Patterns of excitatory and inhibitory synaptic transmission in the rat neostriatum as revealed by 4-AP

1. Synaptic potentials induced by 4-aminopyridine (4-AP) were recorded intracellularly from rat neostriatal neurons in an in vitro slice preparation. EC50 for this 4-AP action was approximately 120 microM. The threshold for activation of synaptic potentials was 5 microM. 2. 4-AP-induced synaptic potentials appeared stochastically. Most were blocked by 1 microM tetrodotoxin or 400 microM Cd2+. Therefore they reflect a release of neurotransmitters dependent on both Ca2+ entry to the terminals and action potential firing. 3. Bicuculline (BIC) (< or = 10 microM), a gamma-aminobuturic acid-A (GABAA) antagonist, blocked about half of the 4-AP-induced synaptic potentials. This suggests that intrinsic inhibitory connections within the neostriatum are activated by 4-AP administration. 4. 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; < or = 10 microM) plus D-2-amino-5-phosphonovaleric acid (D-APV; < or = 100 microM) blocked most of the BIC-resistant 4-AP-induced synaptic potentials. This suggests that 4-AP induced release of glutamate (GLU) from extrinsic glutamatergic afferents. As most glutamatergic afferents are extrinsic, these afferents then would be able to fire spikes and release transmitter for several hours after they are cut from their somata. 5. If CNQX plus D-APV were administered before BIC, neostriatal neurons responded in different ways. In one half of the neurons, all induced synaptic potentials were blocked. This suggests that most GABAergic intrinsic connections between neostriatal neurons are activated indirectly by 4-AP. 4-AP would first activate extrinsic glutamatergic afferents and these in turn would activate GABAergic intrinsic neurons and connections. 6. In the remaining half of the recorded neurons, administration of CNQX plus D-APV blocked most, but not all of the 4-AP-induced synaptic potentials. The synaptic potentials that remained had a characteristic pattern: they were high amplitude, rhythmic, bursts of synaptic potentials. They were blocked by BIC (5 microM) but not by mecamylamine (> 10 microM). This suggests that these bursts of synaptic potentials were GABAergic and generated by intrinsic neurons. Therefore these neurons would not innervate all neostriatal neurons equally but just a subset of them. 7. Records from an identified aspiny neostriatal interneuron, obtained from the same preparation, are shown. This interneuron fired in bursts and its morphologically and physiologically similar to the recently described, fast spiking, parvalbumin immunoreactive, GABAergic, aspiny interneuron is functional in the slice preparation.(ABSTRACT TRUNCATED AT 400 WORDS)     

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The actions of peptidic toxins that work as Ca2+-channel antagonists were investigated on neostriatal glutamatergic transmission. Both intracellularly recorded excitatory postsynaptic potentials (EPSPs) and extracellularly recorded population spikes (PS) evoked by afferent stimulation were evaluated in the presence of 10 microM bicuculline. Percentage of block (mean +/- SEM; n = 4) for these events (EPSP and PS, respectively) was: omega-AgTxIVA (100-200 nM): 35 +/- 2 and 54 +/- 4%; omega-CgTxGVIA (1 microM): 37 +/- 3 and 63 +/- 6%; omega-CgTxMVIIC (500 nM): 40 +/- 4 and 50 +/- 2%; and calciseptine (500 nM): 5 +/- 4 and 9 +/- 6%. When given together, toxins had additive effects. The calciseptine effects were nonsignificant. The toxins were also tested on Ca2+-dependent random synaptic responses induced by 100 microM 4-AP. Each toxin reduced the frequency of spontaneous EPSPs by more than 60% (n = 2). The summed actions of individual toxins yields more than 100% block (superadditivity); suggesting that several terminals may possess more than one channel type. The reduction in frequency was not accompanied by a reduction in amplitude confirming that toxins' actions were presynaptic. It is concluded that at least three different Ca2+-channel subtypes are involved in glutamate release in neostriatal afferents: N-type, P/Q-type, and a type resistant to the toxins used. The L-type Ca2+-channel had little, if any, participation.     

3-Alpha-chloro-imperialine, a potent blocker of cholinergic presynaptic modulation of glutamatergic afferents in the rat neostriatum

