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

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

Ion channels activated by quinolinic acid in cultured rat hippocampal neurons

The membrane responses to quinolinic acid, an excitotoxic brain metabolite, were studied in cultured rat hippocampal neurons with the patch-clamp technique. In the whole-cell recording mode, pressure applications of quinolinic acid elicited inwardly directed membrane currents over a membrane potential range of −60 to −5 mV. The current response reversed at about 0 mV. The current-voltage (I–V) relation of the response had a negative slope conductance at membrane potentials more negative than −40 mV. On removal of Mg²⁺ from the extracellular solution, the current response showed no region of negative slope conductance at potentials more positive than −60 mV. In Mg²⁺-free solution applications of quinolinic acid elicited discrete pulse-like current flows through the outside-out membrane patch. The single channel conductance was 40–46 pS over a membrane potential range of −40 to −80 mV, and 50–55 pS at membrane potentials more positive than +30 mV, showing an outward rectification. These values of the single channel conductance were similar to those of the main conducting state of the channels activated by (NMDA). The responses to quinolinic acid were completely suppressed by the NMDA receptor antagonist (±)-2-amino-5-phosphonovaleric acid. The results indicate that quinolinic acid selectively activates NMDA receptors in the cultured rat hippocampal neurons.     

Synaptic Excitation Triggers Oscillations During NMDA Receptor Activation in Rat Abducens Motoneurons

Rat abducens motoneurons were intracellularly recorded in vivo during synaptic excitation and extracellular microionophoretic application of N -methyl- d -aspartate (NMDA). Trigeminal excitatory post-synaptic potentials (EPSPs) evoked in abducens motoneurons were studied during intracellular current injection. They were not sensitive to hyperpolarization or depolarization of the membrane potential in the range of –75 mV to –55 mV using current pulse intensities between –3 nA and + 1 nA. Microionophoretic applications of aminophosphonovalerate (APV), MK801 and i.v. injections of MK801 (1 – 3 mg/kg) or ketamine (10 mg/kg) did not modify trigeminal EPSPs, suggesting that NMDA receptors are not involved in this synaptic transmission. However, microionophoretic applications of NMDA on abducens motoneurons enhanced trigeminal EPSPs and gave rise to regenerative oscillations. The co-activation of NMDA receptors and trigeminal synapses induced these oscillations. The trigeminal EPSP may delay and reset the oscillations depending on where it was evoked in the oscillatory cycle. Depolarizing current pulses intracellularly applied to abducens motoneurons could trigger a post-hyperpolarization followed by rebound depolarization during NMDA application, confirming the activation of active membrane properties. However, depolarizing current pulses could not trigger oscillations similar to those entrained by the EPSPs. The importance of the location of trigeminal synapses in relation to those of NMDA receptors in the dendritic arborization of abducens motoneurons is discussed. Our results show that the same sensory stimulus may have different post-synaptic effects on abducens motoneurons during the co-activation of NMDA receptors. A complete modification of the motor output during NMDA receptor activation strongly supports an active role of abducens motoneurons provided that NMDA receptors are physiologically activated during motor pattern generation.     

Electrophysiological studies of NMDA receptors

Recent electrophysiological studies of NMDA receptors and of the associated ion channels (NMDA channels) have revealed five properties of this system: (1) the NMDA channels are blocked by Mg²⁺ in a voltage-dependent way; (2) the NMDA channels are permeable to Ca²⁺ as well as to Na⁺ and K⁺; (3) the NMDA channels may adopt multiple conductance states; some of the minor states (small conductances) resemble the major conductance states opened by non-NMDA agonists; (4) continued exposure to NMDA agonists produces short-term and long-term decreases in the sensitivity of the NMDA system; and (5) glycine potentiates the response to NMDA.     

