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.     

Two distinct types of repetitive bursting activity mediated by NMDA in hypothalamic neurons in vitro

Hypothalamic magnocellular dorsal nucleus neurons were recorded from adult guinea pig brain slices with the whole-cell patch-clamp technique to determine the effects of N-methyl-D-aspartate (NMDA) applied in the bath or by iontophoresis. In a majority of cells (59 of 77, 76.6%), rhythmic bursting discharges were evoked by specific activation of NMDA receptors when the membrane was more negative than -60 mV. This endogenous rhythmic activity was resistant to tetrodotoxin. It was suppressed by removal of extracellular Mg2+, indicating the involvement of the voltage-dependent block of the NMDA channel by Mg2+. Application of thapsigargin showed that rhythmic activity did not depend on the release of Ca2+ from reticulum stores. Blockers of Ca2+ conductances Ni2+ and nifedipine had no effects on the bursts. Their repolarization did not involve the activation of a strophantidin- or ouabain-sensitive pump, but partly depended on an apamine-sensitive Ca2+-dependent K+current. In a small subset of cells (9 of 69, 13%), specific activation of NMDA receptors induced another type of bursting activity which consisted of repetitive low-threshold spikes sustaining bursts of action potentials. Rhythmic low-threshold spikes subsisted in the presence of tetrodotoxin but were suppressed by Ni2+. Increasing the amount of NMDA brought about a switch from the rhythmic low-threshold spike burst firing to the rhythmic bursting activity observed for the majority of cells. The present data show for the first time that NMDA receptor activation can induce two independent rhythmic bursting behaviours in the same neuron, probably depending on the strength of the glutamatergic drive.     

Calcium-dependent subthreshold oscillations determine bursting activity induced by N-methyl-D-aspartate in rat subthalamic neurons in vitro

We used whole-cell patch recordings in current clamp to investigate the ionic dependence of burst firing induced by N-methyl-d-aspartate (NMDA) in neurons of the subthalamic nucleus (STN) in slices of rat brain. NMDA (20 microm) converted single-spike firing to burst firing in 87% of STN neurons tested. NMDA-induced bursting was blocked by AP5 (50 microm), and was not mimicked by the non-NMDA receptor agonist AMPA (0.6 microm). Tetrodotoxin (1 microm) converted bursts to oscillations of membrane potential, which were most robust when oscillations ranged between -50 and -70 mV. The NMDA bursts were blocked by an elevated extracellular concentration of Mg(2+), but superfusate containing no added Mg(2+) either reduced or increased burst firing, depending upon the amount of intracellular current injection. Block of K(+) conductances by apamin and tetraethylammonium prolonged burst duration, but iberiotoxin had no effect. NMDA-induced burst firing and membrane oscillations were completely blocked by superfusate containing no added Ca(2+), and they were significantly reduced when patch pipettes contained BAPTA. Selective antagonists for T-type (mibefradil, 10 microm), L-type (nifedipine, 3 microm), and N-type (omega-conotoxin GVIA, 1 micro m) Ca(2+) channels had no effect on NMDA burst firing. Superfusate containing a low concentration of Na(+) (20 mm) completely abolished NMDA-induced burst firing. Flufenamic acid (10 microm), which blocks current mediated by Ca(2+)-activated nonselective cation channels (I(CAN)), reversibly abolished NMDA-depended bursting. These results are consistent with the hypothesis that NMDA-induced burst firing in STN neurons requires activation of either an I(CAN) or a Na(+)-Ca(2+) exchanger.     

Caffeine-dependent stimulus-triggered oscillations in the CA3 region of hippocampal slices from rats chronically exposed to lead