Cortico-thalamic glutamatergic afferents control neuronal activity in the neostriatum. Cholinergic interneurons modulate the activity of medium spiny neurons through both pre- and post-synaptic actions via the activation of muscarinic receptors. The muscarinic pre-synaptic modulation was analyzed electrophysiologically. The transmitter release, induced by 4-AP, was studied and the block of paired pulse facilitation (PPF) by different muscarinic receptor antagonists was analyzed. The GABA(A) antagonist bicuculline isolated the glutamatergic transmission. Muscarinic agonists decreased the frequency of random synaptic potentials induced by 4-AP in about 60% of the cases without changes in input resistance (RN) of the post-synaptic neuron or in the mean amplitude of the synaptic events; indicating a presynaptic action. The administration of both 1 microM carbachol or 20 nM muscarine increased PPF. Muscarinic receptor antagonists blocked this action with a potency order: 3-alpha-chloroimperialine > 4-DAMP>AFDX-116 > or = gallamine > pirenzepine. The IC50's for the first three antagonists were (nM): 0.65, 1.1, and 3.0. Their respective Hill coefficients were: 1.9, 1.4, and 1.3. 3-alpha-Chloroimperialine reduced the PPF almost completely. The M3 and the M2 muscarinic receptor antagonists 4-DAMP and AFDX-116, given at saturating concentrations, consistently blocked only a part of the PPF but had additive effects when given together. These data are consistent with the existence of both M2 and M3 muscarinic receptors in striatal glutamatergic afferents.     

Modulation by substance P of synaptic transmission in the mouse hippocampal slice

The modulatory action of substance P on synaptic transmission of CA1 neurons was studied using intra- or extracellular recording from the mouse hippocampal slice preparation. Bath-applied substance P (2-4 microM) or the selective NK1 receptor agonist substance P methylester (SPME, 10 nM-5 microM) depressed field potentials (recorded from stratum pyramidale) evoked by focal stimulation of Schaffer collaterals. This effect was apparently mediated via NK1 receptors since it was completely blocked by the selective NK1 antagonist SR 140333. The field potential depression by SPME was significantly reduced in the presence of bicuculline. Intracellular recording from CA1 pyramidal neurons showed that evoked excitatory postsynaptic potentials (EPSPs) and evoked inhibitory postsynaptic potentials (IPSPs) were similarly depressed by SPME, which at the same time increased the frequency of spontaneous GABAergic events and reduced that of spontaneous glutamatergic events. The effects of SPME on spontaneous and evoked IPSPs were prevented by the ionotropic glutamate receptor blocker kynurenic acid. In tetrodotoxin (TTX) solution, no change in either the frequency of spontaneous GABAergic and glutamatergic events or in the amplitude of responses of pyramidal neurons to 4 microM alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) or 10 microM N-methyl-D-aspartate (NMDA) was observed. On the same cells, SPME produced minimal changes in passive membrane properties unable to account for the main effects on synaptic transmission. The present data indicate that SPME exerted its action on CA1 pyramidal neurons via a complex network mechanism, which is hypothesized to involve facilitation of a subset of GABAergic neurons with widely distributed connections to excitatory and inhibitory cells in the CA1 area.     

Participation of GABAA-mediated inhibition in ictallike discharges in the rat entorhinal cortex

The spontaneous, synchronous activity induced by 4-aminopyridine (4AP, 50 microM) in the adult rat entorhinal cortex was analyzed with simultaneous field potential and intracellular recordings in an in vitro slice preparation. Four-AP induced isolated negative-going field potentials (interval of occurrence = 27.6 +/- 9.9 (SD) s; n = 27 slices) that corresponded to intracellular long-lasting depolarizations (LLDs), and ictallike epileptiform discharges (interval of occurrence = 10.4 +/- 5.7 min; n = 27 slices) that were initiated by the negative field potentials. LLDs recorded with K-acetate-filled microelectrodes triggered few action potentials of variable amplitude and had a duration of 1.7 +/- 0.8 s (n = 26 neurons), a peak amplitude of 11.8 +/- 5.0 mV (n = 26 neurons) and a reversal potential of -66.2 +/- 3.9 mV (n = 17 neurons). The ictal discharges studied with K-acetate microelectrodes consisted of prolonged depolarizations (duration = 72.9 +/- 44.3 s; peak amplitude = 29.2 +/- 11.4 mV; n = 25 neurons) with action-potential firing during both the tonic and the clonic phase. These depolarizations had a reversal potential of -45.3 +/- 3.8 mV (n = 4 neurons). Intracellular Cl- diffusion from KCl-filled microelectrodes made both LLDs and ictal depolarizations increase in amplitude (30.5 +/- 8.2 mV, n = 8 and 41.8 +/- 9.8 mV, n = 6 neurons, respectively). LLDs recorded with KCl and 2-(trimethyl-amino)N-(2, 6-dimethylphenyl)-acetamide (QX-314) microelectrodesreached an amplitude of 36.3 +/- 5.2 mV, lasted 12.5 +/- 6.5 s, and had a reversal potential of -31.3 +/- 2.5 mV (n = 4 neurons); under these recording procedures the ictal discharge amplitude was 41.5 +/- 5.0 mV and the reversal potential -24.0 +/- 7.0 mV (n = 4 neurons). The N-methyl-D-aspartate (NMDA) receptor antagonist 3,3-(2-carboxy-piperazine-4-yl)-pro-pyl-l-phosphonate (10 microM, n = 5 neurons) alone or concomitant with the nonNMDA receptor antagonist 6-cyano-7-nitro-quinoxaline-2,3-dione (10 microM, n = 4 neurons) abolished ictal discharges, without influencing LLDs. LLDs were blocked by the gamma-aminobutyric acid-A (GABAA) receptor antagonist bicuculline methiodide (BMI, 10 microM, n = 6 neurons) or the mu-opioid receptor agonist (-Ala2-N-Me-Phe, Gly-ol) enkephalin (DAGO, 10 microM, n = 2 neurons). Application of BMI (n = 4 neurons) or DAGO (n = 2 neurons) to control the medium abolished LLDs and ictal discharges but disclosed a novel type of epileptiform depolarization that lasted 3.5 +/- 1.2 s and occurred every 5.2 +/- 2.6 s (n = 6 neurons). Our data indicate that 4AP induces in the rat entorhinal cortex a synchronous, GABA-mediated potential that is instrumental in initiating NMDA-dependent, ictal discharges. Moreover we present evidence for an active role played by GABAA-mediated potentials in the maintenance and termination of these prolonged epileptiform events.     

