Dendritic Spike Saturation of Endogenous Calcium Buffer and Induction of Postsynaptic Cerebellar LTP

The architecture of parallel fiber axons contacting cerebellar Purkinje neurons retains spatial information over long distances. Parallel fiber synapses can trigger local dendritic calcium spikes, but whether and how this calcium signal leads to plastic changes that decode the parallel fiber input organization is unknown. By combining voltage and calcium imaging, we show that calcium signals, elicited by parallel fiber stimulation and mediated by voltage-gated calcium channels, increase non-linearly during high-frequency bursts of electrically constant calcium spikes, because they locally and transiently saturate the endogenous buffer. We demonstrate that these non-linear calcium signals, independently of NMDA or metabotropic glutamate receptor activation, can induce parallel fiber long-term potentiation. Two-photon imaging in coronal slices revealed that calcium signals inducing long-term potentiation can be observed by stimulating either the parallel fiber or the ascending fiber pathway. We propose that local dendritic calcium spikes, evoked by synaptic potentials, provide a unique mechanism to spatially decode parallel fiber signals into cerebellar circuitry changes.     

Tiletamine is a potent inhibitor of N-methyl-aspartate-induced depolarizations in rat hippocampus and striatum

N-methyl-D,L-aspartate (NMA) antagonists are of potential value in the treatment of epilepsy and ischemia, but commonly utilized compounds are of low potency and poorly penetrate the brain. Tiletamine hydrochloride is a lipophilic and potent veterinary anesthetic. This study shows tiletamine to be similar to ketamine and to phencyclidine, agents known to interact with the NMA receptor. Effects of tiletamine on synaptic transmission and on direct excitatory responses to exogenous amino acids were examined in rat hippocampal and striatal slices. In striatal slices, tiletamine inhibited the NMA-mediated, but not the spontaneous, release of [3H]acetylcholine, with an IC50 of 70 nM. In hippocampal CA1 cells, 3 microM tiletamine in the perfusate reversibly blocked the intracellularly recorded responses to ionophoretically applied NMA, but not to glutamate, quisqualate and kainate. Tiletamine, 3 to 100 microM, had no effect on the orthodromically elicited excitatory postsynaptic potential, action potential amplitude or duration, resting membrane potential, or input resistance. In Mg++-free perfusate, the excitatory postsynaptic potential was greatly augmented to give a paroxysmal depolarization shift and was reversibly blocked by 10 microM tiletamine. Our results show that tiletamine is a potent and reversible antagonist of NMA-mediated responses without itself having major effects in low concentrations on normal membrane and synaptic pyramidal cell properties.     

Synaptic transmission in low extracellular calcium is preserved by 3,4-diaminopyridine

Synaptic transmission in the isolated bullfrog sympathetic ganglion was studied during graded reductions in extracellular Ca++, from the normal of 1.8 mM, in the absence and in the presence of different concentrations of 3,4-diaminopyridine (3,4-DAP). In drug-free Ringer's synaptic transmission, measured as the amplitude of the postganglionic compound action potential, failed progressively as Ca++ was reduced from 1.8 to 0.47 mM. This Ca++-dependence curve of synaptic transmission was shifted to the left (lower Ca++) by 3,4-DAP in dose-related fashion with threshold at 0.1 microM and maximum shift at 10 microM 3,4-DAP. At maximum shift (4- to 5-fold) in the Ca++-dependence curve, compound action potential amplitude was normal at 0.33 mM Ca++ then failed progressively as Ca++ was reduced to 0.12 mM. Also 3,4-DAP causes stimulus-bound repetitive postganglionic responses (SBR) to single preganglionic stimuli (Apatoff and Riker, 1982). SBR were selectively abolished as Ca++ was reduced form 1.8 to 0.47 mM. The data reveal that 3,4-DAP facilitates presynaptic influx or binding of Ca++. Furthermore, the high Ca++ requirement for 3,4-DAP-induced SBR, as well as the difference between threshold drug concentrations for preserving transmission (0.1 microM) and for generating SBR (2-5 microM), lead to the speculation that there may be two presynaptic receptors for 3,4-DAP.     