Yoshimura et al. [Yoshimura, H., Sugai, T., Onoda, N., Segami, N., Kato, N., 2002. Age-dependent occurrence of synchronized population oscillation suggestive of a developing functional coupling between NMDA and ryanodine receptors in the neocortex. Dev. Brain Res., 136, 63-68.] have shown that caffeine can elicit synchronized oscillations (10-12 Hz) dependent on calcium-induced calcium release in rat neocortex neurons. In the present work, synchronized oscillations in the CA3 region of rat hippocampus were studied by recording field excitatory postsynaptic potentials (fEPSPs) in vitro. In the presence of 0.1 mM caffeine, in CA3 of 44 of 45 (97.8%) slices from chronic lead-exposed rats, single electrical stimuli triggered a burst of high-frequency oscillations (approximately 230 Hz), whereas in CA3 of caffeine-treated slices from control rats, such oscillations could be elicited in only 2 of 24 (8.3%) slices. The complete (but fully reversible) block of caffeine-dependent oscillations by 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX; 20 microM) indicates that alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptors are necessary for the high-frequency synchronized oscillations. 2-Amino-5-phosphonopentanoate (AP-5; 50 micoM) partially reduced the amplitude of caffeine-dependent oscillations without significantly altering their frequency. Caffeine-dependent oscillations could be abolished by application of AP-5 and 3 mM Mg2+ during the initial period of bursting, indicating that N-methyl-D-aspartate (NMDA) receptors play an important role in the generation of oscillations. The Ca2+ chelator ethylene glycol bis-(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA; 5 mM) added in standard artificial cerebrospinal fluid (ACSF) containing 0.1 mM caffeine fully blocked the oscillations. Caffeine-dependent oscillations are insensitive to an antagonist of gamma-aminobutyric acid (GABAA) receptors (10 microM bicuculline), L-type Ca2+ channels (10 muM nicardipine), L-type and N-type voltage-dependent calcium channels (100 microM Cd2)), and T-type Ca2+ channels (100 microM Ni2+). Previous studies have demonstrated that expression and function of NMDA and AMPA receptors are altered in the hippocampus of chronic lead-exposed rats. We propose that caffeine-dependent stimulus-induced oscillations in CA3 area of hippocampus from chronic lead-exposed rats are mainly mediated by the entry of extracellular Ca2+ through NMDA and non-NMDA receptors, without participation of GABAA receptors. Additionally, the underlying mechanisms are also discussed.     

N-methyl-D-aspartate enhancement of the glycine response in the rat sacral dorsal commissural neurons

The effect of N-methyl-D-aspartate (NMDA) on the glycine (Gly) response was examined in neurons acutely dissociated from the rat sacral dorsal commissural nucleus (SDCN) using the nystatin-perforated patch-recording configuration under voltage-clamp conditions. The application of 100 microM NMDA to SDCN neurons reversibly potentiated Gly-activated Cl- currents (IGly) without affecting the Gly binding affinity and the reversal potential of IGly. A selective NMDA receptor antagonist, APV (100 microM), blocked the NMDA-induced potentiation of IGly, whereas 50 microM CNQX, a non-NMDA receptor antagonist, did not. The potentiation effect was reduced when NMDA was applied in a Ca2+-free extracellular solution or in the presence of BAPTA AM, and was independent of the activation of voltage-dependent Ca2+ channels. Pretreatment with KN-62, a selective Ca2+-calmodulin-dependent protein kinase II (CaMKII) inhibitor, abolished the NMDA action. Inhibition of calcineurin (CaN) further enhanced the NMDA-induced potentiation of IGly. In addition, the GABAA receptor-mediated currents were suppressed by NMDA receptor activation in the SDCN neurons. The present results show that Ca2+ entry through NMDA receptors modulates the Gly receptor function via coactivation of CaMKII and CaN in the rat SDCN neurons. This interaction may represent one of the important regulatory mechanisms of spinal nociception. The results also suggest that GABAA and Gly receptors may be subject to different intracellular modulatory pathways.     

Characterisation of the L- and N-type calcium channels in differentiated SH-SY5Y neuroblastoma cells: calcium imaging and single channel recording.

We have used single cell imaging of [Ca2+]i and single channel cell-attached patch clamp recording to characterise the Ca2+ channels present on the plasma membrane of retinoic acid-differentiated human neuroblastoma (SH-SY5Y) cells. Exposure to raised K+ (45 or 60 mM) for 1 min resulted in a transient rise in [Ca2+]i which was abolished by cadmium (100 microM). The amplitude of the evoked rise varied from cell to cell. Both omega-Conus toxin (500 nM) and nifedipine (10 microM) reduced, but did not abolish, the rise in [Ca2+]i whereas Bay K 8644 (3 microM) potentiated it. In single channel records both L- and N-type Ca2+ channel openings were observed during membrane depolarisations from a holding potential of -90 mV. L-type channel openings (unitary conductance 22.5 pS) were prolonged by S(+)-PN 202-791 (500 nM) and could still be evoked from a depolarised holding potential (-40 mV). N-type channel openings (unitary conductance 12.5 pS) were unaffected by the dihydropyridine agonist but were inactivated at a holding potential of -40 mV. These results indicate that, in contrast to previous observations using whole cell recording, retinoic acid-differentiated SH-SY5Y cells express both L- and N-type Ca2+ channels.     