NMDA receptor-mediated control of presynaptic calcium and neurotransmitter release

Before action potential-evoked Ca2+ transients, basal presynaptic Ca2+ concentration may profoundly affect the amplitude of subsequent neurotransmitter release. Reticulospinal axons of the lamprey spinal cord receive glutamatergic synaptic input. We have investigated the effect of this input on presynaptic Ca2+ concentrations and evoked release of neurotransmitter. Paired recordings were made between reticulospinal axons and the neurons that make axo-axonic synapses onto those axons. Both excitatory and inhibitory paired-cell responses were recorded in the axons. Excitatory synaptic inputs were blocked by the AMPA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 microM) and by the NMDA receptor antagonist 2-amino-5-phosphonopentanoate (AP-5; 50 microM). Application of NMDA evoked an increase in presynaptic Ca2+ in reticulospinal axons. Extracellular stimulation evoked Ca2+ transients in axons when applied either directly over the axon or lateral to the axons. Transients evoked by the two types of stimulation differed in magnitude and sensitivity to AP-5. Simultaneous microelectrode recordings from the axons during Ca2+ imaging revealed that stimulation of synaptic inputs directed to the axons evoked Ca2+ entry. By the use of paired-cell recordings between reticulospinal axons and their postsynaptic targets, NMDA receptor activation was shown to enhance evoked release of transmitter from the axons that received axoaxonic inputs. When the synaptic input to the axon was stimulated before eliciting an action potential in the axon, transmitter release from the axon was enhanced. We conclude that NMDA receptor-mediated input to reticulospinal axons increases basal Ca2+ within the axons and that this Ca2+ is sufficient to enhance release from the axons.     

Isolation and characterization of a persistent potassium current in neostriatal neurons

1. Depolarization-activated, calcium-independent potassium (K+) currents were studied with the use of whole cell voltage-clamp recording from neostriatal neurons acutely isolated from adult (> or = 4 wk old) rats. The whole cell K+ current was composed of transient and persistent components. The aims of the experiments were to isolate the persistent component and then to characterize its voltage dependence and kinetics. 2. Application of 10 mM 4-aminopyridine (4-AP) completely blocked the transient currents while reducing the persistent current by approximately 40% [50% inhibitory concentration (IC50), of blockable current = 125 microM]. The persistent K+ current also was reduced by tetraethylammonium (TEA). Two components to the TEA block were present, having IC50s of 125 microM (23% of the blockable current) and 5.9 mM (77% of the blockable current). Collectively, these results suggested that the persistent components of the total K+ current was pharmacologically heterogeneous. The properties of the 4-AP-resistant, persistent K+ current (IKrp) were subsequently studied. 3. The kinetics of activation and deactivation of IKrp were voltage dependent. Examination of the entire activation/deactivation time constant profile showed that it was bell shaped, with time constants being moderately rapid (tau approximately 50 ms) at membrane potentials corresponding to the resting potential of neostriatal cells (approximately -80 mV), becoming considerably longer (tau approximately 100 ms) at potentials near the cells' spike thresholds (approximately -45 mV), and decreasing to a minimum (tau approximately 5 ms) at potentials associated with the peak of the cells' action potentials (approximately +20 mV). The inactivation kinetics of IKrp also were voltage dependent. The time constants of inactivation varied between 1 and 8 s at potentials between -10 and +35 mV. 4. Unlike persistent K+ currents in many other cell types, IKrp activated at relatively hyperpolarized membrane potentials (approximately -70 mV). The Boltzmann function describing activation had a half-activation voltage of -13 mV and a slope factor of 12 mV. In addition, the Boltzmann function describing the voltage dependence of inactivation of IKrp had a relatively depolarized half-inactivation voltage of -55 and a large slope factor of 19 mV, indicating that this current was available over a broad range of membrane potentials (between -100 and -10 mV). 5. Neostriatal neurons recorded in vivo exhibit subthreshold shifts in membrane potential of variable duration (tens of ms to s) from a hyperpolarized resting state to a depolarized state that is limited in amplitude just below spike threshold. The voltage dependence of activation and inactivation of IKrp indicates that it will be available on depolarization from the hyperpolarized state. However, the slow activation rate of this current suggests that it will contribute little either to limiting the amplitude of the initial depolarization associated with entry into the depolarized state or to depolarizing episodes of short duration (e.g., < 50 ms). However, IKrp should limit the amplitude of membrane depolarizations associated with prolonged excursions into the depolarized state.     

