis at the apical dendrites of CA1 pyramidal neurons

This article reports the cellular mechanisms underlying a form of intracellular "theta-like" (theta-like) rhythm evoked in vitro by microiontophoresis of N-methyl-D-aspartate (NMDA) at the apical dendrites of CA1 pyramidal neurons. Rhythmic membrane potential (Vm) oscillations and action potential (AP) bursts (approximately 6 Hz; approximately 20 mV; approximately 2-5 APs) were evoked in all cells. The response lasted approximately 2 s, and the initial oscillations were usually small (< 20 mV) and below AP threshold. Rhythmic bursts were never evoked by imposed depolarization in the absence of NMDA. Block of Na+ conductance with tetrodotoxin (TTX) (1.5 microM), of non-NMDA receptors with 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) (20 microM) and of synaptic inhibition by bicuculline (50 microM) and picrotoxin (50 microM) did not prevent NMDA oscillation. Inhibition of the voltage dependence of the NMDA conductance in Mg2+-free Ringer's solution blocked oscillations. Preventing Ca2+ influx with Ca2+-free and Co2+ (2-mM) solutions and block of the slow Ca2+-dependent afterhyperpolarization (sAHP) by carbamilcholine (5 microM), isoproterenol (10 microM), and intracellular BAPTA blocked NMDA oscillations. Inhibition of L-type Ca2+ conductance with nifedipine (30 microM) reduced oscillation amplitude. Block of tetraethylammonium (TEA) (10 mM) and 4AP (10 mM)-sensitive K+ conductance increased the duration and amplitude, but not the frequency, of oscillations. In conclusion, theta-like bursts relied on the voltage dependence of the NMDA conductance and on high-threshold Ca2+ spikes to initiate and boost the depolarizing phase of oscillations. The repolarization is initiated by TEA-sensitive K+ conductance and is controlled by the sAHP. These results suggest a role of interactions between NMDA conductance and intrinsic membrane properties in generating the CA1 theta-rhythm.     

The Influence of Magnesium Ions on the NMDA Mediated Responses of Ventral Rhythmic Neurons in the Spinal Cord of Xenopus Embryos

Xenopus embryos immobilized in tubocurarine respond to natural skin stimulation with fictive swimming. This can also occur in saline without Mg2+ and is blocked by NMDA antagonists. Ventral spinal cord neurons which are rhythmically active during swimming are depolarized by bath applied N-methyl-D-aspartate (NMDA) (in 1 microM tetrodotoxin (TTX) to block indirect effects). By using current clamp techniques this depolarization is shown to be partially blocked by 0.5 and 1 mM Mg2+ in a voltage-dependent manner similar to that described in cultured neurons. Mg2+ partially and reversibly reduces the slow NMDA-mediated component of excitatory post-synaptic potentials (EPSPs) in ventral neurons. However, in 1 mM Mg2+ fictive swimming can still be evoked by natural stimulation. The frequency of swimming is slightly lower than in nominally 0 mM Mg2+, but the pattern of ventral root activity and synaptic drive to ventral neurons seems little affected. Fictive swimming can also be induced by applying NMDA to spinal preparations. In 0 mM Mg2+, such rhythmic activity is unstable and transient over a narrow NMDA concentration range. In 0.5 mM Mg2+, continuous rhythmic activity is induced over a wide range of NMDA concentrations. Lower spinal preparations need higher NMDA concentrations to induce activity. We conclude that the neurons rhythmically active in swimming have NMDA receptor channels which show a voltage dependent block in the presence of Mg2+. However, while Mg2+ exerts a powerful stabilizing influence on rhythmic activity induced in spinal embryos by exogenous NMDA, its influence on 'naturally' evoked fictive swimming is less clear. The fictive swimming machinery in the brain and spinal cord can produce stable swimming with or without Mg2+ induced voltage dependency of the NMDA channels.     

