A voltage-clamp analysis of NMDA-induced responses on dopaminergic neurons of the rat subtantia nigra zona compacta and ventral tegmental area

The effects of NMDA-receptor activation on dopaminergic neurons of the rat substantia nigra zona compacta and ventral tegmental area were studied by using in vitro intracellular electrophysiological recordings (current and voltage-clamp). NMDA depolarized the membrane and increased the firing activity. A voltage-dependent inward current and a reduction of the apparent input conductance were observed in voltage-clamp experiments. Interestingly, the peak amplitude of the inward current occurred at approximately - 60 mV. The NMDA-induced responses were reduced by the application of -2-amino-5-phophonovaleric acid (APV). The NMDA-induced current was unaffected by potassium channel blockers, was present in low-sodium solutions or in solutions treated with TTX; but was reduced or blocked in low-calcium solutions containing cobalt. In addition, no reduction of the apparent input conductance was observed either in the solutions without magnesium or in those with low-sodium. Our data indicate that the activation of NMDA receptors produces a powerful excitatory stimulus on the dopaminergic neurons of the ventral mesencephalon and this may be primarily the result of a voltage-dependent influx of calcium ions. The degeneration of the dopaminergic cells after application of neurotoxins may be explained by their peculiar response to NMDA.     

Electrophysiological evidence for the presence of ionotropic and metabotropic excitatory amino acid receptors on dopaminergic neurons of the rat mesencephalon: an in vitro study.

The actions of the ionotropic and metabotropic excitatory amino acid agonists AMPA, quisqualate, kainate, NMDA and trans-ACDP were studied by means of intracellular electrophysiological recordings from dopaminergic neurons of rat mesencephalon in brain slices. It was observed that all these agents evoked an inward current in cells which were voltage-clamped near the resting potential (-50, -60 mV). The membrane responses produced by AMPA, kainate and quisqualate were associated with an increase of the apparent input conductance while the responses induced by NMDA and trans-ACDP were associated with a decrease in the apparent input conductance. Therefore, stimulation of ionotropic and metabotropic amino acid receptors activates inward currents in the dopaminergic cells by different mechanisms.     

Two different mechanisms control inhibition of spike discharge in neurons of cat motor cortex after stimulation of the pyramidal tract

Intracellular recordings were made from neurons of the motor cortex of awake cats while the pyramidal tract (PT) was stimulated at the level of the facial nucleus. In some neurons IPSPs of 35-120 ms peak latency were recorded that diminished in size or reversed with hyperpolarizing current. During these IPSPs a decrease in input resistance reflective of a conductance increase was measured. More often, however, PT stimulation produced IPSPs with comparable latencies that increased in size with hyperpolarizing current. These IPSPs diminished with depolarizing current, and in some instances they appeared to reverse with strong depolarization. During these IPSPs an increased input resistance reflective of a decreased conductance was measured. The results indicate that two different mechanisms control rapid inhibition of spike discharge in neurons of the motor cortex after PT stimulation.     

Excitatory actions of substance P in the rat lateral posterior nucleus

The lateral posterior nucleus (LP) receives inputs from both neocortex and superior colliculus (SC), and is involved with integration and processing of higher‐level visual information. Relay neurons in LP contain tachykinin receptors and are innervated by substance P (SP)‐containing SC neurons and by layer V neurons of the visual cortex. In this study, we investigated the actions of SP on LP relay neurons using whole‐cell recording techniques. SP produced a graded depolarizing response in LP neurons along the rostro‐caudal extent of the lateral subdivision of LP nuclei (LPl), with a significantly larger response in rostral LPl neurons compared with caudal LPl neurons. In rostral LPl, SP (5–2000 nm ) depolarized nearly all relay neurons tested (> 98%) in a concentration‐dependent manner. Voltage‐clamp experiments revealed that SP produced an inward current associated with a decreased conductance. The inward current was mediated primarily by neurokinin receptor (NK)₁ tachykinin receptors, although significantly smaller inward currents were produced by specific NK₂ and NK₃ receptor agonists. The selective NK₁ receptor antagonist RP67580 attenuated the SP‐mediated response by 71.5% and was significantly larger than the attenuation of the SP response obtained by NK₂ and NK₃ receptor antagonists, GR159897 and SB222200, respectively. The SP‐mediated response showed voltage characteristics consistent with a K⁺ conductance, and was attenuated by Cs⁺, a K⁺ channel blocker. Our data suggest that SP may modulate visual information that is being processed and integrated in the LPl with inputs from collicular sources.     