Methylmercury blocks N- and L-type Ca++ channels in nerve growth factor-differentiated pheochromocytoma (PC12) cells.

Effects of methylmercury (MeHg) on whole-cell Ba++ currents in rat pheochromocytoma (PC12) cells were examined. Based on biophysical characteristics and sensitivity to omega-conotoxin GVIA and dihydropyridine agonists and antagonists, voltage-activated Ba++ currents (IBa) in PC12 cells were mediated by N- and L-type Ca++ channels. Addition of MeHg (10 microM) to the extracellular solution caused a rapid and complete block of current carried by 20 mM Ba++. The rate of block of IBa by MeHg increased in a concentration-dependent manner between 1 and 20 microM. Increasing the frequency of stimulation from 0.1 to 0.4 Hz facilitated block of IBa by MeHg. A 2-min application of 10 microM MeHg in the absence of stimulation also reduced IBa by approximately 80%. Thus, block of IBa by MeHg is not state-dependent. Additionally, MeHg blocked IBa when the membrane holding potential was -40, -70 and -90 mV, indicating that both N- and L-type Ca++ channels are blocked by MeHg. Block of IBa by MeHg was voltage-dependent at a membrane holding potential of -40 mV, but not at holding potentials of -70 and -90 mV. Decreasing the extracellular concentration of Ba++ ([Ba++]e) from 20 mM to 10 mM increased the magnitude of block by MeHg from 45.6 to 77.3%. Increasing [Ba++]e to 30 mM caused no further antagonism of block. Block of IBa by MeHg was not reversed by washing with MeHg-free solution. The ionic permeability of PC12 cell Ca++ channels was Ca++ = Sr++ greater than Ba++. In the presence of MeHg, all three divalent cations were equally permeant.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential effects of methylmercury on gamma-aminobutyric acid type A receptor currents in rat cerebellar granule and cerebral cortical neurons in culture

Cerebellar granule cells are particularly sensitive to inhibition by methylmercury (MeHg) on GABA(A) receptor function. This is manifested as a more rapid block of inhibitory postsynaptic currents/inhibitory postsynaptic potentials than for Purkinje cells. The underlying mechanism(s) for differential sensitivity of GABAergic transmission to MeHg in cerebellar neurons is unknown. Differential expression of alpha(6) subunit-containing GABA(A) receptors in cerebellar granule and Purkinje neurons could partially explain this. GABA-evoked currents (I(GABA)) were recorded in response to MeHg in alpha(6) subunit-containing cerebellar granule cells and alpha(6) subunit-deficient cerebral cortical cells in culture. Cortical cells were substituted for Purkinje cells, which do not express alpha(6) subunits. They express the same alpha(1)-containing GABA(A) receptor as Purkinje cells but lack characteristics that enhance Purkinje cell resistance to MeHg. I(GABA) were obtained using whole-cell recording and symmetrical [Cl(-)]. MeHg reduced I(GABA) to complete block in both cell types in a time- and concentration-dependent manner. This effect was faster in granule cells than cortical cells. Effects of MeHg on I(GABA) were recorded in granule cells at various developmental stages (days in vitro 4, 6, and 8) to alter the expression level of alpha(6) subunit-containing GABA(A) receptors. Effects of MeHg on I(GABA) were similar in cells at all days. In human embryonic kidney 293 cells expressing either alpha(6) or alpha(1) subunit-containing GABA(A) receptors, time to block of I(GABA) by MeHg was comparable. Thus, the presence of the alpha(6) subunit alone may not underlie the differential effects of MeHg on I(GABA) observed in cerebellar granule and cortical neurons; other factors are likely to be involved as well.     