Evidence for a specific role for cortical NMDA receptors in slow-wave sleep

Iontophoresis of the N-methyl-D-aspartic acid (NMDA) receptor antagonist 2-amino-5-phosphonovaleric acid (2-APV) was found to suppress spontaneous bursting activity of layer V cortical neurones during stage 3/4 sleep in unrestrained, normally behaving rats. Iontophoresis of NMDA, on the other hand, increased cortical burst durations and increased the number of spikes per burst. 2-APV was found not to alter cells' responses to tactile stimulation or the generation of neuronal spindling activity during stage 2 sleep. These results provide the first evidence that NMDA receptors subserve a specific function in the neocortex of the behaving animal, by gene-rating burst activity in cortical neurones during stage 3/4 of the natural sleep state. The activation of NMDA/2-APV-sensitive cortical receptors by afferents from the anterior intralaminar nuclei in the generation of bursts by cortical cells during stage 3/4 sleep is discussed.     

Differential mechanisms of Ca2+ responses in glial cells evoked by exogenous and endogenous glutamate in rat hippocampus

The mechanisms of Ca2+ responses evoked in hippocampal glial cells in situ, by local application of glutamate and by synaptic activation, were studied in slices from juvenile rats using the membrane permeant fluorescent Ca2+ indicator fluo-3AM and confocal microscopy. Ca2+ responses induced by local application of glutamate were unaffected by the sodium channel blocker tetrodotoxin and were therefore due to direct actions on glial cells. Glutamate-evoked responses were significantly reduced by the L-type Ca2+ channel blocker nimodipine, the group I/II metabotropic glutamate receptor antagonist (S)-alpha-methyl-4-carboxyphenylglycine (MCPG), and the N-methyl-D-aspartate (NMDA) receptor antagonist (+/-)2-amino-5-phosphonopentanoic acid (APV). However, glutamate-induced Ca2+ responses were not significantly reduced by the non-NMDA receptor antagonist 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX). These results indicate that local application of glutamate increases intracellular Ca2+ levels in glial cells via the activation of L-type Ca2+ channels, NMDA receptors, and metabotropic glutamate receptors. Brief (1 s) tetanization of Schaffer collaterals produced increases in intracellular Ca2+ levels in glial cells that were dependent on the frequency of stimulation (> or =50 Hz) and on synaptic transmission (abolished by tetrodotoxin). These Ca2+ responses were also antagonized by the L-type Ca2+ channel blocker nimodipine and the metabotropic glutamate receptor antagonist MCPG. However, the non-NMDA receptor antagonist CNQX significantly reduced the Schaffer collateral-evoked Ca2+ responses, while the NMDA antagonist APV did not. Thus, these synaptically mediated Ca2+ responses in glial cells involve the activation of L-type Ca2+ channels, group I/II metabotropic glutamate receptors, and non-NMDA receptors. These findings indicate that increases in intracellular Ca2+ levels induced in glial cells by local glutamate application and by synaptic activity share similar mechanisms (activation of L-type Ca2+ channels and group I/II metabotropic glutamate receptors) but also have distinct components (NMDA vs. non-NMDA receptor activation, respectively). Therefore, neuron-glia interactions in rat hippocampus in situ involve multiple, complex Ca2+-mediated processes that may not be mimicked by local glutamate application.     

Competitive NMDA receptor antagonists differentially affect dopamine cell firing pattern