Substance P decreases a potassium conductance of spinal cord neurons in cell culture

Substance P (SP) produced membrane depolarization and decreased membrane conductance of mouse spinal cord neurons in primary dissociated cell culture. SP-responses were abolished by intracellular tetraethylammonium suggesting that SP decreased potassium conductance. Reversal of SP-responses was not observed with membrane hyperpolarization suggesting that SP reduced a voltage-dependent potassium conductance that was activated by membrane depolarization.     

Stimulation of a sodium influx by cAMP in Helix neurons

Brief pressure injections of aqueous solutions of cAMP in identified neurons of Helix pomatia caused depolarizations which lasted for tens of seconds. In voltage-clamped neurons an inward current of similar duration was induced which saturated at 10 μA/cm² cell surface. In the range of negative membrane potentials with little voltage-dependent activation, this current was not accompanied by a change in membrane conductance. The inward current was not produced by injection of ATP, ADP, adenosine, inosine or cGMP. cAMP derivatives produced longer-lasting effects. Prolongation of the inward current was also observed after inhibition of the phosphodiesterase by IBMX. Drugs which block active transport had no effect on the response to cAMP injection. The inward current depended on extracellular sodium, and was maximal when all other mono- and divalent cations were replaced by Na⁺. The cAMP-induced current was accompanied by a transient increase in [Na⁺]_i, but there was no change in [Cl⁻]_i. Li⁺ could largely substitute for Na⁺; Ca²⁺ was less effective. Addition of Mg²⁺ or Ca²⁺ to solutions containing a high Na⁺-concentration inhibited the response. Internal acidification with HCl reversibly enhanced the inward current. These data indicate that the depolarizing effect of cAMP can be accounted for by an inward movement of Na-ions, and that the effect is augmented by H⁺-ions.     

Synaptic stimulation of nicotinic receptors in rat sympathetic ganglia is followed by slow activation of postsynaptic potassium or chloride conductances

Two slow currents have been described in rat sympathetic neurons during and after tetanization of the whole preganglionic input. Both effects are mediated by nicotinic receptors activated by native acetylcholine (ACh). A first current, indicated as IAHPsyn, is calcium dependent and voltage independent, and is consistent with an IAHP-type potassium current sustained by calcium ions accompanying the nicotinic synaptic current. The conductance activated by a standard synaptic train was approximately 3.6 nS per neuron; it was detected in isolation in 14 out of a 52-neuron sample. A novel current, IADPsyn, was described in 42/52 of the sample as a post-tetanic inward current, which increased in amplitude with increasing membrane potential negativity and exhibited a null-point close to the holding potential and the cell momentary chloride equilibrium potential. IADPsyn developed during synaptic stimulation and decayed thereafter according to a single exponential (mean tau = 148.5 ms) in 18 neurons or according to a two-exponential time course (tau = 51.8 and 364.9 ms, respectively) in 19 different neurons. The mean peak conductance activated was approximately 20 nS per neuron. IADPsyn was calcium independent, it was affected by internal and external chloride concentration, but was insensitive to specific blockers (anthracene-9-carboxylic acid, 9AC) of the chloride channels open in the resting neuron. It is suggested that gADPsyn represents a specific chloride conductance activatable by intense nicotinic stimulation; in some neurons it is even associated with single excitatory postsynaptic potentials (EPSCs). Both IAHP and IADPsyn are apparently devoted to reduce neuronal excitability during and after intense synaptic stimulation.     

Tachykinins cause inward current through NK1 receptors in bullfrog sensory neurons