Cationic modulation of 5-HT2 and 5-HT3 receptors in rat sensory neurons: the role of K, Ca and Mg

The effects of changes in external K⁺, Ca²⁺, and Mg²⁺ concentrations on 5-HT₂- and 5-HT₃ receptor-mediated depolarizations of the resting membrane potential in rat dorsal root ganglion (DRG) cells was studied. In cells exhibiting a 5-HT₂-mediated response, 5-HT and α-methyl 5-HT depolarized the resting membrane potential (RMP) and increased the slope of the current–voltage (I/V) relationship. The equilibrium potential (E_r) for the depolarization was linearly related to the logarithm of the [K⁺]_o, indicating the depolarization resulted from a decrease in resting K⁺ conductance. In a subpopulation of large-diameter acutely dissociated DRG neurons recorded from using the whole-cell patch-clamp configuration, 5-HT produced an inward shift in the current required to hold cells at −60 mV. This inward shift in holding current was associated with a reduction in membrane conductance and reversed near E_k. This data suggests that the 5-HT₂ receptor-mediated depolarization and increase in R_in seen in intact DRG preparation is produced by blockade of an outward K⁺ leak current. Increases in [K⁺]_o reduced the increase in R_in and depolarization induced by 5-HT with 50% inhibition of the depolarization occurring at 8.3 mM of [K⁺]_o. Half-normal Ca²⁺ (1.2 mM) produced a downward shift of the 5-HT concentration–response curve, reducing the maximal response by 40%, with minimal effect on the half-maximal response. Mg²⁺ ions did not affect this 5-HT response. In cells exhibiting a 5-HT₃ receptor response, 5-HT and 2-methyl-5-HT produced depolarization with decreased R_in. The E_r for this depolarizing response (−30.2±1.8 mV) became less negative (−11.5 mV) in 10 mM [K⁺]_o with minimal effect on the amplitude of the depolarization. In Na⁺-free superfusate, the 5-HT-induced depolarization was converted to hyperpolarization. This indicated the 5-HT₃ response increased a mixed Na⁺/K⁺ conductance. Elevated Ca²⁺ or Mg²⁺ markedly reduced the 5-HT₃ response. Incubation with 3.5 mM Ca²⁺ shifted the 5-HT concentration–response curve downward and to the right, decreasing the maximal response by 49% and increasing the EC₅₀ by 10-fold. Elevated Mg²⁺ produced similar effects. In cells where both 5-HT₂- and 5-HT₃-mediated responses could be demonstrated, the elevation of K⁺ or the reduction of Ca²⁺ converted a 5-HT₂ response to a 5-HT₃ response. The above data suggest that elevation of [K⁺]_o or reduction of [Ca²⁺]_o produced by rapid firing rates of sensory neurons will favor the expression of 5-HT₃ responses over 5-HT₂ responses.     

Serotonin 5‐HT1B receptor‐mediated calcium influx‐independent presynaptic inhibition of GABA release onto rat basal forebrain cholinergic neurons

Modulatory roles of serotonin (5‐HT) in GABAergic transmission onto basal forebrain cholinergic neurons were investigated, using whole‐cell patch‐clamp technique in the rat brain slices. GABA_A receptor‐mediated inhibitory postsynaptic currents (IPSCs) were evoked by focal stimulation. Bath application of 5‐HT (0.1–300 μm ) reversibly suppressed the amplitude of evoked IPSCs in a concentration‐dependent manner. Application of a 5‐HT_1B receptor agonist, CP93129, also suppressed the evoked IPSCs, whereas a 5‐HT_1A receptor agonist, 8‐OH‐DPAT had little effect on the evoked IPSCs amplitude. In the presence of NAS‐181, a 5‐HT_1B receptor antagonist, 5‐HT‐induced suppression of evoked IPSCs was antagonised, whereas NAN‐190, a 5‐HT_1A receptor antagonist did not antagonise the 5‐HT‐induced suppression of evoked IPSCs. Bath application of 5‐HT reduced the frequency of spontaneous miniature IPSCs without changing their amplitude distribution. The effect of 5‐HT on miniature IPSCs remained unchanged when extracellular Ca²⁺ was replaced by Mg²⁺. The paired‐pulse ratio was increased by CP93129. In the presence of ω‐CgTX, the N‐type Ca²⁺ channel blocker, ω‐Aga‐TK, the P/Q‐type Ca²⁺ channel blocker, or SNX‐482, the R‐type Ca²⁺ channel blocker, 5‐HT could still inhibit the evoked IPSCs. 4‐AP, a K⁺ channel blocker, enhanced the evoked IPSCs, and CP93129 had no longer inhibitory effect in the presence of 4‐AP. CP93129 increased the number of action potentials elicited by depolarising current pulses. These results suggest that activation of presynaptic 5‐HT_1B receptors on the terminals of GABAergic afferents to basal forebrain cholinergic neurons inhibits GABA release in Ca²⁺ influx‐independent manner by modulation of K⁺ channels, leading to enhancement of neuronal activities.     