Alterations in the firing pattern of mesencephalic dopamine (DA) neurons appear to constitute a physiological mechanism through which these cells modify their effects on target neurons. Several lines of evidence suggest that the activity patterns exhibited by DA cells in vivo are contingent on tonic activation of N-methyl-D-aspartate (NMDA) receptors. In the present series of experiments, extracellular single unit recording techniques were used to assess the effects of the centrally acting, competitive NMDA receptor antagonists CGS-19755, (±)-CPP, NPC-12626 and NPC-17742 on the firing properties of nigral DA neurons in the chloral hydrate-anesthetized rat. Each of the drugs tested produced a modest increase in firing rate accompanied by a significant regularization of neuronal firing pattern. Although the number of bursts and the percentage of spikes in bursts were reduced, the proportion of cells operationally defined as bursting was not appreciably altered. This appeared to be due to the ability of these drugs to reduce the number of spike doublets without affecting the incidence of longer bursts. Although generally consistent with the notion that NMDA receptors modulate DA neuronal firing pattern, the present data do not support the contention that tonic activation of these receptors is solely responsible for the expression of bursting activity in vivo. Synapse 25:234–242, 1997. © 1997 Wiley-Liss, Inc.     

Presynaptic site of action underlying the ethanol-induced inhibition of norepinephrine release evoked by stimulation of N-methyl-D-aspartate (NMDA) receptors in rat cerebral cortex.

Rat brain cortex synaptosomes pre-incubated with [3H]norepinephrine were used (1) to provide evidence that part of the NMDA receptors mediating stimulation of norepinephrine (NE) release are located on the noradrenergic varicosities themselves, (2) to characterize these receptors and (3) to examine whether ethanol specifically inhibits the NMDA-evoked NE release via a presynaptic site of action. In synaptosomes superfused with Mg(2+)-free Krebs-Henseleit solution, NMDA (2-min exposure) stimulated tritium overflow in a concentration- and glycine-dependent manner. The stimulatory effect of NMDA was not altered by tetrodotoxin but was abolished by omission of Ca2+ from the superfusion fluid and was considerably reduced in the presence of 1.2 mM Mg2+. DL-(E)-2-Amino-4-methyl-5-phosphono-3-pentanoic acid (CGP 37849; a competitive NMDA receptor antagonist) produced a parallel shift of the concentration-response curve for NMDA to the right, whereas dizocilpine (MK-801; an antagonist at the phencyclidine, PCP, recognition site of the NMDA-gated ion channel) reduced the maximum effect of NMDA. Ethanol inhibited the NMDA-evoked tritium overflow in a concentration-dependent manner. In contrast, in synaptosomes superfused with Ca(2+)-free Krebs-Henseleit solution containing 15 mM K+ throughout, ethanol did not affect the tritium overflow evoked by 2 min introduction of 75 microM Ca2+ into the superfusion fluid. This Ca(2+)-evoked overflow was also not altered by tetrodotoxin and dizocilpine, but was inhibited by the inorganic Ca2+ channel antagonist Cd2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of the organization of background activity of the septal neurons of the guinea pig in vitro

Activity of medial septum-diagonal band neurons (MS-DB-neurons) was investigated extracellularly in slices of the guinea pig septum. Four groups of units were differentiated on the basis of pattern of discharges and coefficient of variation (CV) of interspike intervals: highly regular (CV less than 0.3); regular (CV greater than 0.3 less than 0.7); irregular (CV greater than 0.7); and rhythmically bursting group. Activity of the 1st group was highly resistant to superfusion of medium with low Ca2+, high Mg2+ (or Co2+) concentration, while synaptic effects were blocked under this condition. The same was true for some cells with rhythmic bursts. The activity of Mg2+-resistant rhythmically bursting cells did not change its pattern also under the influence of GABA- and acetylcholine antagonists. It is concluded that the MS-DB contains units with properties of regular and bursting endogenous pacemakers.     