The effects of tachykinins on primary afferent neurons of bullfrog dorsal root ganglia (DRG) were examined by using whole-cell patch-clamp methods. Neurokinin A (NKA) caused inward current (I_NKA) in a concentration-dependent manner. Concentration-response curve showed that the EC₅₀ for NKA was 6 nM. The I_NKA showed strong tachyphylaxis, when NKA was continuously applied for more than 1 min. Substance P (SP) also produced inward current with potency similar to that of NKA. Neurokinin B (NKB) was less effective in producing the inward current. The order of agonist potency was NKA = SP NKB. Spantide ([D-Arg¹, D-Trp^7,9, Leu¹¹]SP), non-selective peptide antagonist at tachykinin receptors, reduced the tachykinin-induced current. CP-99,994, a selective non-peptide antagonist for neurokinin-1 (NK₁) receptor, inhibited the inward currents produced by NKA and SP. The I_NKA was associated with decrease in K⁺ conductance. NKA suppressed both a voltage-dependent K⁺ current, the M-current (I_M), and a voltage-independent background K⁺ current, I_K(B). Intracellular dialysis with GTPγS (100 nM) or GDPβS (100 μM) depressed the I_NKA. Pre-treatment of DRG neurons with pertussis toxin (PTX) did not prevent the I_NKA. Depletion of intracellular ATP depressed the I_NKA. These results suggest that the tachykinin-induced inward current is mediated through the NK₁ receptor which mainly couples to PTX-insensitive G-protein in bullfrog primary afferent neurons.     

Contrasting properties of K+ conductances induced by baclofen and gamma-aminobutyric acid in slices of the guinea pig hippocampus

Properties of membrane K+ conductances induced by baclofen and gamma-aminobutyric acid (GABA) in the hippocampus were investigated by using guinea-pig brain slices. Baclofen hyperpolarized the membrane and decreased the input resistance of pyramidal cells through the activation of a membrane K+ conductance. GABA caused a biphasic response in pyramidal cells, consisting of hyperpolarizing and depolarizing components. Combined application of picrotoxin and bicuculline eliminated the major part of the depolarizing component of the biphasic response and produced a relatively pure hyperpolarizing response which was also mediated by an increase in K+ conductance. The K+ conductance change induced by baclofen showed prominent inward rectification. However, the K+ conductance induced by GABA did not show an obvious rectifying property. The K+ conductance activated by baclofen was strongly antagonized by a low concentration (5 x 10(-6) M) of 4-aminopyridine (4-AP). In contrast, the K+ conductance activated by GABA was insensitive to 4-AP even at a high concentration of 10(-3) M. The slow inhibitory postsynaptic potential (slow i.p.s.p.) evoked by stimulation of the mossy fibres was totally suppressed by a low concentration of baclofen (5 x 10(-6) M). Whereas GABA (10(-3) M) decreased the amplitude of the slow i.p.s.p., the reduction of the amplitude was proportional to the decrease in the amplitude of the electrotonic potentials produced by constant inward current injections. These results suggest that the hyperpolarizations induced by GABA and baclofen may be generated by K+ conductances of different kinetic and pharmacologic properties.     

Increase in sodium conductance decreases firing rate and gain in model neurons

We studied the effects of increased sodium conductance on firing rate and gain in two populations of conductance-based, single-compartment model neurons. The first population consisted of 1000 model neurons with differing values of seven voltage-dependent conductances. In many of these models, increasing the sodium conductance threefold unexpectedly reduced the firing rate and divisively scaled the gain at high input current. In the second population, consisting of 1000 simplified model neurons, we found that enhanced sodium conductance changed the frequency-current (FI) curve in two computationally distinct ways, depending on the firing rate. In these models, increased sodium conductance produced a subtractive shift in the FI curve at low firing rates because the additional sodium conductance allowed the neuron to respond more strongly to equivalent input current. In contrast, at high input current, the increase in sodium conductance resulted in a divisive change in the gain because the increased conductance produced a proportionally larger activation of the delayed rectifier potassium conductance. The control and sodium-enhanced FI curves intersect at a point that delimits two regions in which the same biophysical manipulation produces two fundamentally different changes to the model neuron's computational properties. This suggests a potentially difficult problem for homeostatic regulation of intrinsic excitability.     

Synaptically Activated Low-threshold Muscarinic Inward Current Sustains Tonic Firing in Rabbit Prevertebral Sympathetic Neurons