Dopamine modulates inwardly rectifying hyperpolarization-activated current (Ih) in cultured rat olfactory receptor neurons

The presence of dopamine receptors in olfactory receptor neurons (ORNs) suggests that odor sensitivity may be modulated by neurotransmitters at the level of primary sensory neurons. Using standard patch-clamp techniques on rat ORNs, we found that 1 microM dopamine, 500 microM SQ 22536 (SQ, an adenylyl cyclase inhibitor), 20 and 50 microM quinpirole (a selective dopamine D2 receptor agonist), and 1 mM adenosine 3', 5'-cyclic monophosphate (cAMP) modulate the hyperpolarization-activated current Ih. On hyperpolarizing from a holding potential of -58 mV, a small Cs+-sensitive inwardly rectifying current (Ih) was observed. Increases in extracellular K+ increased Ih amplitude without shifting its voltage dependence of activation, whereas increases in temperature produced an increase in Ih amplitude and a hyperpolarizing shift in the activation curve. Application of 1 microM dopamine reversibly shifted Ih activation to more negative potentials and decreased Ih current amplitudes. These effects were blocked by concomitant application of dopamine with sulpiride, a selective dopamine D2 receptor antagonist. The effects of dopamine were mimicked by quinpirole. Quinpirole (20 microM) decreased Ih current amplitude, but was without effect on Ih voltage dependence of activation. However, 50 microM quinpirole produced both a reduction of Ih peak currents and a hyperpolarizing shift in the activation curve for Ih. External application of the adenylyl cyclase inhibitor SQ 22536 produced a reversible decrease in peak currents but had no effect on Ih voltage dependence of activation, whereas internal application of cAMP shifted Ih activation to more depolarized potentials. Because Ih modulates cell excitability and spike frequency adaptation, our findings support a role for dopamine in modulating the sensitivity and output of rat ORNs to odorants.     

Heterogeneity in use-dependent depression of inhibitory postsynaptic potentials in the rat neostriatum in vitro

"Minimal stimulation" was applied to evoke responses in an "all-or-none" fashion in presumed medium spiny neurons of rat neostriatal slices in the presence of antagonists for glutamatergic excitation. For comparison, responses were evoked in the same cells by compound stimulation. Bicuculline (30 microM) blocked responses evoked by minimal stimulation, indicating that they were gamma-aminobutyric acid-A (GABAA)-receptor-mediated inhibitory postsynaptic potentials (IPSPS), whereas responses evoked by compound stimulation were only reduced in amplitude. Likewise, R(-)baclofen (1-20 microM) blocked IPSPS evoked by minimal stimulation in all but one cell. On the contrary, responses evoked by compound stimulation were always reduced in amplitude but never blocked. Paired-pulse depression (PPD) of averaged responses to minimal and compound stimulation was observed at a stimulus interval of 300 ms. The GABAB receptor antagonist CGP55845A (0.5 microM) had no effect on PPD evoked by compound stimulation but abolished PPD evoked by minimal stimulation. In a second set of experiments, the two stimulation paradigms were used to evoke responses in neostriatal slices continuously bathed in R(-)baclofen (10-20 microM). In R(-)baclofen a strong PPD was evoked by minimal and by compound stimulation. The amplitude of the response to compound stimulation increased on application of CGP55845A (0.5 microM). At the same time, PPD evoked by compound stimulation decreased. On the contrary, IPSP amplitude and PPD evoked by minimal stimulation remained unchanged. We conclude that two types of GABAergic terminals exist in the rat neostriatum, only one of which is regulated by GABAB receptors. However, the other class of terminals, not regulated by GABAB receptors, displays a much more pronounced PPD.     