Developmental and circadian changes in Ca2+ mobilization mediated by GABAA and NMDA receptors in the suprachiasmatic nucleus

The hypothalamic suprachiasmatic nucleus (SCN) develops as the circadian pacemaker during postnatal life. Although both GABAA and NMDA receptors are expressed in the majority of SCN neurons, postnatal development of their functions has not been analysed. Thus, we studied the receptor-mediated Ca2+ responses in mouse hypothalamic slices prepared on postnatal days (P) 6-16. The NMDA-induced Ca2+ flux was prominent in the SCN and maximal Ca2+ responses in Mg2+-free conditions had no day-night variations in P14-16 mice. At P6-7, extracellular Mg2+ reduced the NMDA-induced Ca2+ flux irrespective of the circadian time whereas, after P9-10, Mg2+ produced a larger reduction at night than during the daytime. Muscimol also significantly increased Ca2+ in the developing SCN. Voltage-sensitive Ca2+ channel blockers inhibited the muscimol-induced Ca2+ increase whereas tetrodotoxin had no effect, suggesting that stimulation of postsynaptic GABAA receptors depolarizes SCN neurons to increase Ca2+. Macroscopic imaging analysis demonstrated a developmental reduction in the muscimol-induced Ca2+ increase preferentially in the nighttime group older than P9-10. The day-night variation in the magnitude of the Ca2+ response was due to two cell populations, one of which exhibited an increase and the other a decrease in Ca2+ in response to muscimol. Because the critical developmental stages for exhibiting day-night variations in the receptor-mediated Ca2+ responses overlapped the maturation of firing rhythms in SCN neurons, the Ca2+ signalling may be necessary for or regulated by the mature circadian clock.     

Modulation of N-methyl-D-aspartate receptor-mediated increases in cytosolic calcium in cultured rat cerebellar granule cells.

The concentration of intracellular free Ca2+ ([Ca2+]i) was measured in rat cerebellar granule cells using the fluorescent indicator fura-2. Culturing the cells as monolayers on plastic squares which could be placed into cuvettes allowed measurements of [Ca2+]i to be performed on large and homogeneous populations of CNS neurons. Granule cells so cultured maintained low levels of [Ca2+]i (around 90 nM) which increased promptly upon the addition of various excitatory amino acids including N-methyl-D-aspartate (NMDA). Increases in [Ca2+]i elicited by NMDA were inhibited by Mg2+ (1 mM) and often potentiated by glycine (1 microM). The addition of TTX or strychnine (5 microM each) did not alter responses to NMDA or NMDA plus glycine. Cytosolic Ca2+ responses to NMDA/glycine were dependent on the presence of extracellular Ca2+ and were unaffected by concentrations of nifedipine or verapamil that blocked increases in [Ca2+]i elicited by K+ depolarization. Responses elicited by NMDA/glycine were inhibited competitively by 2-amino-5-phosphonovalerate or 3-((+-)-2-carboxypiperazin-4-yl)-propyl-1- phosphonic acid and non-competitively by MK-801 or Mg2+. HA-966 and 7-chlorokynurenate inhibited responses to NMDA alone and blocked competitively the potentiating effects of glycine. The results demonstrate NMDA-mediated increases in [Ca2+]i in cerebellar granule cells that arise solely from influx of extracellular Ca2+ through dihydropyridine-insensitive channels. The strict dependence of the NMDA-evoked response on extracellular Ca2+ provides little evidence for a coupling of NMDA receptors to inositol phosphate metabolism and mobilization of intracellular Ca2+. The effect of various agents on NMDA/glycine-induced increases in [Ca2+]i parallels their effects on ligand binding to or current flow through the NMDA receptor-channel complex. The measurement of cytosolic Ca2+ in this preparation of neuronal cells thus appears especially well suited for assessing, on a functional level, the regulation of NMDA receptors in the CNS.     