Mg dependence of membrane resistance increases evoked by NMDA in hippocampal neurones

The response of granule cells and CA1 pyramidal neurones to NMDA was studied in the presence of and absence of Mg²⁺ using an in vitro slice preparation. In the absence of Mg²⁺ the depolarizing response of hippocampal neurones to NMDA is accompanied by a decrease in input resistance. In the presence of Mg²⁺ ions, however, the response to NMDA is always associated with an apparent increase in input resistance. These results indicate that the action of NMDA is by a classical mechanism of conductance increase and are in agreement with the suggestion that the apparent increase in input resistance associated with NMDA depolarizations is the result of voltage-dependent channel block by Mg²⁺ of the NMDA evoked current.     

Effects of magnesium on fictive locomotion induced by activation of N-methyl-d-aspartate (NMDA) receptors in the Lamprey spinal cord in vitro

It has previously been demonstrated that an activation of N-methyl-d-aspartate (NMDA) receptors can induce fictive locomotion as well as tetrodotoxin (TTX)-resistant membrane potential oscillations in certain types of neurone in the in vivo preparation of the lamprey spinal cord. These oscillations in individual neurons depend on voltage-sensitive properties of NMDA-activated channels which are only manifested in the presence of Mg²⁺. To evaluate the role of these pacemaker-like oscillations in the generation of locomotion, the motor patterns induced by N-methyl-d,l-aspartate (NMA) before and after removal of Mg²⁺ were compared. It was found that the ventral root burst pattern of fictive locomotion was more irregular after removal of Mg²⁺, particularly at low burst rates. This suggests that the membrane properties underlying the NMDA-induced TTX-resistant membrane potential oscillations are of importance for the generation of a stable and regular locomotor activity in particular at low rates of fictive locomotion. When fictive locomotion was induced instead by an activation of kainate receptors a removal of Mg²⁺ had no effect on the motor pattern. The effects of the two K⁺-channel blockers, tetraethylammonium (TEA) and gallamine were also tested on NMA-induced fictive locomotion. Both compounds caused an increase in the burst frequency. The Mg²⁺-dependent NMDA-induced bistable membrane properties thus appear to be of importance for the operation of the network which generates the locomotor pattern.     

Channel properties of NMDA receptors on magnocellular neuroendocrine cells cultured from the rat supraoptic nucleus

Application of N-methyl-d-aspartate (NMDA) to the supraoptic nucleus of the hypothalamus (SON) generates clustered firing that may be important in hormone release. However, synaptically evoked EPSPs recorded from SON neurons exhibit varying contributions from NMDA receptors. We used the high resolution of single-channel recording to examine the receptor and ion channel properties of NMDA receptors expressed by SON neurons in `punch' culture. Biocytin introduced into individual neurons during patch clamp recording revealed large (32.1±3.3 μm), oblong somas and bipolar extensions typical of magnocellular neuroendocrine cells (MNCs). Rapid application of NMDA (100–300 μM) in the presence of 10 μM glycine to outside-out macropatches resulted in openings with an average conductance of 46.9 pS and reversal potential of +3.9 mV. Increasing glycine from 0.03 to 30 μM increased the apparent frequency, duration and occurrence of overlapping NMDA-elicited openings. NMDA responses were inhibited by Mg²⁺ in a voltage-dependent manner and by the NMDA-site antagonist, d-(−)-2-amino-5-phosphonovaleric acid (D-APV). Application of saturating NMDA or glycine alone with the glycine-site antagonist, 5,7-dichlorokynurenate (DCK) or with D-APV, respectively, did not result in agonist-induced openings. NR1 immunoreactivity was observed in large neurons (>25 μm) with MNC-like morphology. These single-channel and immunocytochemical data confirm the presence of functional NR1-containing NMDA receptors in MNCs.     