Comparative effects of divalent cations on the methylmercury-induced alterations of acetylcholine release

Bath application of methylmercury (MeHg) at the murine neuromuscular junction blocks synchronous evoked release of acetylcholine (ACh) and then increases spontaneous release of ACh effects observed electrophysiologically as cessation of EPPS, and increased MEPP frequency (MEPPf), respectively. The objectives of the present study were to test whether the effect of MeHg on spontaneous release was Ca++-specific by substituting Sr++ or Ba++ for Ca++, whether the time course of MeHg-induced block of synchronous evoked release was altered by varying Ca++ concentrations or substituting Sr++ and whether the processes involved in the decay of elevated MEPPf after repetitive stimulation (asynchronous evoked release) were altered by MeHg. MEPPf was recorded continuously from the rat hemidiaphragm using conventional methods during pretreatment with 2 mM Ca++, 2 mM Sr++ or 0.5 mM Ba++ and subsequently with the cation plus 100 microM MeHg. The time to peak MEPPf in MeHg was not different under any condition; however, peak MEPPf was lower in Sr++ solutions than in Ca++ or Ba++ solutions. EPPs were recorded from the rat hemidiaphragm cut muscle preparation during pretreatment with either 2, 4 or 8 mM Ca++ or 2 or 4 mM Sr++ and subsequently with the cation plus 100 microM MeHg. The latency to block of the EPP in 4 and 8 mM Ca++ was not significantly different from the latency in 2 mM Ca++. The latency to block in 2 or 4 mM Sr++ was also not different from that in Ca++. In addition, under all conditions EPP amplitude remained virtually unchanged from pretreatment values until block occurred after 8 to 9 min exposure to MeHg.(ABSTRACT TRUNCATED AT 250 WORDS)     

Heterogeneity of nicotine actions in the rat cerebellum: an in vivo electrophysiologic study

Much controversy surrounds the actions of nicotine on mammalian central neurons, especially with respect to the question of presence of multiple nicotine receptors and with respect to similarities of responses to those induced by acetylcholine (ACh). To resolve some of these complexities, the authors determined the effects of pressure-ejected nicotine on identified neurons in the cerebellar cortex of Sprague-Dawley rats under urethane anesthesia. Purkinje cells and interneurons were identified by their anatomical localization, discharge characteristics and responses to electrical stimulation of superficial parallel fibers. Locally applied nicotine altered single-unit activity in a manner strictly dependent on cell type. Pressure-ejected nicotine inhibited Purkinje cells (50/51) and excited cerebellar interneurons (22/22). The effects of nicotine on cell discharge rate were probably receptor mediated because "classical" nondepolarizing nicotinic antagonists selectively blocked the agonistic actions of nicotine. A curare-sensitive site (neuromuscular type) was found to mediate the excitatory effects of nicotine, and a hexamethonium-sensitive site (ganglionic type) was found to mediate the inhibitory effects of nicotine. ACh mimicked the effects of nicotine on both cell classes although muscarinic interactions were also observed. The inhibitory effects of ACh on Purkinje cells were antagonized by the ganglionic blocker hexamethonium only if muscarinic receptors were simultaneously blocked via systemic administration of scopolamine. The excitatory effects of ACh on interneurons, on the other hand, were antagonized by the neuromuscular blocker curare even in the absence of a muscarinic antagonist. No interactions of curare and hexamethonium were observed with the amino acid transmitters gamma-aminobutyric acid (Purkinje cells) and glutamate (interneurons).     

Block of 45Ca uptake into synaptosomes by methylmercury: Ca++- and Na+-dependence