NMDA receptor mediates dopamine release in the striatum of unanesthetized rats as measured by brain microdialysis.

We have studied the characteristics associated with the activation of the N-methyl-D-aspartate (NMDA) subtype of the glutamate receptor on the release of dopamine (DA) in the striatum of awake rats as measured by brain microdialysis technique. NMDA dose-dependently stimulated the striatal DA release in Mg(2+)-free Ringer's solution. The stimulation was significant at 90 microM and the maximum observed effect was at the highest concentration tested (800 microM). The selective NMDA receptor antagonist, 2-amino-5-phosphonovalerate (AP5; 300 microM), blocked the stimulatory effect of NMDA. The NMDA-induced release of DA was reduced by 1.2 mM Mg2+ and totally blocked by 2.5 mM of the cation. Glycine (200 microM) potentiated the response evoked by 300 microM NMDA while 7-chloro-kynurenate (100 microM), an antagonist of the glycine site, reduced markedly this response. Neither atropine (100 microM) nor tetrodotoxin (TTX) (5 microM) prevented the stimulatory effect of NMDA. These results suggest that glutamate released from corticostriatal terminals presynaptically stimulates the release of DA via an NMDA receptor.     

Glutamate-induced inhibition of paired pulse facilitation of monosynaptic excitatory post-synaptic potentials in frog spinal motoneurons.

To evaluate actions of glutamate on excitatory synaptic transmission in the central nervous system, we examined glutamate-induced changes in the paired pulse facilitation of monosynaptic excitatory post-synaptic potentials evoked by stimulation of the lateral column fibers (LC-EPSPs) on lumbar motoneurons in the frog spinal cord. Glutamate (1 mM) depolarized motoneurons both in the presence and absence of Mg2+. In most cells perfused with Mg(2+)-free or high Ca(2+)-Mg2+ solutions, the glutamate potential was accompanied by a reduction in peak amplitude of EPSPs, although the degree of change varied with the cells. Glutamate enhanced the EPSP amplitude in a few cells with Mg(2+)-free and high Ca(2+)-Mg2+ solutions, and in most cells with high Mg2+ medium. In 3/5 cells tested, the paired pulse facilitation of EPSPs was reduced by glutamate when the EPSP amplitude either increased or decreased. NMDA (50 microM), kainate (50-100 microM), quisqualate (5-50 microM) and L-2-amino-4-phosphonobutyrate (L-AP4, 1 mM) also decreased the facilitation in about half of the cells tested. The glutamate-induced decrease in the facilitation was observed in both the presence and absence of Mg2+ and was not affected by the concomitant application of glutamate and antagonists for non-NMDA or NMDA receptors, such as 6-cyano-7-nitro-quinoxalinediones (CNQX, 60 microM) or 2-amino-5-phosphonovalerate (APV, 250 microM). Glutamate reduced the facilitation of excitatory post-synaptic currents (EPSCs) recorded at a constant membrane potential under voltage clamp, when the EPSC amplitude either increased or decreased and when the input conductance either increased or decreased.(ABSTRACT TRUNCATED AT 250 WORDS)     

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)     

Redox modulation of NMDA receptor-mediated Ca2+ flux in mammalian central neurons

NMDA receptor-mediated Ca2+ flux was studied in cultured rat retinal ganglion cells and neocortical neurons. Intracellular free calcium ([Ca2+]i was measured with fura-2 fluorescence imaging. Baseline [Ca2+]i was 59 +/- 5 nM. In low [Mg2+]o, 200 microM NMDA reversibly increased [Ca2+]i to 421 +/- 70 nM. This rise in [Ca2+]i was blocked by the NMDA antagonists APV (200 microM) or [Mg2+]o (1 mM), but only slightly inhibited by the non-NMDA antagonist CNQX (10 microM). Chemical reduction with dithiothreitol (DTT) had no effect on resting [Ca2+]i. However, DTT increased the NMDA-induced rise in [Ca2+]i approximately 1.6-fold; the oxidizing agent dithiobisnitrobenzoic acid (DTNB) reversed this effect. In patch-clamp experiments, DTT increased NMDA-activated whole-cell conductance approximately 1.7-fold in low and high [Ca2+]o. The Ca2+/Na+ permeability ratio of approximately 7 for NMDA channels remained unaltered by chemical reduction. Thus, redox modulation of the NMDA receptor/channel complex results in a dramatic alteration in current magnitude but no change in ionic permeabilities.     