Effect of zinc on NMDA receptor-mediated channel currents in cortical neurons.

Recent data have indicated that the divalent cation Zn2+ can selectively block central neuronal excitation mediated by N-methyl-D-aspartate (NMDA) receptors. The present experiments were conducted to determine the action of Zn2+ at the single-channel level. Outside-out membrane patches were prepared from cultured murine cortical neurons. Glutamate, 3 microM, in the presence of 5 microM glycine activated channels with a main conductance state of about 50 pS which were blocked in a voltage-dependent manner by Mg2+. Zn2+ appeared to have 2 effects on these NMDA receptor-activated channels. First, at concentrations as low as 1-10 microM, Zn2+ produced a concentration-dependent reduction in channel open probability, insensitive to membrane voltage between -60 and +40 mV; about 50% reduction in open probability was produced by 3 microM Zn2+. This reduction was mostly due to a decrease in opening frequency and only weakly mimicked by Mg2+. Second, at higher concentrations (10-100 microM) and negative membrane voltages, Zn2+ additionally produced an apparent reduction in single-channel amplitude, associated with an increase in channel noise, suggestive of a fast channel block. The amplitude reduction was voltage-dependent, with a delta of 0.51; amplitude distribution analysis suggested that this voltage dependence was primarily contributed by the "on" blocking rate constant, with little contribution from the "off" rate constant. The channel block produced by Zn2+ was faster than that of Mg2+, which at 100 microM and negative membrane voltages induces flickering of the NMDA receptor-activated channel without changing apparent channel amplitude.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMDA Actions on Rat Abducens Motoneurons

Intracellular recordings were made from rat abducens motoneurons in vivo during local extracellular micro-ionophoretic application of N-methyl-d-aspartate (NMDA) and NMDA receptor antagonists. Typical NMDA responses, at a resting potential of -60 mV, consisted of a slow depolarization with an apparent increase in membrane resistance, bursts of action potentials followed by stable repetitive firing. Ionophoretic applications of aminophosphonovalerate (APV), kynurenate or MK801 reduced or blocked the NMDA-induced responses. The NMDA responses were voltage-dependent. NMDA responses induced by short (< 30 s) NMDA application pulses were blocked by hyperpolarizing the neuron. Long duration (> 30 s) NMDA applications induced rhythmic plateau potentials in hyperpolarized abducens motoneurons. The rhythmic depolarizations (15 - 30 mV) were modulated in both frequency and duration by current injection. They were abolished by further hyperpolarization or replaced by stable repetitive firing when hyperpolarization was removed. Our data show that NMDA receptors are present in rat abducens motoneurons and may be involved in the induction of rhythmic activities. The voltage-dependent blockade of somatic NMDA receptor-associated ion channels by cell hyperpolarization may be important for these oscillations. It is suggested that the rhythmic behaviour is due to the activation of dendritic NMDA receptors.     