Block of Ca++ influx into isolated nerve terminals by the neurotoxicant methylmercury (MeHg) was studied for its dependence on extracellular Ca++ and Na+. Depolarization-independent entry of 45Ca++ was determined in rat forebrain synaptosomes incubated in 5 mM K+ solution. 45Ca++ uptake was similarly measured after 1 ("fast" phase) or 10 sec ("total") of elevated K+ (41.25 mM)-induced depolarization or after 10 sec of elevated K+-induced depolarization after synaptosomes had been predepolarized for 10 sec in Ca++- and MeHg-free solutions ("slow" phase). In 5 mM K+ solutions, MeHg concentrations of 125 microM and greater significantly reduced synaptosomal 45Ca++ uptake measured during 1 or 10 sec of incubation. In K+-depolarized synaptosomes, the estimated IC50 for block of total, fast and slow 45Ca++ uptake by MeHg is 75 microM; 250 microM MeHg reduced uptake by approximately 90%. The reversibility of block by extracellular Ca++ was tested by increasing the extracellular Ca++ concentration from 0.01 to 1.15 mM. When compared to control, 50 microM MeHg reduced total uptake of 45Ca++ by greater than or equal to 70% and reduced fast uptake by 20 to 60% at all concentrations of extracellular Ca++ tested. At Ca++ concentrations of 0.01 to 0.15 mM, MeHg (50 microM) reduced slow uptake by 75 to 90%, but did not affect slow uptake at higher Ca++ concentrations (greater than or equal to 0.30 mM). When the dependence of block of 45Ca++ uptake on extracellular Na+ was tested, equivalent levels of inhibition were caused by MeHg (25 microM) for fast uptake by synaptosomes in Na+-containing and Na+-free solutions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of local anesthetic QX-314 on the membrane properties of hippocampal pyramidal neurons

The quaternary lidocaine derivative QX-314 was applied internally to CA1 pyramidal neurons of the guinea-pig hippocampal slice preparation. This local anesthetic blocked both fast, Na+-dependent action potentials and the voltage-dependent, non-inactivating Na+ conductance. Partially blocked Na+ spikes exhibited pronounced frequency-dependent depression at rates as low as 0.2 Hz. Ca++-dependent electrogenesis, synaptic potentials and glutamate-induced depolarizations were apparently unaffected even after large doses of QX-314. The results indicate that the cellular mechanisms of local anesthetics on central neuronal membranes are similar to those described for peripheral axons. The frequency-dependence of spike blockade may account for some of the effects of local anesthetics on the central nervous system in vivo. Additionally, the localized intracellular application of QX-314 may prove useful as a specific pharmacological tool in studies of central neurons.     

Effects of lidocaine and on slow response and depressed fast response action potentials of canine cardiac Purkinje fibers

Disease may decrease resting potential of cardiac fibers, thereby depressing the upstroke velocity of the action potential, causing slow conduction and reentry. A decrease in resting potential may also cause automaticity. We studied the effects of lidocaine (5 and 20 mg/l) on canine Purkinje fibers with reduced membrane potentials with either depressed Na+-dependent upstrokes (depressed fast responses) or with slow inward (Ca++) current-dependent upstrokes (slow responses). Depressed fast responses were produced by elevating [K+]0 in the perfusate, reducing membrane potential to around -60 mV, without abolishing excitability. Slow responses were produced by either perfusing fibers with a Na+-free, Ca++-rich solution, or by perfusing them with a high [K+]0 Tyrode's solution containing norepinephrine. Lidocaine had a marked depressant effect on depressed fast response action potentials. The drug markedly decreased Vmax and conduction velocity. It sometimes decreased action potential amplitude and caused conduction block. Resting potential was not changed. On the other hand, lidocaine had little effect on slow response action potentials. Resting potential, Vmax and action potential amplitude were not altered nor was conduction changed. The rate of spontaneous impulse initiation was slightly reduced by 5 mg/l of lidocaine but not by 20 mg/l. We conclude that lidocaine does not exert its antiarrhythmic effect by directly depressing the slow inward current but may be antiarrhythmic because it depresses an already depressed fast inward current and can cause conduction block.     

Effect of calcium antagonist drugs on calcium currents in mammalian skeletal muscle fibers

The calcium channel-inhibiting drugs diltiazem, verapamil and nitrendipine represent three general classes of organic calcium antagonists. In the present study, the effect of these drugs on calcium currents (ICa++) in rabbit sternomastoid muscle fibers was examined. ICa++ were recorded at room temperature using a vaseline gap voltage clamp. ICa++ measured had similar kinetics to those reported in rat skeletal muscle, were partially blocked by 0.5 mM CdCI2 and could be reduced by substitution of Mg++ for Ca++. Diltiazem reversibly blocked ICa++ in a concentration-dependent manner with the 50% inhibitory concentration (IC50) being 63 microM. Verapamil was slightly more potent with approximately 50% block of ICa++ occurring at 10 microM. In contrast, nitrendipine at concentrations from 1 to 10 microM had no blocking action on ICa++, even after 20 min of exposure. Thus, although Ca++ channels in mammalian skeletal muscle fibers are readily blocked by cadmium, diltiazem and verapamil, these channels appear to be insensitive to the dihydropyridine compound nitrendipine.     