Whole-cell patch-clamp experiments were performed on non-dissociated rabbit coeliac sympathetic neurons in the presence of nicotinic blockers. Coeliac neurons were classified as either silent or spontaneously active (pacemaker) cells. Under voltage-clamp conditions, pacemaker cells exhibited a steady-state N-shaped current-voltage relationship due to the presence of a persistent voltage-dependent inward current in the potential range of -100 to —20 mV. This inward current sustained the regular firing activity of pacemaker cells and was absent from quiescent neurons. It disappeared in the presence of tetrodotoxin and in low Ca²⁺-high Mg²⁺ external solutions and was enhanced by eserine. Splanchnic nerve stimulation induced slow regenerative depolarizations and firing discharges in silent neurons by activating a low-threshold voltage-sensitive inward current. The synaptic current had a U-shaped voltage-dependence from —96 to —20 mV and exhibited the dynamic properties of the muscarinic voltage-dependent inward current l _{Na, M}. It gave the current-voltage relationship an N shape similar to that observed in spontaneously active cells. The muscarinic antagonists atropine and pirenzepine abolished the inward current present in pacemaker cells and that induced by nerve stimulation in silent neurons. These data provide evidence that both spontaneous firing activity and nerve-evoked depolarizing responses in coeliac neurons are sustained by the activation of the muscarinic Na, M current. The tonic activation of l _{Na, M} in spontaneously firing cells results from a sustained Ca²⁺-dependent tetrodotoxin-sensitive release of acetylcholine. This study provides evidence that the role of the muscarinic receptors is not purely a neuromodulatory one, but that these receptors are directly involved in ganglionic neurotransmission.     

2 types of neuronal reactions of the medulla oblongata to microstimulation of the locomotor and inhibitory points of the brain stem

Synaptic responses (postsynaptic potentials and action potentials) of medial and lateral bulbar neurons were evoked by stimulation of the medullary locomotor point (LP) and pontine inhibitory point (IP) by current up to 20 microA in mesencephalic decerebellated cats. Some neurons responded even to single (2-5/s) stimuli. Other neurons responded only to rhythmic (30-60/s) stimulation. Both groups of medial neurons were more susceptible to the input from IP. Lateral neurons which responded even to single stimuli were more reactive to the input from LP, whereas those which responded only to rhythmic stimulation were under the predominant influence from IP. Many neurons (both lateral and medial) with the background activity did not generate responses which were time-locked to a stimulus, however the background activity was either enhanced or inhibited under the rhythmic stimulation. These reactions were encountered more often during stimulation of LP. Partial redistribution of target neurons which accompanied the elevation of the frequency of the presynaptic input may be very significant in the motor control.     

Passive electrical membrane properties of rat neostriatal neurons in an in vitro slice preparation

The passive electrical membrane properties of rat neostriatal neurons were studied in in vitro slice preparations. The data are only from neurons having stable resting membrane potentials of more than 50 mV and able to generate action potentials of amplitudes greater than 70 mV evoked by local or intracellular stimulation. All neurons measured for current-voltage relationship (n = 52) showed non-linearity of the input resistance in the hyperpolarizing direction. The mean input resistance at the resting membrane potential was 16.6 M omega. Depolarizing postsynaptic potentials evoked by local stimulation were decreased both in their amplitude and half-decay time by inward current injections exceeding more than 1 nA due to the strong membrane rectification at these levels of hyperpolarization. The mean membrane time constant (tau 0) was 5.3 ms, as measured from the semilogarithmic plots of transmembrane potential shift produced by small hyperpolarizing current pulses. In some neurons, the equalizing term (tau 1) could be determined as well and had a mean value of 1.0 ms. Measurement of (tau 0) using the strength-latency relation showed a similar value (5.0 ms) to that measured from the voltage transients. Intracellular labeling of the recorded neurons with horseradish peroxidase suggested that the recordings were obtained from medium spiny neurons.     

Phase-dependent Modulation of a Cutaneous Sensory Pathway by Glycinergic Inhibition from the Locomotor Rhythm Generator in Xenopus Embryos