Protective effect of MgSO4 infusion on NMDA receptor binding characteristics during cerebral cortical hypoxia in the newborn piglet

This study tests the hypothesis that magnesium, a selective non-competitive antagonist of the NMDA receptor, will attenuate hypoxia-induced alteration in NMDA receptors and preserve MK-801 binding characteristics during cerebral hypoxia in vivo. Anesthetized, ventilated and instrumented newborn piglets were divided into three groups: normoxic controls were compared to untreated hypoxic and Mg²⁺-treated hypoxic piglets. Cerebral hypoxia was induced by lowering the FiO₂ to 5–7% and confirmed biochemically by a decrease in the levels of phosphocreatine (82% lower than control). The Mg²⁺-treated group received MgSO₄ 600 mg/kg over 30 min followed by 300 mg/kg administered during 60 min of hypoxia. Plasma Mg²⁺ concentrations increased from1.6 ± 0.1mg/dl to17.7 ± 3.3mg/dl.³H-MK-801 binding was used as an index of NMDA receptor modification. TheB_max in control, hypoxic and Mg²⁺-treated hypoxic piglets was1.09 ± 0.17, 0.70 ± 0.25and0.96 ± 0.14pmoles/mg protein, respectively. TheK_d for the same groups were10.02 ± 2.04, 4.88 ± 1.43and8.71 ± 2.23nM, respectively. TheB_max andK_d in the hypoxic group were significantly lower compared to the control and Mg²⁺-treated hypoxic groups, indicating a preservation of NMDA receptor number and affinity for MK-801 during hypoxia with Mg²⁺. The activity of Na⁺, K⁺ ATPase, a marker of neuronal membrane function, was lower in the hypoxic group compared to the control and Mg²⁺-treated hypoxic groups. These findings show that MgSO₄ prevents the hypoxia-induced modification of the NMDA receptor and attenuates neuronal membrane dysfunction. We suggest that the administration of Mg²⁺ prior to and during hypoxia may be neuroprotective in vivo, possibly by reducing the NMDA receptor-mediated influx of calcium.     

Pacemaker potentials of serotonergic dorsal raphe neurons: Contribution of a low-threshold Ca2+ conductance

Neurons of the dorsal raphe nucleus exhibit intrinsic pacemaker potentials (gradual interspike depolarizing ramps) enabling them to sustain spontaneous rhythmic activity in the absence of synaptic interactions. A depolarizing prepotential (PP) has been observed in these cells, which appears to trigger the spike toward the end of the pacemaker cycle. The purposes of this study, carried out in the rat dorsal raphe nucleus brain slice preparation, were to (1) determine the ionic nature of the PP, (2) investigate its time- and voltage-dependent properties, and (3) investigate the possible modulation of the underlying conductance by the α₁-agonist phenylephrine and by serotonin (5-HT), agents that modify dorsal raphe pacemaker activity. During intracellular recording under current clamp, PPs were completely and reversibly blocked by divalent cations indicating that Ca²⁺ carries a significant portion of the current causing the PPs. Ni²⁺ specifically inhibited the PP with no effect on high-threshold (?40 mV) Ca²⁺ spikes or the Ca²⁺ activated K⁺ conductance in these neurons. Activation threshold for the PP was found to be approximately ?60 mV. Priming by hyperpolarization allowed removal of inactivation (de-inactivation) of the PP in a time- and voltage-dependent manner, with maximal PPs accompanying hyperpolarizing pulses to ?90 mV and the de-inactivation beginning to occur between ?65 and ?75 mV. Single-electrode voltage-clamp experiments demonstrated a region of negative-slope conductance between ?60 and ?50 mV, which corresponds to the range of PP activation. The results of this study are consistent with a lowthreshold Ca²⁺ conductance underlying the PP whose role is to enable the membrane potential to rebound to action potential threshold at the end of the pacemaker cycle; neither phenylephrine nor 5-HT directly affected this inward current.     