Voltage Oscillations in Xenopus Spinal Cord Neurons: Developmental Onset and Dependence on Co-activation of NMDA and 5HT Receptors

The development of intrinsic, N-methyl-D-aspartate (NMDA) receptor-mediated voltage oscillations and their dependence on co-activation of 5-hydroxytryptamine (5HT) receptors was explored in motor neurons of late embryonic and early larval Xenopus laevis. Under tetrodotoxin, 100 μM NMDA elicited a membrane depolarization of around 20 mV, but did not lead to voltage oscillations. However, following the addition of 2–5 μM 5HT, oscillations were observed in 12% of embryonic and 70% of larval motor neurons. The voltage oscillations depended upon co-activation of NMDA and 5HT receptors since they were curtailed by selectively blocking NMDA receptors with D-2-amino-5-phosphonovaleric acid (APV) or by excluding Mg²⁺ from the experimental saline. 5HT applied in the absence of NMDA also failed to elicit oscillations. Oscillations could be induced by the non-selective 5HT_1a receptor agonist, 5-carboxamidotryptamine (5CT) and both 5HT- and 5CT-induced oscillations were abolished by pindobind-5HT₁, a selective 5HT_1a receptor antagonist. To test whether 5HT enables voltage oscillations by modulating the voltage-dependent block of NMDA channels by Mg²⁺, membrane conductance was monitored under tetrodotoxin. Although 5HT caused membrane hyperpolarization of 4–8 mV, there was little detectable change in conductance. NMDA application caused an approximate 20 mV depolarization and an ‘apparent’ decrease in conductance, presumably due to the conductance pulse bringing the membrane into a voltage region where Mg²⁺ blocks the NMDA ionophore. 5HT further decreased conductance, which we propose is due to its enhancement of the voltage-dependent Mg²⁺ block. When the membrane potential was depolarized by ～20 mV via depolarizing current injection (to mimic the NMDA-induced depolarization), 5HT increased rather than decreased membrane conductance. Furthermore, 5HT did not affect the increase in membrane conductance following NMDA applications in zero Mg²⁺ saline. The results suggest that intrinsic, NMDA receptor-mediated voltage oscillations develop in a brief period after hatching, and that they depend upon the co-activation of 5HT and NMDA receptors. The enabling function of 5HT may involve the facilitation of the voltage-dependent block of the NMDA ionophore by Mg²⁺ through activation of receptors with 5HT_1a-like pharmacology.     

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)     

Firing pattern of rat hippocampal neurons: a perforated patch clamp study

To test whether the slow afterhyperpolarization (sAHP) underlies the filter function of hippocampal granule cells (GCs), we compared the sAHP and spike frequency adaptation between granule cells and CA3 pyramidal cells (PCs) in hippocampal slices employing gramicidin perforated patch clamp recordings to best preserve the physiological cytoplasmic constitution. sAHPs were evoked in GCs and PCs with trains of action potentials in current clamp mode and showed comparable kinetics in both types of cells. The threshold frequency (500 ms firing) triggering a detectable sAHP was approximately 10 Hz and approximately 3 Hz in GCs and PCs, respectively. Half maximal sAHPs were reached at 30 Hz and 8 Hz in GCs and PCs, respectively. Maximal amplitude of sAHPs in GCs amounted to approximately 3.5 mV, was approximately 2-fold smaller than in PCs and could not be further increased with higher firing frequencies. The time course of sAHP activation was investigated with 50 Hz trains of action potentials applied for increasing durations. In both types of cells, the sAHP amplitude increased with a time constant of approximately 400 ms. Nevertheless, sAHP never exceeded 4 mV in GCs but rose to approximately 12 mV in PCs when cells fired for 3 s. The repetitive firing pattern of GCs and PCs was compared by injecting current amplitudes adjusted to provoke an initial firing frequency of 50 Hz. In GCs firing frequency declined slower (tau = 229 ms) and leveled off at a higher tonic firing frequency (28 Hz) when compared to PCs (tau = 126 ms, 18 Hz). We conclude that the intrinsic excitability of GCs cannot be primarily regulated by the sAHP. The sAHP in GCs is minimal most likely due to a small sAHP-channel density as well as to a more rigid control of intracellular Ca(2+) levels.     