In immobilized Xenopus laevis embryos two classes of sensory interneuron are excited by mechanosensory Rohon - Beard neurons and rhythmically inhibited during fictive swimming. Dorsolateral commissural (DLC) interneurons are inhibited in time with rhythmic motor discharge on the same side as their soma, while unidentified dorsolateral (DLX) interneurons are inhibited in the opposite phase of the swimming rhythm. The inhibition is abolished by bath application of strychnine sulphate at 1 - 10 microM, but not by the gamma-aminobutyric acid antagonists bicuculline (20 - 40 microM) or curare (70 - 100 microM). The inhibitory postsynaptic potentials (IPSPs) involve an increase in chloride conductance since they are reversed in sign to become depolarizing following intracellular injection of chloride ions. The conductance increase during inhibition was able to block impulses evoked by intracellular current in a phase-dependent manner, suggesting that postsynaptic inhibition is sufficient to account for the gating of afferent input to the spinal cord during swimming. An interneuron receives IPSPs that are predominantly in one phase of the rhythm, but most interneurons are also inhibited sporadically in the opposite phase. The amplitude and time course of the IPSPs closely follow the frequency of the swimming rhythm, with maximal inhibition occurring near the starts of episodes, when swimming frequency is at its highest. Towards the end of an episode, when swimming frequency declines, the level of inhibition is low, the membrane potential of the interneurons returns to rest between cycles, and IPSPs often fail to occur. Inhibition suppresses sensory excitation in a phase-dependent manner (cf. Sillar and Roberts, Nature, 331, 262 - 265, 1988). Sensory interneurons fire a single impulse in response to a brief sensory stimulus, but they will usually fire multiple impulses when depolarized with sufficient intracellular current. In some sensory interneurons a short-latency IPSP follows the impulse evoked by skin stimulation that could curtail impulse activity. However, when the inhibition is blocked by strychnine, sensory interneurons still fire a single short-latency impulse, favouring the conclusion that brief, synchronized afferent excitation elicits a single impulse in neurons that are capable of firing multiply. Since the inhibition of DLC interneurons occurs in phase with activity on the same side it probably originates from spiking in ipsilateral glycinergic commissural interneurons which have ipsilateral as well as contralateral projections. The inhibition of DLX interneurons in the opposite phase probably originates from the contralateral projections of commissural interneurons.     

Changes in body temperature and thermosensitivity of preoptic and septal neurons during hippocampal stimulation

The effects of electrical stimulation of the ventral subiculum (VSB) of hippocampal formation on the thermosensitivity of neurons in the preoptic area and the septum (PO/SP) were studied in the urethane-anesthetized rat. The local thermosensitivity of sixteen of 28 PO/SP thermosensitive neurons (25 warm-units and 3 cold-units) was reduced or lost during low frequency (4.8 Hz) stimulation of VSB. The extent of reduction was positively correlated to the magnitude of inhibition by the VSB stimulation. Thermosensitivity did not change in 11 of the remaining 12 warm-units and increased slightly in one warm-unit during VSB stimulation. In the conscious rat, bilateral stimulation (5 Hz) of VSB for 10 min caused a rise in rectal temperature of 0.28-0.87 degrees C in a warm environment (30-33 degrees C) and a fall of 0.18-0.57 degrees C in a cold (9.4-9.8 degrees C) environment. The results suggest that the predominantly inhibitory input from VSB decreases the thermosensitivity of PO/SP warm- and cold-sensitive neurons and thereby reduces the ability of thermoregulation.     

Two Signal Transduction Mechanisms of Substance P-induced Depolarization in Locus Coeruleus Neurons

Effects of substance P on cultured neurons of the locus coeruleus of the rat were studied using the whole-cell patch clamp technique. In some cells substance P produced a decrease in a K conductance which showed an inwardly rectifying property. In other cells substance P produced an initial inward current which was accompanied by a conductance increase. The rest of the cells showed responses which were mixtures of the above two responses. The measurement of the reversal potential of the initial inward current after suppressing the voltage-gated Ca and K conductances suggests that it is caused by an increase in a non-selective ionic conductance. In cells loaded with 260 μM GTPγS, application of substance P produced an irreversible reduction of the K conductance, while the initial inward current could still be recorded, suggesting that the former is mediated by a G protein, whereas the latter may be activated by a different signal transduction mechanism. The initial inward current was not eliminated by external application of high concentrations of tetrodotoxin, d -tubocurarine or amiloride. Nor was it affected by the intracellular application of cyclic GMP or cyclic AMP.     