A purinergic component of the excitatory postsynaptic current mediated by P2X receptors in the CA1 neurons of the rat hippocampus

The pyramidal neurons in the CA1 area of hippocampal slices from 17- to 19-day-old rats have been investigated by means of patch clamp. Excitatory postsynaptic currents (EPSCs) were elicited by stimulating the Schaffer collateral at a frequency below 0.2 Hz. It was found that inhibition of glutamatergic transmission by 20 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 100 microM 2-amino-5-phosphonovaleric acid (D-APV) left a small component of the EPSC uninhibited. The amplitude of this residual EPSC (rEPSC) comprised 25 +/- 11% of the total EPSC when measured at a holding potential of -50 mV. The rEPSC was blocked by selective P2 blocker pyridoxal phosphate-6-azophenyl-2'-4'-disulphonic acid (PPADS) 10 microM and bath incubation with non-hydrolysable ATP analogues, ATP-gamma-S and alpha, beta-methylene-ATP at 50 and 20 microM, respectively. The rEPSC was dramatically potentiated by external Zn2+ (10 microM). In another series of experiments exogenous ATP was applied to the CA1 neurons in situ. An inward current evoked by ATP was inhibited by PPADS to the same extent as the rEPSC. It is concluded that, depending on membrane voltage, about one-fifth to one-quarter of the EPSC generated by the excitatory synaptic input to the hippocampal CA1 neurons of rat is due to the activity of P2X receptors.     

Central vestibular networks in the guinea-pig: functional characterization in the isolated whole brain in vitro

The isolated, in vitro whole brain of guinea-pig was used to assess some of the main physiological and pharmacological properties of the vestibulo-ocular pathways in this species. Extracellular and intracellular recordings were obtained from the vestibular, abducens and oculomotor nuclei, as well as from the abducens and oculomotor nerves, while inputs from the vestibular afferents, the visual pathways and the spinal cord were activated. The three main types of medial vestibular nucleus neurons (A, B and B+LTS), previously described on slices, were also identified in the isolated brain. They had similar membrane properties in both preparations. Eighty-five per cent of cells recorded in the vestibular nucleus responded with monosynaptic, excitatory postsynaptic potentials (latency 1.05-1.9 ms) to stimulation of the ipsilateral vestibular nerve, and were thus identified as second-order vestibular neurons. In addition, stimulation of the contralateral vestibular afferents revealed in most cases a disynaptic or trisynaptic, commissural inhibition. Second-order vestibular neurons displayed in the isolated brain a high degree of variability of their spontaneous activity, as in alert guinea-pigs. Type A neurons always exhibited a regular firing, while type B and B+LTS cells could have very irregular patterns of spontaneous discharge. Thus, type A and type B neurons might correspond, respectively, to the tonic and phasic vestibular neurons described in vivo. The regularity of spontaneous discharge was positively correlated with the amplitude of spike after hyperpolarization, and there was a trend for irregular neurons to be excited from ipsilateral vestibular afferents at shorter latencies than regular units. Synaptic activation could trigger subthreshold plateau potentials and low-threshold spikes in some of the second-order vestibular neurons. As a second step, the pharmacology of the synaptic transmission between primary vestibular afferents and second-order neurons was assessed using specific antagonists of the glutamatergic receptors. Both the synaptic field potentials and excitatory postsynaptic potentials elicited in the medial vestibular nucleus by single shock stimulation of the ipsilateral vestibular nerve were largely or, sometimes, totally blocked by 6-cyano-7-nitroquinoxaline-2,3-dione, indicating a dominating role of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor-mediated glutamatergic transmission. The remaining component of the responses was completely or partially suppressed by DL-2-amino-5-phosphonovaleric acid in 35% of the cases, suggesting a concomitant, moderate involvement of N-methyl-D-asparate receptors. In addition, a synaptic response resistant to both antagonists, but sensitive to a zero Ca2+/high Mg(2+)-containing solution, was often observed. Finally, recordings from abducens and oculomotor complexes confirmed the existence in the guinea-pig of strong bilateral, disynaptic excitatory and inhibitory inputs from vestibular afferents to motoneurons of extraocular muscles, which contribute to generation of the vestibulo-ocular reflex. The functional integrity of vestibular-related pathways in the isolated brain was additionally checked by stimulation of the spinal cord and optic tract. Stimulation of the spinal cord evoked, in addition to antidromic responses in the vestibular nucleus, short-latency synaptic responses in both the vestibular nucleus and abducens motoneurons, suggesting possible recruitment of spinal afferents. Activation of visual pathways at the level of the optic chiasm often induced long latency responses in the various structures under study. These results demonstrate that the in vitro isolated brain can be readily used for detailed, functional studies of the neuronal networks underlying gaze and posture control.     

