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.     

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.     

A pre- and postsynaptic modulatory action of 5-HT and the 5-HT2A, 2C receptor agonist DOB on NMDA-evoked responses in the rat medial prefrontal cortex.

Intracellular recordings were made from pyramidal neurons in layers V and VI of the rat medial prefrontal cortex in slice preparations to investigate the effect of the serotonin 5-HT2A,2C receptor agonist (-)-1-2,5-dimethoxy-4-bromophenol-2-aminopropane (DOB) and 5-hydroxytryptamine (5-HT) on N-methyl-D-aspartate (NMDA)-induced responses. Bath application of either DOB or 5-HT [in the presence of antagonists to 5-HT1A, 5-HT3 and gamma-aminobutytric acid (GABA) receptors] produced a concentration-dependent biphasic modulation of the NMDA responses. They facilitated and inhibited NMDA responses at low (相似文献   

Substance P augments a persistent slow inward calcium-sensitive current in voltage-clamped spinal dorsal horn neurons of the rat

Rat spinal dorsal horn neurons in slice preparations perfused with Ringer solution containing 0.5-1 microM TTX and/or 10-20 mM tetraethylammonium at 29 degrees C, were studied by using a single microelectrode voltage-clamp technique. Slow persistent inward currents were recorded during depolarizing voltage commands to membrane potentials positive to about -40 mV. The inward current was depressed by removing external Ca, or by adding 0.1-0.2 mM Cd, 5 mM Co or 0.1 mM verapamil, and was increased by adding Ba or Bay-K 8644. Substance P (SP) augmented a persistent slow inward Ca-sensitive current in a dose-dependent manner. It is suggested that this effect may be instrumental in generating the SP-evoked slow depolarization, increase in membrane excitability, and the 'bursting' behavior in the immature rat dorsal horn neurons. In addition, in some neurons SP reduced the M-like current, which effect may contribute to, but not explain, generation of the SP-induced slow depolarization.     

Antihistamine terfenadine potentiates NMDA receptor-mediated calcium influx, oxygen radical formation, and neuronal death

We previously reported that the histamine H1 receptor antagonist terfenadine enhances the excitotoxic response to N-methyl-D-aspartate (NMDA) receptor agonists in cerebellar neurons. Here we investigated whether this unexpected action of terfenadine relates to its antihistamine activity, and which specific events in the signal cascade coupled to NMDA receptors are affected by terfenadine. Low concentrations of NMDA (100 microM) or glutamate (15 microM) that were only slightly (<20%) toxic when added alone, caused extensive cell death in cultures pre-exposed to terfenadine (5 microM) for 5 h. Terfenadine potentiation of NMDA receptor response was mimicked by other H1 antagonists, including chlorpheniramine (25 microM), oxatomide (20 microM), and triprolidine (50 microM), was prevented by histamine (1 mM), and did not require RNA synthesis. Terfenadine increased NMDA-mediated intracellular calcium and cGMP synthesis by approximately 2.4 and 4 fold respectively. NMDA receptor-induced cell death in terfenadine-treated neurons was associated with a massive production of hydrogen peroxides, and was significantly inhibited by the application of either (+)-alpha-tocopherol (200 microM) or the endogenous antioxidant melatonin (200 microM) 15 min before or up to 30 min after receptor stimulation. This operational time window suggests that an enduring production of reactive oxygen species is critical for terfenadine-induced NMDA receptor-mediated neurodegeneration, and strengthens the importance of antioxidants for the treatment of excitotoxic injury. Our results also provide direct evidence for antihistamine drugs enhancing the transduction signaling activated by NMDA receptors in cerebellar neurons.     

Prostaglandin E2 increases calcium conductance and stimulates release of substance P in avian sensory neurons.