Swelling-activated calcium signalling in cultured mouse primary sensory neurons

The effects of hypo-osmotic membrane stretch on intracellular calcium concentration ([Ca(2+)](i)), cell volume and cellular excitability were investigated in cultured mouse primary sensory trigeminal neurons. Hypotonic solutions (15--45%) led to rapid cell swelling in all neurons. Swelling was accompanied by dose-dependent elevations in [Ca(2+)](i) in a large fraction of neurons. Responses could be classified into three categories. (i) In 57% of the neurons [Ca(2+)](i) responses had a slow rise time and were generally of small amplitude. (ii) In 21% of the neurons, responses had a faster rise and were larger in amplitude. (iii) The remaining cells (22%) did not show [Ca(2+)](i) responses to hypo-osmotic stretch. Slow and fast [Ca(2+)](i) changes were observed in trigeminal neurons of different sizes with variable responses to capsaicin (0.5 microM). The swelling-induced [Ca(2+)](i) responses were not abolished after depletion of intracellular Ca2+ stores with cyclopiazonic acid or preincubation in thapsigargin, but were suppressed in the absence of external Ca(2+). They were strongly attenuated by extracellular nickel and gadolinium. Hypotonic stimulation led to a decrease in input resistance and to membrane potential depolarization. Under voltage-clamp, the [Ca(2+)](i) elevation produced by hypotonic stimulation was accompanied by the development of an inward current and a conductance increase. The time course and amplitude of the [Ca(2+)](i) response to hypo-osmotic stimulation showed a close correlation with electrophysiological properties of the neurons. Fast [Ca(2+)](i) responses were characteristic of trigeminal neurons with short duration action potentials and marked inward rectification. These findings suggest that hypo-osmotic stimulation activates several Ca(2+)-influx pathways, including Gd(3+)-sensitive stretch-activated ion channels, in a large fraction of trigeminal ganglion neurons. Opening of voltage-gated Ca(2+) channels also contributes to the response. The pattern and rate of Ca(2+) influx may be correlated with functional subtypes of sensory neurons.     

The effects of serotonin on N-methyl-D-aspartate and synaptically evoked depolarizations in rat neocortical neurons

The effects of serotonin (5-HT) on neuronal responses to the excitatory amino acid agonist N-methyl-D-aspartate (NMDA) were examined in neocortical slices of the Fischer rat using current-clamp and single-electrode voltage-clamp techniques. Layer V neocortical neurons responded to application of NMDA by depolarization with no change or an apparent increase in input resistance. Following perfusion with 10(-5) M 5-HT, the response of these neurons to NMDA was significantly increased in both amplitude and duration, whereas neuronal responses to quisqualic acid and acetylcholine were not altered by 5-HT. Furthermore, the enhanced response to NMDA in 5-HT was long-lasting, and could not be reversed during the course of the experiment. Resting membrane potential and the postspike train afterhyperpolarization were not significantly altered by 5-HT, although the input resistance was decreased by 5-HT. Excitatory postsynaptic potentials (EPSPs) were usually not affected or reversibly decreased by 5-HT. However, in a few cells exhibiting a complex voltage-dependent EPSP, 5-HT produced a long-lasting enhancement in the amplitude of the EPSP. Under voltage-clamp conditions, with Na+- and K+-channels blocked, 5-HT enhanced the inward current stimulated by application of NMDA. It is suggested that 5-HT selectively enhances the voltage- and Ca2+-dependent NMDA response.     

Ionic mechanisms of cholinergic excitation in mammalian hippocampal pyramidal cells

Intracellular recordings from CA1 hippocampal pyramidal neurons were obtained using the in vitro hippocampal slice preparation. Responses to ACh were monitored in the presence of blockers of voltage-dependent conductances including Mn2+, TTX and Ba2+. When Mn2+ was used to block voltage-dependent Ca conductance and possible indirect presynaptic cholinergic actions, ACh still induced a significant voltage-sensitive increase in apparent input resistance (Ra) (29%), but only an insignificant depolarization of membrane potential (Vm). When both voltage-dependent Ca and Na conductances were blocked by application of Mn2+ and TTX, respectively, ACh produced voltage-dependent increases in Ra (31%) without significant depolarization. In solutions containing TTX alone, ACh produced voltage-sensitive increases in Ra (32%) as well as a significant depolarization (6.2 +/- 3.1 mV (S.D.)). ACh transiently blocked the conductance increase which followed presumed Ca spikes, suggesting an action on the Ca-activated K-dependent conductance. The effects of Ba2+ application (100-200 microM) on Ra mimicked those of ACh. When ACh was applied to neurons in the presence of Ba2+, Ra remained unchanged, although Vm depolarization of 5-15 mV was still seen. The data indicate that ACh decreases both a voltage-dependent K conductance (distinct from that of the delayed rectifier) and a Ca-activated K conductance. Muscarinic cholinergic depolarization occurs as a result of blockade of K conductance, and is mediated by voltage-dependent Ca and Na conductances, and perhaps by presynaptic actions.     

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)