Electrophysical properties, synaptic transmission and neuromodulation in serotonergic caudal raphe neurons

1. We studied electrophysiological properties, synaptic transmission and modulation by 5-hydroxytryptamine (5-HT) of caudal raphe neurons using whole-cell recording in a neonatal rat brain slice preparation; recorded neurons were identified as serotonergic by post-hoc immunohistochemical detection of tryptophan hydroxylase, the 5-HT-synthesizing enzyme. 2. Serotonergic neurons fired spontaneously (approximately 1 Hz), with maximal steady state firing rates of < 4 Hz. 5-Hydroxytryptamine caused hyperpolarization and cessation of spike activity in these neurons by activating inwardly rectifying K+ conductance via somatodendritic 5-HT1A receptors. 3. Unitary glutamatergic excitatory post-synaptic potentials (EPSP) and currents (EPSC) were evoked in serotonergic neurons by local electrical stimulation. Evoked EPSC were potently inhibited by 5-HT, an effect mediated by presynaptic 5-HT1B receptors. 4. In conclusion, serotonergic caudal raphe neurons are spontaneously active in vitro; they receive prominent glutamatergic synaptic inputs. 5-Hydroxytryptamine regulates serotonergic neuronal activity of the caudal raphe by decreasing spontaneous activity via somatodendritic 5-HT1A receptors and by inhibiting excitatory synaptic transmission onto these neurons via presynaptic 5-HT1B receptors. These local modulatory mechanisms provide multiple levels of feedback autoregulation of serotonergic raphe neurons by 5-HT.     

Electrophysiology of the neuroprotective agent riluzole on striatal spiny neurons

Striatal spiny neurons are selectively vulnerable in Huntington's disease (HD). No effective treatment is available to limit neuronal death in this pathological condition. In an experimental model of HD, a beneficial effect has recently been reported by the neuroprotective agent riluzole. We performed intracellular recordings in order to characterize the electrophysiological effects of this compound on striatal spiny neurons. Riluzole (0.1-100 microM) affected neither the resting membrane potential nor the input resistance/membrane conductance of the recorded cells. Bath application of this pharmacological agent produced a dose-dependent reduction of the number of spikes evoked by long-lasting depolarizing pulses. The EC50 value for this effect was 0.5 microM. Low doses of riluzole selectively reduced the firing frequency in the last part of the depolarizing pulse suggesting a use-dependent action at low concentrations of this compound. Riluzole produced a dose-dependent reduction of the amplitude of the corticostriatal glutamatergic excitatory post-synaptic potentials (EPSPs) with an extrapolated EC50 value of 6 microM. This effect was reversible and maximal at a concentration of 100 microM. Paired-pulse facilitation (PPF) was not affected by riluzole suggesting that the reduction of excitatory transmission was not only caused by a decrease of presynaptic release. Accordingly, riluzole also reduced the amplitude of membrane depolarization induced by exogenous glutamate. The modulatory action of riluzole on the activity of striatal spiny neurons might support the use of this drug in experimental models of excitotoxicity and in the neurodegenerative disorders involving the striatum.     

Nerve terminal currents induced by autoreception of acetylcholine release

The activation of autoreceptors is known to be important in the modulation of presynaptic transmitter secretion in peripheral and central neurons. Using whole-cell recordings made from the free growth cone of myocyte-contact motoneurons of Xenopus cell cultures, we have observed spontaneous nerve terminal currents (NTCs). These spontaneous NTCs are blocked by d-tubocurarine (d-TC) and alpha-bungarotoxin (alpha-BuTx), indicating that endogenously released acetylcholine (ACh) can produce substantial membrane depolarization in the nerve terminals. Local application of NMDA to the growth cone increased the frequency of spontaneous NTCs. When the electrical stimulations were applied at the soma to initiate evoked-release of ACh, evoked ACh-induced potentials were recorded in the nerve terminals, which were inhibited by d-TC and hexamethonium but not by atropine. Replacement of normal Ringer's solution with high-Mg2+, low-Ca2+ solution also reversibly inhibited evoked ACh-induced potentials. The possible regulatory role of presynaptic nicotinic autoreceptors on the synaptic transmission was also examined. When the innervated myocyte was whole-cell voltage-clamped to record synaptic currents, application of hexamethonium inhibited the amplitude of evoked synaptic currents at a higher degree than that of iontophoretic ACh-induced currents. Furthermore, hexamethonium markedly reduced the frequency of spontaneous synaptic currents at high-activity synapses. Pretreatment of neurons with alpha-BuTx also inhibited the evoked synaptic currents in manipulated synapses. These results suggest that ACh released spontaneously or by electrical stimulation may act on the presynaptic nicotinic autoreceptors of the same nerve terminals to produce membrane potential change and to regulate synaptic transmission.     

Glutamate receptor activity is required for normal development of tectal cell dendrites in vivo

Glutamatergic retinotectal inputs mediated principally by NMDA receptors can be recorded from optic tectal neurons early during their morphological development in Xenopus tadpoles. As tectal cell dendrites elaborate, retinotectal synaptic responses acquire an AMPA receptor-mediated synaptic component, in addition to the NMDA component. Here, we tested whether glutamatergic activity was required for the elaboration of dendritic arbors in Xenopus optic tectal neurons. In vivo time-lapse imaging of single DiI-labeled neurons shows that the NMDA receptor antagonist APV (100 microM) blocked the early development of the tectal cell dendritic arbor, whereas the AMPA receptor antagonist CNQX (20 microM) or the sodium channel blocker TTX (1 microM) did not. The decreased dendritic development is attributable to failure to add new branches and extend preexisting branches. These observations indicate that NMDA-type glutamatergic activity promotes the initial development of the dendritic arbor. At later stages of tectal neuron development when AMPA receptor-mediated synaptic transmission is strong, both APV and CNQX decrease dendritic arbor branch length, consistent with a role for glutamatergic synaptic transmission in maintaining dendritic arbor structure. These results indicate that AMPA and NMDA receptors can differentially influence dendritic growth at different stages of neuronal development, in correlation with changes in the relative contribution of the receptor subtype to synaptic transmission.     