Prostaglandins are known to lower activation threshold to thermal, mechanical, and chemical stimulation in small-diameter sensory neurons. Although the mechanism of prostaglandin action is unknown, agents known to elevate intracellular calcium produce a sensitization that is similar to that produced by prostaglandins. Consistent with the idea of prostaglandin-induced elevations in calcium, prostaglandins might also stimulate the release of neurotransmitter from sensory neurons. We therefore examined whether prostaglandin E2 (PGE2) could enhance the release of the putative sensory transmitter substance P (SP) from isolated neurons of the avian dorsal root ganglion grown in culture. Utilizing the whole-cell patch-clamp recording technique, we also examined whether PGE2 could alter calcium currents in these cells. Exposure of sensory neurons to PGE2 produced a dose-dependent increase in the release of SP. One micromolar PGE2 increased release approximately twofold above basal release, whereas 5 and 10 microM PGE2 increased release by about fourfold. The release evoked by these higher concentrations of PGE2 was similar in magnitude to the release induced by 50 mM KCl. Neither arachidonic acid (10 microM), prostaglandin F2 alpha (10 microM), nor the lipoxygenase product leukotriene B4 (1 microM) significantly altered SP release. The addition of 1 microM PGE2 increased the peak calcium currents by 1.8-fold and 1.4-fold for neurons held at potentials of -60 and -90 mV, respectively. The action of PGE2 was rapid with facilitation occurring within 2 min. As with release studies, arachidonic acid, prostaglandin F2 alpha, and leukotriene B4 had no significant effect on the amplitude of the calcium current. These results suggest that PGE2 can stimulate the release of SP through the activation or facilitation of an inward calcium current. The capacity of PGE2 to facilitate the calcium current in these sensory neurons may be one mechanism to account for the ability of prostaglandins to sensitize sensory neurons to physical or chemical stimuli.     

Isoflurane preconditioning decreases glutamate receptor overactivation-induced Purkinje neuronal injury in rat cerebellar slices

A brain slice model was used to test the hypothesis that preconditioning with isoflurane, a commonly used volatile anesthetic in clinical practice, reduces neuronal injury caused by overstimulation of glutamate receptors. Glutamate receptors were stimulated by various concentrations of glutamate for 20 min, N-methyl-d-aspartate (NMDA) for 15 min or alpha-amino-3-hydroxy-5-methyl-4-isoxazol propionic acid (AMPA) for 15 min. Morphology of Purkinje neurons in the cerebellar slices of adult male Sprague-Dawley rats was evaluated 5 h after the agonist stimulation. Glutamate, NMDA and AMPA induced a dose-dependent decrease in the percentage of morphologically normal Purkinje neurons. The concentration to induce the maximal neurotoxic effect was 300 microM for glutamate, 300 microM for NMDA and 30 microM for AMPA. Isoflurane preconditioning (2% isoflurane for 30 min and then a 15-min rest period before the agonist stimulation) significantly reduced the neurotoxicity induced by 300 microM glutamate, 300 microM NMDA or 30 microM AMPA. Isoflurane preconditioning-induced protection against glutamate neurotoxicity was abolished by two protein kinase C (PKC) inhibitors, calphostin C (0.5 microM) and chelerythrine (5 microM), or a nitric oxide synthase (NOS) inhibitor, l-nitro(G)-arginine methyl ester (l-NAME, 1.5 mM), but was not affected by an adenosine A1 receptor inhibitor, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 300 nM), or a Gi protein inhibitor, pertussis toxin (PTX, 200 ng/ml). Isoflurane preconditioning-induced protection against NMDA neurotoxicity was also abolished by calphostin C, chelerythrine or l-NAME. Thus, isoflurane preconditioning reduced glutamate receptor overstimulation-induced neuronal injury/death. This neuroprotection may be PKC- and NOS-dependent.     

Substance P release in the dorsal horn assessed by receptor internalization: NMDA receptors counteract a tonic inhibition by GABA(B) receptors