Dendritic glutamate-induced bursting in the prefrontal cortex: further characterization and effects of phencyclidine

To understand the role of N-methyl-d-aspartate (NMDA) receptors in the prefrontal cortex (PFC) and to investigate how the psychotomimetic drug phencyclidine (PCP) may alter PFC function, we made whole-cell recordings from PFC neurons in rat brain slices. Our result showed that most deep layer pyramidal neurons in the PFC were regular spiking cells. They could fire repetitive bursts, however, when activated by glutamate focally applied to the apical dendrite. Application of NMDA to the same dendritic spot also induced bursting, whereas application of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) evoked single spikes only. Coapplication of AMPA with NMDA evoked more single spikes and decreased NMDA-induced bursting. Experiments with NMDA and AMPA antagonists further showed that dendritic glutamate (dGlu)-induced bursting required NMDA receptor activation and was enhanced when AMPA receptors were blocked. At subanesthetic concentrations, PCP decreased dGlu-induced bursting and altered the temporal characteristics of the bursts by decreasing spikes per burst and increasing interspike intervals within bursts. The latter two changes were not observed when AMPA receptors were blocked, suggesting that they are secondary to the increased AMPA receptor contribution to glutamate responses evoked in the presence of PCP. These results suggest that NMDA receptors are essential for PFC pyramidal cells to fire in bursts in response to dGlu input and that PCP suppresses dGlu-induced bursting. Since bursting is necessary for pyramidal cells to activate GABA interneurons, the suppression effect of PCP may further lead to a weakening of the connections from pyramidal cells and GABA interneurons, thereby contributing to PCP's psychotomimetic effects.     

Irreversible suppression of calcium entry into nerve terminals by methylmercury

Measurements of uptake of 45Ca into rat forebrain synaptosomes depolarized with high K+ and EPP amplitudes at the rat neuromuscular junction were used to assess the effects of methylmercury (MeHg) on voltage-dependent Ca++ uptake and subsequent transmitter release at model central and peripheral synapses. The objectives were to: determine whether MeHg altered uptake of 45Ca into purified synaptosomes depolarized by high K+; compare its effects with those produced by HgCl2; ascertain whether the "fast" or "slow" components of Ca++ uptake were affected preferentially by MeHg; and determine whether a functional correlate to the effects on 45Ca uptake could be observed electrophysiologically at the mammalian neuromuscular junction. HgCl2 (10-500 microM) produced a concentration-dependent decrease of total depolarization-induced 45Ca uptake. Peak inhibition occurred at 200 microM Hg++ which suppressed nerve terminal Ca++ uptake to approximately 5% of Hg-free control values, a result similar to that obtained previously by others. Similarly, MeHg also suppressed total 45Ca uptake although the maximal inhibition produced (70% at 200 microM MeHg) was less than that produced by HgCl2. The effect of MeHg was apparent both in nonpreviously depolarized synaptosomes after a 1-sec depolarization ("fast uptake") and after 10-sec incubation in synaptosomes predepolarized with 41 mM K+ in Ca-free solutions before addition of MeHg and 45Ca ("slow uptake"). A significant decrease in the slow phase of 45Ca uptake occurred with 200 and 500 microM MeHg.(ABSTRACT TRUNCATED AT 250 WORDS)     

Analysis of the effects of cannabinoids on synaptic transmission between basket and Purkinje cells in the cerebellar cortex of the rat