Pharmacologically and functionally distinct calcium currents of stomatogastric neurons

Previous studies have suggested the presence of different types of calcium channels in different regions of stomatogastric neurons. We sought to pharmacologically separate these calcium channel types. We used two different preparations from different regions of stomatogastric neurons to screen a range of selective calcium channel blockers. The two preparations were isolated cell bodies in culture, in which calcium current was measured directly, and isolated neuromuscular junction, in which synaptic transmission was the indirect assay for presynaptic calcium influx. The selective blockers were two different dihydropyridines, omega-Agatoxin IVA, and omega-Conotoxin GVIA. Cultured cell bodies possessed both high-threshold calcium current and calcium-activated outward current, similar to intact neurons. The calcium current had transient and maintained components, but both components had the same voltage dependence of activation and inactivation. Dihydropyridines at >/=10 microM blocked both high-threshold calcium current and calcium-activated outward current. Nanomolar doses of omega-Agatoxin IVA did not block calcium current, but micromolar doses did. omega-Conotoxin GVIA did not block either current. In contrast, at the neuromuscular junction, dihydropyridines reduced the amplitude of postsynaptic potentials by only a modest amount, whereas omega-Agatoxin IVA at doses as low as 64 nM reduced the amplitude of postsynaptic potentials almost entirely. These effects were presynaptic. omega-Conotoxin GVIA did not change the amplitude of postsynaptic potentials. The different pharmacological profiles of the two isolated preparations suggest that there are at least two different types of calcium channel in stomatogastric neurons and that omega-Agatoxin IVA and dihydropridines can be used to pharmacologically distinguish them.     

Post-receptor mechanisms underlying striatal long-term depression

Extracellular and intracellular recordings were obtained from striatal neurons in a brain slice preparation in order to characterize the post-receptor mechanisms underlying striatal posttetanic long-term depression (LTD). Striatal LTD was blocked in neurons intracellularly recorded either with 1,2-bis (o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid (BAPTA) or with EGTA, calcium (Ca2+) chelators. Intracellular injection of QX-314, a lidocaine derivative that has been shown to block voltage-dependent sodium channels, abolished action potential discharge and blocked striatal LTD. However, under this condition, striatal LTD was restored when, immediately before the delivery of the tetanus, the cell was depolarized at a membrane potential ranging between -30 mV and -20 mV by injecting continuous positive current. Nifedipine (10 microM), a blocker of voltage-dependent L-type Ca2+ channels, blocked striatal LTD. Nifedipine by itself altered neither cortically evoked EPSPs nor input resistance and firing properties of most of the recorded cells. Striatal LTD was also reduced or blocked by incubation of the slices in the presence of the following inhibitors of Ca(2+)-dependent protein kinases: staurosporine (10-50 nM), 1-(5-isoquinolinesulfonyl)-2- methylpiperazine (H-7; 10-50 microM), and calphostin C (1 microM). Our findings suggest that generation of striatal LTD requires a Ca2+ influx through voltage-dependent nifedipine-sensitive Ca2+ channels and a sufficient intracellular free Ca2+ concentration. Furthermore, this form of synaptic plasticity seems to involve the activation of Ca(2+)-dependent protein kinases. Different drugs, acting at receptor and/or post-receptor level, may affect this form of synaptic plasticity and might alter the formation of motor memory.     

Neonatal irradiation prevents the formation of hippocampal mossy fibers and the epileptic action of kainate on rat CA3 pyramidal neurons