Inhibitory amino acids have antinociceptive actions in the spinal cord that may involve inhibition of neurotransmitter release from primary afferents. Rat spinal cord slices with dorsal roots were used to study the effect of GABA and glycine on substance P release, assessed by the internalization of neurokinin 1 receptors. After electrical stimulation of the dorsal root at 100 Hz, about half of neurokinin 1 receptor-immunoreactive neurons in laminae I-IIo showed internalization. This internalization was inhibited by GABA (100 microM) and the GABA(B) agonist R-baclofen (10 microM), but not by the GABA(A) agonist muscimol (20 microM) or glycine (100 microM). The GABA(B) antagonist 2-hydroxysaclofen (100 microM) reversed the inhibitory effect of GABA, but not the GABA(A) antagonist bicuculline (100 microM). These findings demonstrate that GABA(B) receptors, but not GABA(A) or glycine receptors, inhibit substance P release induced by dorsal root stimulation. In contrast, R-baclofen did not inhibit the internalization produced by NMDA (100 microM), indicating that the stimulatory effect of NMDA receptors on substance P release is able to surmount the inhibitory effect of GABA(B) receptors. In the presence of the GABA(B) antagonist 2-hydroxysaclofen (100 microM), but not in its absence, stimulation of the dorsal root at 1 or 10 Hz was able to elicit internalization, which was not inhibited by the NMDA receptor antagonist AP-5 (50 microM) or the channel blocker MK-801 (10 microM). Therefore, inhibition of substance P release by GABA(B) receptors is tonic, and in its absence SP release no longer requires NMDA receptor activation.     

The role of intrinsic and agonist-activated conductances in determining the firing patterns of preoptic area neurons in the guinea pig

Whole-cell and intracellular recordings were made in coronal hypothalamic slices prepared from ovariectomized female guinea pigs. 62% of preoptic area (POA) neurons fired action potentials in a bursting manner, and exhibited a significantly greater afterhyperpolarization (AHP) than did non-bursting POA neurons. The majority (70%) of POA neurons (n=76) displayed a time-dependent inward rectification (I(h)) that was blocked by CsCl (3 mM) or by ZD 7288 (30 microM). In addition, 51% of the cells expressed a low-threshold spike (LTS) associated with a transient inward current (I(T)) that was blocked by NiCl(2) (200 microM). A smaller percentage of POA neurons (29%) expressed a transient outward, A-type K(+) current that was antagonized by a high concentration of 4-aminopyridine (3 mM). Moreover, POA neurons responded to bath application of the mu-opioid receptor agonist DAMGO (93%) or the GABA(B) receptor agonist baclofen (83%) with a membrane hyperpolarization or an outward current. These responses were accompanied by a decrease in input resistance or an increase in conductance, respectively, and were attenuated by BaCl(2) (100 microM). In addition, the reversal potential for these responses closely approximated the Nernst equilibrium potential for K(+). These results suggest that POA neurons endogenously express to varying degrees an AHP, an I(h), an I(T) and an A-type K(+) current. The vast majority of these neurons also are inhibited upon mu-opioid or GABA(B) receptor stimulation via the activation of an inwardly-rectifying K(+) conductance. Such intrinsic and transmitter-activated conductances likely serve as important determinants of the firing patterns of POA neurons.     

Human immunodeficiency virus type 1 Tat protein directly activates neuronal <Emphasis Type="Italic">N</Emphasis>-methyl-<Emphasis Type="SmallCaps">d</Emphasis>-aspartate receptors at an allosteric zinc-sensitive site

The human immunodeficiency virus type 1 (HIV-1) regulatory protein Tat is neurotoxic and may be involved in the neuropathogenesis of HIV-1 dementia, in part via N-methyl-D-aspartate (NMDA) receptor activation. Here, in acutely isolated rat hippocampal neurons, Tat evoked inward currents reversing near 0 mV, with a negative slope conductance region characteristic of NMDA receptor activation. Although the NMDA receptor antagonist ketamine blocked Tat's actions, competitive glutamate- and glycine-binding site antagonists were ineffective (AP-5 and 5,7-dichlorokynurenate, respectively). Evidence for Tat acting at a distinct modulatory site on the NR1 subunit of NMDA receptors was provided by findings that 1 microM Zn(2+) abolished Tat-evoked responses in all neurons tested. Thus, Tat appears to excite neurons via direct activation of the NMDA receptor at an allosteric Zn(2+)-sensitive site.     