The hypothesis of the present work was that activation of CB1 cannabinoid receptors inhibits GABAergic neurotransmission between basket and Purkinje cells in the cerebellar cortex. The aim was to test this hypothesis under near-physiological conditions. Action potentials of basket cells and spontaneous inhibitory postsynaptic currents (sIPSCs) in synaptically coupled Purkinje cells were recorded simultaneously in rat brain slices. The cannabinoid agonists (R)-(+)-[2,3-dihydro-5-methyl-3-[(morpholinyl) methyl] pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl)methanone mesylate (WIN 55212-2) and (-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)-phenyl]-trans-4-(3-hydroxy-propyl)-cyclohexanol (CP55940) decreased the amplitude of sIPSCs occurring simultaneously with basket cell action potentials and lowered the success rate of synaptic transmission. These effects were prevented by the CB1 receptor antagonist N-piperidino-5-(4-chlorophenyl)-1-(2,4-dichloro-phenyl)-4-methyl-3-pyrazole-carboxamide (SR141716). Depolarization of Purkinje cells also led to suppression of neurotransmission; prevention of this suppression by CP55940 and SR141716 indicates that endocannabinoids released from Purkinje cells were involved. WIN 55212-2 lowered the amplitude of autoreceptor currents recorded in basket cells (autoreceptor currents are due to the action of GABA released from axon terminals on GABAA autoreceptors of the same axon terminals); this is novel proof of the presynaptic action of cannabinoids. Autoreceptor current experiments also indicated that endogenous cannabinoids are not released by basket cell axon terminals. A presynaptic action is additionally supported by the observation that WIN 55212-2 lowered the frequency of miniature IPSCs recorded in the presence of tetrodotoxin and the calcium ionophore ionomycin. In conclusion, activation of CB1 receptors by exogenous cannabinoids and by endogenous cannabinoids released by Purkinje cells presynaptically inhibits GABAergic neurotransmission between basket and Purkinje cells. This was demonstrated under near-physiological conditions: transmitter release was elicited by action potentials generated by spontaneously firing intact presynaptic neurons.     

Effects of activation of sodium and calcium entry on spontaneous release of acetylcholine induced by methylmercury

The effect of ionophores and channel activators for Ca and Na on the time course and magnitude of methylmercury (MeHg)-induced increase in spontaneous release of neurotransmitter was studied at the murine neuromuscular junction using intracellular microelectrode recording techniques. The goal was to test whether chemicals that increase entry of Na+ or Ca++ into nerve terminals would shorten the latent period that precedes the onset of MeHg-induced increase in MEPP frequency. Administration of MeHg (100 microM) with A23187 (25 microM), a calcium ionophore, caused a more rapid time to peak induced increase in MEPP frequency than "control" MeHg preparations. This effect also occurred in solutions to which no extracellular Ca++ was added. Use of monensin, a Na+ ionophore (25-100 microM), did not shorten the time to peak increase of MEPP frequency. The dihydropyridine Ca++ channel agonist Bay K 8644 (750 nM) produced the most marked shortening of the time to peak MEPP frequency for MeHg. This effect also occurred in solutions deficient in extracellular Ca++. Veratridine (20 microM), a sodium channel activator, decreased the time to peak MEPP frequency when used in conjunction with MeHg in both Ca++-containing and Ca++-deficient solutions. Replacement of sodium in the extracellular perfusion solution with methylamine, which does not penetrate axon sodium channels, did not prevent the MeHg-induced increase in MEPP frequency although it did prolong the time to peak increase and decreased the maximal MEPP frequency induced by MeHg compared with experiments conducted in sodium-containing solutions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrophysiologic effects of amiloride in canine Purkinje fibers: evidence for a delayed effect on repolarization