1. The effects of unilateral gamma-ray irradiation at birth on the properties of adult CA3 pyramidal neurons have been studied in hippocampal slices. 2. Neonatal gamma-ray irradiation reduced by 80% the number of granule cells and prevented the formation of mossy fiber synapses without reducing the number of CA3 pyramidal cells. The destruction of the mossy fibers was also confirmed with extracellular recordings. 3. Excitatory and inhibitory postsynaptic potentials (EPSPs and IPSPs) evoked by stimulation of the stratum radiatum had similar properties in nonirradiated and irradiated hippocampi: the EPSP reversed polarity near 0 mV, was reduced in amplitude by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10 microM) and D(-)-2-amino-5-phosphonovalerate (APV, 50 microM); the fast and slow IPSPs reversed at -75 and -100 mV, were blocked by bicuculline (10 microM), and reduced by phaclofen (0.5 mM), respectively. 4. Bath application of kainate (300-500 nM) evoked epileptiform activity in 81.5% of nonirradiated hippocampal CA3 regions and only in 29% of the irradiated CA3 regions. In contrast, bath application of high potassium (7 mM) and bicuculline (10 microM) generated spontaneous and evoked epileptiform activity in both nonirradiated and irradiated CA3 regions. 5. In nonirradiated and irradiated CA3 regions, kainate (200-300 nM) reduced the amplitude of the fast and slow IPSPs, reduced spike accommodation, and increased the duration of the action potential generated by a depolarizing pulse. 6. The postsynaptic responses of CA3 neurons to bath application of glutamatergic agonists were similar in nonirradiated and irradiated hippocampi in terms of amplitude, reversal potential, and pharmacology. 7. It is concluded that the most conspicuous effect of neonatal gamma-ray irradiation is to prevent the epileptic action of kainate. We propose that kainate generates epileptiform activity in the intact CA3 region by activating high-affinity binding sites located on the mossy fiber terminals.     

Activation of muscarinic receptors during blockade of GABA(A)-mediated inhibition induces synchronous epileptiform activity in immature rat hippocampus

We investigated the effects of the cholinergic agonist carbachol (25 microM) on the synaptic potentials recorded extracellularly and intracellularly from the CA3 area of immature hippocampal slices of the rat (postnatal days 10-20). In control conditions, carbachol reduced the amplitude of evoked synaptic responses (n=8) and did not induce any spontaneous synchronous activity (n=12); the depressant effect of carbachol was mimicked by acetylcholine (100 microM, in eserine 10 microM, n=5) and was reversed by the muscarinic antagonist atropine (1 microM, n=2). The GABA(A)-receptor antagonist bicuculline (10 microM) enhanced the amplitude and duration of the evoked synaptic responses and induced infrequent (0.016-0.045 Hz) spontaneous synchronous discharges in 23/37 of the slices. Application of carbachol in the presence of bicuculline reduced the amplitude of the evoked synaptic responses (n=21) and in addition induced synchronous discharges with rates of occurrence 0.075-0.225 Hz, in 64/68 slices. Both effects were mimicked by acetylcholine and eserine, and antagonized by atropine. The specific muscarinic antagonists pirenzepine (M1-type), tripitramine (M2-type), 4-diphenylacetoxy-N-methylpiperidine methiodide (M3-type) and tropicamide (M4-type) (all tested at 0.1-1 microM) reversibly reduced the frequency of synchronous carbachol-induced discharges. In addition, these discharges were reversibly blocked by high Ca2+ perfusion medium (7 mM CaCl2, n=4) and by the glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (10 microM, n=7). Synchronous epileptiform discharges were recorded from both CA1 and CA3 areas in intact slices (n=3), but only from CA3 following disruption of the CA1-CA3 synaptic connections (n=3). These experiments suggest that activation of muscarinic receptors during blockade of GABA(A)-mediated potentials, may enhance synchronous epileptiform activity in immature (postnatal days 10-20) hippocampus, through activation of local excitatory circuits and that endogenous acetylcholine may be sufficient to play this role.     

Direct recording of nicotinic responses in presynaptic nerve terminals

Nicotinic acetylcholine receptors are widely expressed in the nervous system, but their functions remain poorly understood. One attractive hypothesis is that the receptors act presynaptically to modulate synaptic transmission. We provide a direct demonstration of presynaptic nicotinic receptors in situ by using whole-cell patch-clamp techniques to record currents in large presynaptic calyces that midbrain neurons form on ciliary neurons. Bath application of nicotine induced inward currents in the calyces capable of generating action potentials that overrode the limited space clamp achievable. The inward currents reversed near 0 mV and showed inward rectification common for neuronal nicotinic receptors. Tetrodotoxin (TTX) blocked the action potentials but not the inward currents. alpha-Bungarotoxin blocked both, consistent with the presynaptic receptors containing alpha7 subunits. Recording from the postsynaptic ciliary neurons during nicotine exposure revealed EPSCs that TTX blocked, presumably by blocking presynaptic action potentials. The postsynaptic cells also displayed bimodal inward currents caused by their own nicotinic receptors; the bimodal currents were not blocked by TTX but were blocked partially by alpha-bungarotoxin and completely by D-tubocurarine. Dye-filling with Lucifer yellow from the recording pipette confirmed the identity of patched structures and showed no dye transfer between calyx and ciliary neuron. When calyces or ciliary neurons were labeled en mass with neurobiotin and biocytin through nerve roots, dye transfer was rarely observed. Thus, electrical synapses were infrequent and unlikely to influence calyx responses. Immunochemical analysis of preganglionic nerve extracts identified receptors that bind alpha-bungarotoxin and contain alpha7 subunits. The results unambiguously document the existence of functional presynaptic nicotinic receptors.