An immunocapture/scintillation proximity analysis of Gαq/11 activation by native serotonin (5‐HT)2A receptors in rat cortex: Blockade by clozapine and mirtazapine

Though transduction mechanisms recruited by heterologously expressed 5‐HT_2A receptors have been extensively studied, their interaction with specific subtypes of G‐protein remains to be directly evaluated in cerebral tissue. Herein, as shown by an immunocapture/scintillation proximity analysis, 5‐HT, the prototypical 5‐HT_2A agonist, DOI, and Ro60,0175 all enhanced [³⁵S]GTPγS binding to Gαq/11 in rat cortex with pEC₅₀ values of 6.22, 7.24 and 6.35, respectively. No activation of Go or Gs/olf was seen at equivalent concentrations of DOI. Stimulation of Gαq/11 by 5‐HT (30 μM) and DOI (30 μM) was abolished by the selective 5‐HT_2A vs. 5‐HT_2C/5‐HT_2B antagonists, ketanserin (pK_B values of 9.11 and 8.88, respectively) and MDL100,907 (9.82 and 9.68). By contrast, 5‐HT‐induced [³⁵S]GTPγS binding to Gαq/11 was only weakly inhibited by the preferential 5‐HT_2C receptor antagonists, RS102,221 (6.94) and SB242,084 (7.39), and the preferential 5‐HT_2B receptor antagonist, LY266,097 (6.66). The antipsychotic, clozapine, which had marked affinity for 5‐HT_2A receptors, blocked the recruitment of Gαq/11 by 5‐HT and DOI with pK_B values of 8.54 and 8.14, respectively. Its actions were mimicked by the “atypical” antidepressant and 5‐HT_2A receptor antagonist, mirtazapine, which likewise blocked 5‐HT and DOI‐induced Gαq/11 protein activation with pK_B values of 7.90 and 7.76, respectively. In conclusion, by use of an immunocapture/scintillation proximity strategy, this study shows that native 5‐HT_2A receptors in rat frontal cortex specifically recruit Gαq/11 and that this action is blocked by clozapine and mirtazapine. Quantification of 5‐HT_2A receptor‐mediated Gαq/11 activation in frontal cortex should prove instructive in characterizing the actions of diverse classes of psychotropic agent. Synapse 63:95–105, 2009. © 2008 Wiley‐Liss, Inc.     

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.     

A Study of the Barium-sensitive and -insensitive Components of the Action of Thyrotropin-releasing Hormone on Lumbar Motoneurons of the Rat Isolated Spinal Cord

The electrophysiological action of thyrotropin-releasing hormone (TRH) on rat spinal motoneurons was studied in vitro using single-electrode voltage- and current-clamp techniques. In current-clamp conditions TRH elicited a slowly developing depolarization, associated with a large input resistance increase and sustained neuronal firing; the primary metabolites of TRH were ineffective. Under voltage-clamp conditions in the presence of tetrodotoxin, TRH evoked a large inward current (I_TRH; peaking at approximately –40 mV) associated with a large input conductance fall. Only 44% of cells displayed I_TRH reversal; when the chord conductance values of these cells were plotted against membrane potential, a bell-shaped relation occurred, indicating voltage-dependent block by TRH of a persistent conductance active over a wide range of membrane potentials. I_TRH reversal values were shifted to more positive levels in high K⁺ solution in Nernstian fashion; hence a large proportion of the TRH response is suggested to be mediated by the block of a K⁺ conductance (I_K(T)). I_K(T) (and its voltage-dependent block by TRH) was resistant to certain K⁺ channel antagonists (tetraethylammonium, Cs⁺, 4-aminopyridine or apamin), but was depressed by Ba²⁺. The Ba²⁺-resistant fraction of I_TRH was attenuated by Cd²⁺, Mn²⁺ or Co²⁺, indicating that it probably involved a Ca²⁺-sensitive inward current. Concomitant application of Ba²⁺ and Cd²⁺ induced a near-total block of the response to TRH. It is suggested that suppression of I_K(T), associated with the onset of a Ca²⁺-sensitive current, can explain the excitatory effect of TRH on rat spinal motoneurons.     