Amiloride, a potassium-sparing diuretic, inhibits Na+ transport, Na+-H+ exchange and possibly Na+-Ca++ exchange in a variety of cellular and epithelial tissues. Similar membrane ion transport mechanisms exist in cardiac tissue, yet there are little data on possible interference by amiloride with ion transport in the heart. Given recent evidence for a delay in amiloride uptake into erythroid cells, we studied the electrophysiologic effects of amiloride after prolonged drug exposure in canine Purkinje fibers using standard microelectrode techniques. Amiloride (1-10 microM) led to a progressive lengthening of action potential duration with a tau of 1.8 +/- 0.5 hr (n = 15). At long cycle lengths (greater than or equal to 2000 msec) early afterdepolarizations and oscillations around the plateau were seen. To determine the etiology of the afterdepolarizations, Purkinje fibers treated for 2 hr with 10 microM amiloride were then exposed to tetrodotoxin, manganese and nisoldipine. Tetrodotoxin (7.8 X 10(-7) M) reversed completely all amiloride effects rapidly and reversibly. MnCl2 (4 mM) increased the afterdepolarizations, and arrest occurred at the plateau potential routinely. Nisoldipine (10(-6) M), a more selective blocker of slow inward current, shortened action potential duration somewhat but did not reverse fully the effects of amiloride. We conclude that amiloride has a pronounced effect on repolarization in the canine Purkinje fiber and this effect is manifest only after prolonged exposure to the drug.     

Interactions of MK-801 with glutamate-, glutamine- and methamphetamine-evoked release of [3H]dopamine from striatal slices.

The interactions of MK-801 [(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d] cyclohepten-5,10-imine], glutamate and glutamine with methamphetamine (METH)-evoked release of [3H]dopamine were assessed in vitro to determine whether MK-801 inhibition of METH neurotoxicity might be mediated presynaptically, and to evaluate the effects of glutamatergic stimulation on METH-evoked dopamine release. MK-801 inhibition of glutamate- or METH-evoked dopamine release might reduce synaptic dopamine levels during METH exposure and decrease the formation of 6-hydroxydopamine or other related neurotoxins. Without Mg++ present, 40 microM and 1 mM glutamate evoked a N-methyl-D-aspartate receptor-mediated [3H]dopamine and [3H]metabolite (tritium) release of 3 to 6 and 12 to 16% of total tritium stores, respectively, from striatal slices. With 1.50 mM Mg++ present, 10 mM glutamate alone or in combination with the dopamine uptake blocker nomifensine released only 2.1 or 4.2%, respectively, of total tritium stores, and release was only partially dependent on N-methyl-D-aspartate-type glutamate receptors. With or without 1.50 mM Mg++ present, 0.5 or 5 microM METH evoked a substantial release of tritium (5-8 or 12-21% of total stores, respectively). METH-evoked dopamine release was not affected by 5 microM MK-801 but METH-evoked release was additive with glutamate-evoked release. Without Mg++ present, 1 mM glutamine increased glutamate release and induced the release of [3H]dopamine and metabolites. Both 0.5 and 5 microM METH also increased tritium release with 1 mM glutamine present. When striatal slices were exposed to 5 microM METH this glutamine-evoked release of glutamate was increased more than 50%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Noncontractile acetylcholine receptor-operated Ca++ mobilization: suppression of activation by open channel blockers and acceleration of desensitization by closed channel blockers in mouse diaphragm muscle

The effects of various blockers of the nicotinic acetylcholine receptor-activated ionic channel on noncontractile slow Ca++ mobilization were investigated at the neuromuscular synapse of aequorin-injected diaphragm muscles of mice. Intracellular Ca++ mobilization (Ca++ transients) was evoked in the presence of neostigmine (0.3 microM) by nerve stimulation. Bupivacaine, an open channel blocker, decreased the peak amplitude, whereas chlorpromazine, a closed channel blocker, shortened the duration. Phencyclidine, an open and closed channel blocker, decreased both peak amplitude and duration. beta-Eudesmol, a compound of Atractylodes lancea, clearly and specifically shortened the duration but had little effect on peak amplitude. All the above channel blockers, when given in the same concentration ranges, also blocked the total amount of contractile Ca++ transients. The effects of bupivacaine, chlorpromazine and phencyclidine on noncontractile Ca++ transients were not affected by 5 mM [Ca++]o, whereas the effect of beta-eudesmol was enhanced. Geographutoxin II (0.3 microM), a skeletal muscle Na+ channel blocker, selectively and partly reversibly blocked contractile Ca++ transients without affecting noncontractile ones. These results suggest that: 1) the activation of noncontractile Ca++ mobilization is suppressed by open channel blockers, whereas its desensitization is accelerated by closed channel blockers and 2) activation of the muscle Na+ channel and subsequent release of Ca++ from sarcoplasmic reticulum is not involved in the mechanism of noncontractile Ca++ mobilization. It may reflect the steps of the desensitization process.     