A hyperpolarization-activated inward current in heart interneurons of the medicinal leech

Heart interneurons (HN cells) in isolated ganglia of the medicinal leech were voltage-clamped with single microelectrodes. Hyperpolarizing voltage steps elicited a slow inward current (Ih), which underlies the characteristic depolarizing response of HN cells to injection of prolonged hyperpolarizing current pulses (Arbas and Calabrese, 1987a). The conductance underlying Ih begins to activate near -mV and is fully activated between -70 and -80 mV. The activation kinetics of Ih are slow and voltage dependent. The activation time constant (tau h) ranges from approximately 2 sec at -60 mV to near 700 msec at -100 mV. Ih persists in low Ca2+ (0.1 mM), 5 mM Mn2+ saline and exhibits a reversal potential of -21 +/- 5 mV. The reversal potential is shifted by altering [Na+]o or [K+]o but is unaffected by changes in [Cl-]o. Ih is blocked by extracellular Cs+ (1-5 mM) but not Ba2+ (5 mM) or TEA (25 mM). Low concentrations of Cs+ (100-200 microM) cause a partial block that exhibits strong voltage dependence. Temperature changes were also shown to affect Ih. Both the rate of activation and the steady-state amplitude of Ih are enhanced by temperature increases. HN cells are interconnected by inhibitory chemical synapses, and their normal electrical activity consists of bursts of action potentials separated by periods of inhibition. During the inhibitory phase of rhythmic bursting activity, HN cells hyperpolarize to a voltage range where Ih is activated. Block of Ih with extracellular Cs+ (4 mM) disrupted the normal bursting activity of HN cells. These results are consistent with the hypothesis that Ih contributes to escape from inhibitory inputs during normal bursting activity.     

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.     

Suppression of epileptiform activity by GABA(B) receptors in wild type and weaver hippocampus 'in vitro'

Inhibition by GABA(B) receptors comprises activation of K(+) conductance and inhibition of Ca(2+) conductance, thereby reducing action potential dependent transmitter release and silencing neuronal activity. We compared epileptiform activity and its inhibition by the activation of GABA(B) receptors in homozygous weaver (wv/wv) and wild type (+/+) CA3 neurons disinhibited by GABA(A) receptor blockade. In wv/wv mice GABA(B) receptors have lost their ability to activate K(+) conductance (J. Neurosci. 18 (1998) 4001). Spontaneous synchronous burst discharges in elevated [K(+)](o) displayed only subtle differences in +/+ and wv/wv slices, except that the GABA(B) receptor agonist R-baclofen in low concentration (0.1 microM) strongly reduced the frequency of synchronous bursts in +/+ CA3 neurons, but not in wv/wv CA3 neurons. A high affinity GABA(B) antagonist, CGP55845A (0.5 microM) promoted the incidence of bursts in low [K(+)](o). Concentration dependence of the reduction of evoked EPSCs was identical in wv/wv and +/+ neurons (IC(50)=0.3 microM). Amplitudes of evoked IPSCs were reduced by 0.01 microM R-baclofen in +/+, but not in wv/wv CA3 neurons. The effect of the low concentration was abolished by Ba(2+), which is known to block Kir conductance. The data suggest that activation of Kir conductance is important for the control of GABA release by GABA(B) autoreceptors in the CA3 network. We conclude that the loss of a contribution of Kir conductance to GABA(B) receptor-mediated autoinhibition reduces the inclination towards spontaneous bursts of wv/wv CA3 pyramidal neurons.     

T-type CaV3.3 calcium channels produce spontaneous low-threshold action potentials and intracellular calcium oscillations

The precise contribution of T-type Ca2+ channels in generating action potentials (APs), burst firing and intracellular Ca2+ signals needs further elucidation. Here, we show that CaV3.3 channels can trigger repetitive APs, generating spontaneous membrane potential oscillations (MPOs), and a concomitant increase in the intracellular Ca2+ concentration ([Ca2+]i) when overexpressed in NG108-15 cells. MPOs were dependent on CaV3.3 channel activity given that they were recorded from a potential range of -55 to -70 mV, blocked by nickel and mibefradil, as well as by low external Ca2+ concentration. APs of distinct duration were recorded: short APs (sAP) or prolonged APs (pAP) with a plateau potential near -40 mV. The voltage-dependent properties of the CaV3.3 channels constrained the AP duration and the plateau potential was supported by sustained calcium current through CaV3.3 channels. The sustained current amplitude decreased when the resting holding potential was depolarized, thereby inducing a switch of AP shape from pAP to sAP. Duration of the [Ca2+]i oscillations was also closely related to the shape of APs. The CaV3.3 window current was the oscillation trigger as shown by shifting the CaV3.3 window current potential range as a result of increasing the external Ca2+ concentration, which resulted in a corresponding shift of the AP threshold. Overall, the data demonstrate that the CaV3.3 window current is critical in triggering intrinsic electrical and [Ca2+]i oscillations. The functional expression of CaV3.3 channels can generate spontaneous low-threshold calcium APs through its window current, indicating that CaV3.3 channels can play a primary role in pacemaker activity.     