Effects of substance P on excitability and ionic currents of normal and axotomized rat dorsal root ganglion neurons

Substance P (SP) may act within dorsal root ganglia (DRG) to modulate the transmission of nociceptive information. Because peripheral nerve injury (axotomy) alters the peptide content of sensory neurons, we used whole-cell recording to examine the effects of sciatic nerve section on the sensitivity of rat lumbar DRG neurons to SP (0.3--1 microM). At 1 microM, SP increased the excitability of 'small', putative nociceptive neurons but had little effect on the excitability of 'large' neurons. Two-four weeks after sciatic nerve section, however, the effect of SP on 'large' axotomized neurons was increased and its effect on 'small' neurons was decreased. SP did not affect Ca(2+) channel currents in control or axotomized neurons. The effects of SP on the current-voltage (I--V) relationship of 77% of neurons involved increased inward current at potentials below -30 mV and suppressed outward current at potentials above -20 mV. The effects of SP on the I--V relationship were similar in control and in axotomized neurons and the altered sensitivity of 'small' and 'large' cells could not be attributed to axotomy-induced changes in input resistance or membrane potential. The possible relevance of alterations in sensitivity, of 'large' DRG neurons to SP, to the generation of neuropathic pain is discussed.     

l-Homocysteate Preferentially Activates N-methyl-D-aspartate Receptors to CA1 Rat Hippocampal Neurons

Intracellular recordings and current and single-electrode voltage-clamp techniques were used to study the membrane responses of CA1 pyramidal neurons to bath application of l-homocysteic acid (l-HC) in the rat hippocampal slice preparation. In control artificial cerebrospinal fluid (ACSF), l-HC (25 - 250 microM) depolarized the membrane and induced a burst-like firing pattern. Both the membrane depolarization and the burst firing were blocked by the N-methyl-d-aspartic acid (NMDA) receptor antagonists d-(-)-2-amino-5-phosphonovaleric acid (AP-5, 50 microM), d-(-)-2-amino-7-phosphonoheptanoic acid (AP-7, 50 microM) and (+/-)-3-(2-carboxy-piperazin-4-yl)-propyl-1-phosphonic acid (CPP, 20 microM). In ACSF containing tetrodotoxin (1 microM), l-HC (100 - 300 microM) induced at resting membrane potential a depolarization which was associated with a small increase in input conductance. These effects were unaffected by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 - 20 microM) but were fully blocked by AP-5, AP-7 (50 microM) and CPP (10 - 20 microM). In voltage-clamp experiments, l-HC induced slow inward currents which were voltage-dependent between - 70 and - 30 mV and reversed polarity near 0 mV. The l-HC-induced inward current was unaffected by CNQX (10 - 20 microM) but was strongly reduced by AP-5 or AP-7 (50 microM). The l-HC-induced inward current was temperature-dependent. Between - 60 and - 70 mV, its amplitude increased by 320% when the temperature was lowered from 33 to 22 degrees C. The l-HC-induced current was also potentiated by the specific l-HC uptake blocker beta-p-chlorophenylglutamate (Chlorpheg, 0.5 - 2 mM). These data suggest that l-HC preferentially activates NMDA receptors in CA1 hippocampal neurons.     

GABA mediated excitation in immature rat CA3 hippocampal neurons

Intracellular recordings from rat hippocampal neurons in vitro during the first postnatal week revealed the presence of spontaneous giant depolarizing potentials (GDPs). These were generated by the synchronous discharge of a population of neurons. GDPs reversed polarity at -27 and -51 mV when recorded with KCl or K-methylsulphate filled electrodes, respectively. GDPs were blocked by the GABAA receptor antagonist bicuculline (10 microM). Iontophoretic or bath applications of GABA (10-300 microM) in the presence of tetrodotoxin (1 microM), induced a membrane depolarization or in voltage clamp experiments an inward current which reversed polarity at the same potential as GDPs. The response to GABA was blocked in a non-competitive manner by bicuculline (10 microM) and did not desensitize. GABA mediated GDPs were presynaptically modulated by N-methyl-D-aspartate (NMDA) and non-NMDA receptors. Their frequency was reduced or blocked by NMDA receptor antagonists and by the rather specific non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). The frequency of GDPs was enhanced by glycine and D-serine (10-30 microM) in a strychnine insensitive manner. This effect was blocked by AP-5, suggesting that it was mediated by the allosteric modulatory site of the NMDA receptor. These observations suggest that most of the 'excitatory' drive in immature neurons is mediated by GABA acting on GABAA receptors; furthermore excitatory amino acids modulate the release of GABA by a presynaptic action on GABAergic interneurons.     