Synaptic facilitation by 3-aminopyridine and its antagonism by verapamil and diltiazem

The effects of 3-aminopyridine (3-AP) on synaptic transmission in bullfrog sympathetic ganglion were studied in normal Ringer's solution and during graded reductions in extracellular Ca++ by means of intra- and extracellular recording techniques. 3-AP caused a single orthodromic stimulus to generate a brief burst of repetitive postganglionic discharges (SBR). In the absence of 3-AP, synaptic transmission, measured as the amplitude of the postganglionic compound action potential, failed progressively as Ca++ was reduced from 1.8 to 0.47 mM. This Ca++ dependence curve of synaptic transmission was shifted to the left (lower Ca++) by 3-AP in dose-related fashion, with maximum shift (4- to 5-fold) at 1 mM 3-AP. The magnitude of the maximum shift produced by 3-AP was precisely the same as that produced by 3,4-diaminopyridine and 4-aminopyridine. Although 3-AP could prevent transmission failure at otherwise suboptimal Ca++ levels, its ability to generate SBR failed progressively as Ca++ was reduced from normal (1.8 mM) to 0.5 mM. Thus, there was a wide difference between the Ca++ dependence domains of synaptic transmission and of SBR in the presence of 3-AP. To confirm this difference in Ca++ dependence domains by a method other than reduction of [Ca++]0, we investigated the interactions between 3-AP and two Ca++ entry blockers, verapamil and diltiazem. 3-AP SBR was abolished by verapamil and by diltiazem at concentrations significantly below those required to block synaptic transmission in the presence of 3-AP. The results thus demonstrate a competitive interaction between aminopyridines and Ca++ entry blockers and further confirm the Ca++ dependent nature of the synaptic actions of aminopyridines.     

Low concentrations of pyridostigmine prevent soman-induced inhibition of GABAergic transmission in the central nervous system: involvement of muscarinic receptors

This study was designed to investigate the effects of the cholinesterase inhibitors soman and pyridostigmine bromide (PB) on synaptic transmission in the CA1 field of rat hippocampal slices. Soman (1-100 nM, 10-15 min) decreased the amplitude of GABAergic postsynaptic currents (IPSCs) evoked by stimulation of Schaffer collaterals and recorded from CA1 pyramidal neurons. It also decreased the amplitude and frequency of spontaneous IPSCs recorded from pyramidal neurons. Whereas the maximal effect of soman on evoked GABAergic transmission was observed at 10 nM, full cholinesterase inhibition was induced by 1 nM soman. After 10-15-min exposure of hippocampal slices to 100 nM PB, GABAergic transmission was facilitated and cholinesterase activity was not significantly affected. At nanomolar concentrations, soman and PB have no direct effect on GABA(A) receptors. The effects of soman and PB on GABAergic transmission were inhibited by the m2 receptor antagonist 11-[[[2-diethylamino-O-methyl]-1-piperidinyl] acetyl]-5,11-dihydrol-6H-pyridol[2,3-b][1,4]benzodiazepine-6- one (1 nM) and the m3 receptor antagonist 4-diphenylacetoxy-N-methyl-piperidine (100 nM), respectively, and by the nonselective muscarinic receptor antagonist atropine (1 microM). Thus, changes in GABAergic transmission are likely to result from direct interactions of soman and PB with m2 and m3 receptors, respectively, located on GABAergic fibers/neurons synapsing onto the neurons under study. Although the effects of 1 nM soman and 100 nM PB were diametrically opposed, they only canceled one another when PB was applied to the neurons before soman. Therefore, PB, acting via m3 receptors, can effectively counteract effects arising from the interactions of soman with m2 receptors in the brain.