NMDA-dependent, network-driven oscillatory activity induced by bicuculline or removal of Mg2+ in rat olfactory bulb neurons

Spontaneous, low-frequency voltage oscillations (LFOs) were observed in the neurons of rat olfactory bulb upon disinhibition with GABAA antagonists and/or removal of Mg2+ from external saline. Ordinarily, LFOs presented a highly organized temporal structure, with bursts recurring regularly at about 0.05 Hz. Slow depolarizing shifts with similar frequencies were observed in all types of bulbar neurons. Simultaneous recordings from mutually independent neurons showed that LFOs were highly synchronized in distinct cells. The occurrence of LFOs was prevented by NMDA, but not AMPA/kainate, receptor antagonists. The oscillations were also halted by Ca2+ antagonists and tetrodotoxin. The pace of the oscillations was reset by stimulation of the olfactory nerve but not by direct injection of depolarizing current into the oscillating cell. Removal of the outer portion of the slice with a cut along the external plexiform layer provided crucial evidence that the bursting activity first initiated in the glomerular region and propagated synaptically downstream towards the inner layers, suggesting an organizing role for olfactory glomeruli.     

Immunohistochemical differentiation of electrophysiologically defined neuronal populations in the region of the rat hypothalamic paraventricular nucleus.

Intracellular recording and labeling were combined with neurophysin immunohistochemistry to study neurons in the paraventricular nucleus region of the rat hypothalamus. Neuronal membrane properties were examined in hypothalamic slices, and cells were labeled by injecting biocytin or Lucifer yellow. Slices were then embedded, sectioned, and immunohistochemically processed for neurophysin. Immunoreactivity patterns, and in some cases counterstaining, enabled determinations of the cytoarchitectonic positions of recorded cells to be made. Recorded cells were divided into three types according to their electrophysiological characteristics. The first type lacked low-threshold Ca2+ spikes and displayed linear current-voltage relations, a short time constant, and evidence for an A current. These were relatively large cells that were typically immunoreactive for neurophysin and were situated near other neurophysin-positive neurons. The second type had relatively small low-threshold potentials that did not generate bursts of Na+ spikes. These cells had heterogeneous current-voltage relations and intermediate time constants. They did not label for neurophysin, and most were located in the parvicellular subregion of the paraventricular nucleus. The third type had large low-threshold Ca2- spikes that generated bursts of Na+ spikes, and these cells had nonlinear current-voltage relations and long time constants. These neurons were dorsal or dorsolateral to the paraventricular nucleus and were not immunoreactive for neurophysin. These results indicate that paraventricular magnocellular neurons lack low-threshold potentials, whereas paraventricular parvicellular neurons display low-threshold potentials that generate one or two action potentials. Neurons that fire spike bursts from low-threshold potentials are adjacent to the paraventricular nucleus, confirming earlier reports.     

Glutamate stimulation of tyrosine hydroxylase is mediated by NMDA receptors in the rat striatum.

We have studied the action of glutamate on striatal tyrosine hydroxylase activity and determined which type of glutamate receptors are involved. Glutamate stimulated (EC50 = 4 +/- 2 microM) the activity of tyrosine hydroxylase in slices of rat neostriatum. The selective N-methyl-D-aspartate (NMDA) receptor antagonist 2-amino-5-phosphonovalerate (10 microM) blocked the stimulation; however, both the non-NMDA receptor antagonist glutamate diethyl ester (10 microM) and the general excitatory amino acid antagonist kynurenate (10 microM) had no effect. NMDA was even more potent than glutamate in stimulating tyrosine hydroxylase activity. Quisqualate (100 microM) only slightly stimulated the enzyme, and kainate had practically no effect. Omission of Mg2+ from the incubation medium potentiated the glutamate stimulation. Neither tetrodotoxin nor atropine prevented the stimulation. These results suggest that glutamate stimulates striatal tyrosine hydroxylase activity via NMDA receptors. The lack of effect of tetrodotoxin and atropine suggests that glutamate acts on NMDA receptors located on the dopaminergic nigrostriatal terminal. The stimulation may involve the entry of Ca2+ into the terminal through the NMDA receptor ionophore, since a Ca(2+)-free medium or cadmium totally blocked the stimulation of the enzyme by glutamate.     