Substance P mobilizes intracellular calcium and activates a nonselective cation conductance in rat spiral ganglion neurons

We demonstrate the expression of functional tachykinin receptors in rat spiral ganglion neurons (SGNs) using calcium signal measurement and whole-cell patch clamp recording. Substance P (SP; 10 microm, 1 s application) induced a transient increase in intracellular calcium. The SP dose-response study showed an EC50 of 18.8 microm and a Hill slope of 0.77. Comparison between specific agonists for the three tachykinin receptor (NKR) types showed the potency NKR3 > NKR1 > NKR2 at 10 microm. The Ca2+ response could be evoked in Ca2+-free medium and was blocked by N-ethylmaleimide and U-73122, indicating that Ca2+ was released from intracellular stores via a G-protein and phospholipase C pathway. Under whole-cell voltage clamp recording at a holding potential of -50 mV, SP (10 microm, 1 s) evoked a slowly developing transient inward current. The current reversed near to 0 mV and ionic permeability experiments revealed a cation nonselective conductance also permeable to large organic cations such as N-methyl-D-glucamine and tetraethylammonium. Neither removing extracellular calcium nor chelating intracellular calcium with 10 mm BAPTA could block the SP-evoked current. This conductance appeared coupled to G-protein activation as intracellular GDP-betaS blocked the SP-evoked current. Mutual desensitization and occlusion studies with acetylcholine and ATP showed that the SP-evoked conductance share effector channels and/or intracellular processes with the purinergic/cholinergic conductance. In SGNs, SP could have both a trophic action, via a calcium response, and a neuromodulatory role, by a depolarizing action through the activation of nonselective cation channels.     

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.     

Antinociceptive involvement of substance P in the spinal cord of mice: dose effects of substance P on the behavior elicited by intrathecally administered NMDA

The functional interaction between substance P (SP) and N-methyl-d-aspartate (NMDA) was studied to clarify the diversity of the roles of SP in nociceptive processes at the spinal level in mice. Behavioral responses elicited by intrathecal co-administration of NMDA (0.25 nmol) with various doses of SP (0.3–12 pmol) were observed for 1 min. The high dose of SP (12 pmol) potentiated NMDA-induced responses, which consisted of caudally directed licking and biting, while the low dose of SP (1 pmol) significantly reduced the responses by 40% compared to control mice administered NMDA alone. The antinociceptive effect of the low dose of SP was negated by co-administration of the opioid receptor antagonist naloxone. Furthermore, the antinociception produced by SP was present in mice pre-treated with systemic administration of capsaicin during the neonatal period. These results suggest that one of the roles SP plays at the spinal level is an involvement in antinociception. The activities of excitatory dorsal horn neurons are considered to be inhibited by endogenous opioid peptides released from inhibitory dorsal horn neurons directly stimulated by SP.     

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.     

NMDA receptor activation enhances diazepam binding inhibitor and its mRNA expressions in mouse cerebral cortical neurons

Effects of N-methyl-D-aspartate (NMDA) on diazepam binding inhibitor (DBI) and its mRNA expression in mouse cerebral cortical neurons were examined. A significant increase in DBI mRNA expression was observed 1 day after the exposure to 0.1 microM NMDA and the maximal expression occurred 2 days after the exposure, whereas transient exposure to 0.1 microM NMDA for 15 min, 1 and 3 h produced no changes in the expression. Similarly, no changes in the expression were found by the concomitant exposure to NMDA and MK-801, a NMDA receptor antagonist, for 72 h subsequent to the incubation with NMDA alone for 3 h. Such NMDA-induced increases in DBI mRNA expression were dose-dependently inhibited by MK-801. Moreover, neuronal DBI content significantly increased by treatment with NMDA, which was completely abolished by MK-801. These results indicate that continuous activation of NMDA receptors is an essential factor for increasing DBI expression in the neurons.