Zinc (Zn) blocks voltage gated calcium channels in cultured rat dorsal root ganglion cells

Dorsal root ganglion cells (DRGs) exhibit 3 types of voltage-dependent calcium channels. We have cultured DRGs from 2- to 4-day-old rat pups and obtained whole-cell patch-clamp recordings of calcium-channel currents after 1–5 days in culture. The calcium-channel currents (carried by barium) were recorded with tetrodotoxin (TTX) in the external solution. A cesium-based solution containing Na-ATP, HEPES and EGTA was used in the recording pipette. Cells were held at −80 mV and calcium channel currents were evoked by stepping to depolarized voltages. The divalent cation zinc (Zn²⁺) blocked sustained and transient voltage sensitive calcium channel currents. Onset of the blockade was fast and a steady-state was reached within 5–15 min, depending upin the concentration used. The IC₅₀ for inhibition of the peak current evoked by a step depolarization from −80 mV to 0 mV (N plus L channels) for 80 ms was 69 μM Zn²⁺ and the Hill slope about 1. The calcium current evoked by a voltage step from −80 mV to voltages between −40 mV and −15 mV (T-type current) was more sensitive (> 80% block with 20 μM Zn²⁺). During wash the effect was only partly reversible in 50% of the neurons. Thus, Zn²⁺ is a potent blocker of voltage dependent calcium currents in mammalian neurons, especially of T-type currents.     

The effect of glutamate and inhibitors of NMDA receptors on postdenervation decrease of membrane potential in rat diaphragm

The early postdenervation depolarization of rat diaphragm muscle fibers (8–10 mV within 3 h in vitro) is substantially smaller (3 mV) when muscles are bathed with 1×10^?3 M l-glutamate (Glu) or 1×10^?3 M N-methyl-d-aspartate (NMDA). The effects of Glu and NMDA are inhibited in a dose-dependent manner by competitive inhibitor 2-amino-5-phosphonovaleric acid (APV) withK _i 6.3×10^?4 M, by 2×10^?7 M MK-801, which acts as an open channel inhibitor, by 2–3×10^?4 Zn²⁺, which reacts with surface-located sites of the NMDA subtype of the glutamate receptor, and also by glycine-free solutions and 7-Cl-kynurenic acid, which inhibits the glycine binding sites on NMDA receptors. It follows that the effect of glutamate on early postdenervation depolarization is mediated by the NMDA subtype of glutamate receptor with similar pharmacological properties to those found in neurons. The only exception found was the glutamate-like action of 1×10^?7 M MK-801, which partially prevented the early postdenervation depolarization when present in the muscle bath during the first 3 h after nerve section.     

Anti‐dyskinetic mechanisms of amantadine and dextromethorphan in the 6‐OHDA rat model of Parkinson’s disease: role of NMDA vs. 5‐HT1A receptors

Amantadine and dextromethorphan suppress levodopa (L‐DOPA)‐induced dyskinesia (LID) in patients with Parkinson’s disease (PD) and abnormal involuntary movements (AIMs) in the unilateral 6‐hydroxydopamine (6‐OHDA) rat model. These effects have been attributed to N‐methyl‐d ‐aspartate (NMDA) antagonism. However, amantadine and dextromethorphan are also thought to block serotonin (5‐HT) uptake and cause 5‐HT overflow, leading to stimulation of 5‐HT_1A receptors, which has been shown to reduce LID. We undertook a study in 6‐OHDA rats to determine whether the anti‐dyskinetic effects of these two compounds are mediated by NMDA antagonism and/or 5‐HT_1A agonism. In addition, we assessed the sensorimotor effects of these drugs using the Vibrissae‐Stimulated Forelimb Placement and Cylinder tests. Our data show that the AIM‐suppressing effect of amantadine was not affected by the 5‐HT_1A antagonist WAY‐100635, but was partially reversed by the NMDA agonist d ‐cycloserine. Conversely, the AIM‐suppressing effect of dextromethorphan was prevented by WAY‐100635 but not by d ‐cycloserine. Neither amantadine nor dextromethorphan affected the therapeutic effects of L‐DOPA in sensorimotor tests. We conclude that the anti‐dyskinetic effect of amantadine is partially dependent on NMDA antagonism, while dextromethorphan suppresses AIMs via indirect 5‐HT_1A agonism. Combined with previous work from our group, our results support the investigation of 5‐HT_1A agonists as pharmacotherapies for LID in PD patients.