Facilitation of Ca2+ Store-Dependent Noradrenaline Release After an N-Methyl-d-Aspartate Receptor Antagonist in the Rat Supraoptic Nucleus

We examined the role of N-methyl-d-aspartate (NMDA) receptors in the control of noradrenaline release in the supraoptic nucleus (SON) using a microdialysis method in urethane-anaesthetized rats. Local application of 0.5 mm NMDA into the SON by retrodialysis decreased noradrenaline content in the dialysate from the SON. On the other hand, MK-801, a channel blocker of NMDA receptors, or D(-)2-amino-5-phosphonopentanoic acid (AP-5), a competitive NMDA receptor antagonist, increased the basal noradrenaline content. Tetrodotoxin did not completely block the noradrenaline increase after NMDA antagonists. Infusion of Ca2+-free solution containing Ni2+ and Cd2+, or a mixture of omega-agatoxin IVA and omega-conotoxin GVIA, voltage-sensitive Ca2+ channels blockers, did not block noradrenaline increase after AP-5, but blocked noradrenaline increase after high K+. Infusion of intracellular Ca2+ blockers, thapsigargin or TMB-8, impaired noradrenaline increase after AP-5 but not that after high K+. These data are consistent with the hypothesis that activation of an NMDA receptor inhibits an intracellular Ca2+ store-dependent noradrenaline release from nerve terminals in the SON.     

N-methyl-D-aspartate induces recurrent synchronized burst activity in immature hippocampal CA3 neurones in vitro

Slices of hippocampus prepared from rats aged 1-10 days have been used to examine the chemosensitivity of CA3 pyramidal neurones to N-methyl-D-aspartate (NMDA). Superfusion of NMDA excited all neurones tested at all ages including the first day postnatal. In the majority of neurones this excitation was associated with the induction of a period of burst firing which disappeared on removal of NMDA. These bursts took the form of paroxysmal depolarizing shifts (PDSs) with a large amplitude depolarization and a high frequency discharge of spikes. The amplitude but not the frequency of occurrence of the PDSs was influenced by changes in the membrane potential and they could be abolished by either a high divalent cation medium or tetrodotoxin. Their occurrence was synchronous with an extracellularly recorded discharge. The NMDA induced excitation and the induction of the PDSs was attenuated by selective NMDA receptor antagonists D-aminophosphonovalerate (10-50 microM) and D,L-aminophosphonoheptanoate (20-30 microM). The results indicate that chemosensitivity to NMDA develops prenatally and that activation of NMDA receptors can in immature CA3 pyramidals induce recurrent synchronized burst activity.     

Differential responses to NMDA receptor activation in rat hippocampal interneurons and pyramidal cells may underlie enhanced pyramidal cell vulnerability

Hippocampal interneurons are generally more resistant than pyramidal cells to excitotoxic insults. Because NMDA receptors play a crucial role in neurodegeneration, we have compared the response to exogenous NMDA in CA1 pyramidal cells and interneurons of the stratum oriens using combined whole-cell patch-clamp recording and ratiometric Ca2+ imaging. In voltage-clamp, current-clamp or in nominally Mg2+-free medium, NMDA (10 microM; 3-5 min exposure in the presence of tetrodotoxin) induced a markedly larger inward current and Ca2+ rise in pyramidal cells than in interneurons. Pyramidal cells also showed a more pronounced voltage dependence in their response to NMDA. We hypothesized that this enhanced response to NMDA receptor activation in pyramidal cells could underlie their increased vulnerability to excitotoxicity. Using loss of dye as an indicator of degenerative membrane disruption, interneurons tolerated continuous exposure to a high concentration of NMDA (30 microM) for longer periods than pyramidal cells. This acute neurodegeneration in pyramidal cells was independent of intracellular Ca2+, because high intracellular BAPTA (20 mM) did not prolong survival time. Thus, a plausible explanation for the enhanced sensitivity of pyramidal neurons to excitotoxic insults associated with cerebral ischemia is their greater response to NMDA receptor activation, which may reflect differences in NMDA receptor expression and/or subunit composition.