In vivo intrahippocampal microinfusion of carbachol and bicuculline induces theta-like oscillations in the septally deafferented hippocampus.

In their laboratory the authors have previously demonstrated that hippocampal slices could be induced to generate trains of "theta-like" oscillations by whole-bath perfusions of carbachol. Until recently, it has not been possible to generate similar activity in the septally deafferented hippocampus of an otherwise intact brain by microinfusions of carbachol. This study presents a full report of the first demonstration of a theta-like oscillation in the in vivo, septally deafferented hippocampal formation. Rats were anesthetized with urethane and implanted with microinfusion cannulae in the region of the medial septum/vertical limb of the diagonal band of Broca (MS/vDBB) and at single or multiple sites in the stratum moleculare of the fascia dentata. The MS/vDBB was microinfused with procaine hydrochloride to produce a reversible suppression lasting for approximately 20 minutes. Intrahippocampal microinfusions of carbachol or bicuculline alone (in the postprocaine condition of the MS/vDBB) failed to produce any theta-like oscillations. The combination of carbachol and bicuculline produced trains of theta-like oscillations during suppression of the MS/vDBB very similar to those seen in the slice preparations. The oscillations were blocked by intravenous administration of atropine sulfate, and they had the same depth profile as that of theta. Theta-on cells were shown to discharge in rhythmic bursts in synchrony with the oscillations. Thus, it would appear that the essential nature of the medial septal input to the hippocampal formation, for the generation of theta field activity in the intact brain, consists of a critical balance between cholinergic and GABAergic circuitry.     

Activation of N-methyl-D-aspartate (NMDA) receptors augments repolarizing responses in lamprey spinal neurons

Current- and voltage-clamp techniques were used to analyze the mechanisms underlying the repolarization during N-methyl-D-aspartate (NMDA)-induced, tetrodotoxin-resistant pacemaker-like oscillations in lamprey spinal neurons. Long-lasting depolarizing current pulses (15-40 mV, 50-400 ms, tetrodotoxin and tetraethylammonium present) were followed by hyperpolarizing afterpotentials even when NMDA receptors were blocked, but they were markedly enhanced by application of N-methyl-D,L-aspartate (NM(DL)A). The afterpotentials were depressed by replacing Ca2+ with Ba2+. During voltage-clamp NM(DL)A enhanced a Ba2+-sensitive outward tail current following voltage steps of 15-40 mV. The outward current remained after injection of Cl-, as did the NMDA-induced membrane potential oscillations observed under current-clamp. These results suggest that the repolarization during NMDA-induced oscillations is due to Ca2+ entry both via NMDA-gated channels and conventional voltage-gated Ca2+ channels, leading to an activation of Ca2+-dependent K+ channels. The afterhyperpolarization following single action potentials, which is also due to Ca2+-dependent K+ channels, was not significantly altered by NMDA receptor activation, suggesting a different location of the Ca2+ entry during the two conditions in relation to the location of the activated Ca2+-dependent K+ channels.     

Intracellular cAMP regulates the response to NMDA in hippocampal neurons.

We have studied the effects of intracellular cyclic AMP (cAMP) on the response to N-methyl-D-aspartate (NMDA) in hippocampal cultured neurons by loading them with 2'-o-dibutyryladenosine 3', 5'-cyclic monophosphate (dcAMP) and have obtained evidence for regulation of Ca2+ release from intracellular stores by cAMP. Extracellular Ringer's solution was either Ca(2+)-containing or Ca(2+)-free. A brief initial stimulation with NMDA (10-100 microM, 12 s) was required before the neurons were loaded with dcAMP to potentiate the changes in intracellular Ca2+ concentration induced by the second stimulus of NMDA. Forskolin (10 microM) mimicked dcAMP, but to a lesser extent. A phorbol ester 12-o-tetradecanoylphorbol-13-acetate (100 nM) inhibited the effect of dcAMP.