Cultured rat sensory neurones express functional tachykinin receptor subtypes 1, 2 and 3.

The neuropeptide substance P (SP) is known to play a key role in peripheral nociceptive processes. We investigated the in vitro pharmacological characteristics of functional tachykinin receptors expressed in dorsal root ganglia (DRG) sensory neurones by analysing intracellular free calcium concentration changes induced after stimulation by SP or specific tachykinin agonists. We observed that about 37% of the tested neurones were responsive to either SP or an NK1-, NK2- or NK3-specific agonist. Tachykinin-responsive neurones had a small soma diameter (<20 microm) and were sensitive to capsaicin. These results suggest the presence of NK1, NK2 and NK3 receptors in noxious sensory neurones.

Electrophysiological evidence for multiple glycinergic inputs to neonatal rat sympathetic preganglionic neurons in vitro.

The time pattern of glycinergic inhibitory postsynaptic currents (IPSCs) in sympathetic preganglionic neurons was studied in thin transverse spinal cord slices of neonatal (1-10 days postnatal) rats by means of the patchclamp technique. Three time patterns could be distinguished: (i) large events [mostly > 400 pA (30-36 degrees C)] occurring at regular intervals, (ii) small events occurring at irregular intervals, and (iii) small events occurring in transient (1.5-10 s), high-frequency (> 15 Hz) bursts of synaptic activity. The large regular events had uniform kinetics which was consistent with the idea of a proximal site of origin for all of these events. They were reversibly inhibited in amplitude and frequency by extracellular application of a high concentration of acetylcholine (200 microM) or the specific nicotinic acetylcholine receptor agonist dimethylphenylpiperazinium iodide (DMPP; 1 mM), but unaffected by glutamate (100 microM). IPSCs occurring in bursts had slower and less uniform kinetics, suggesting a more diverse site of origin. The frequency of events decreased during a burst. Similar bursts could be induced by extracellular application of glutamate receptor agonists. These results indicate that sympathetic pregnanglionic neurons in a thin, transverse spinal cord slice receive at least two different glycinergic inputs.

Tachykinin neurokinin-1 and neurokinin-3 receptor-mediated responses in guinea-pig substantia nigra: an in vitro electrophysiological study.

The effects of tachykinin receptor agonists and antagonists were investigated using intra- and extracellular recordings on spontaneously firing nigral neurons in guinea-pig brain slices. In 70 of 76 electrophysiologically identified dopaminergic neurons, a concentration-dependent increase in firing rate was induced by the selective neurokinin-3 tachykinin agonist senktide and by the natural tachykinin agonists neurokinin B and substance P, with EC50 values of 14.7, 31.2 and 12200 nM respectively. These responses were inhibited in a concentration- and time-dependent manner by the selective non-peptide neurokinin-3 receptor antagonist SR 142801 (1-100 nM; n=23), but neither by its S-enantiomer SR 142806 (100 nM; n=4) nor by selective antagonists of neurokinin-1 (SR 140333) or neurokinin-2 (SR 48968) receptors (both at 100 nM; n=3). The selective neurokinin-1 agonist [Sar9,Met(O2)11]substance P (30-100 nM; n=23) and the selective neurokinin-2 agonist [Nle10]neurokinin A(4-10)(30-100 nM; n=13) were without any effect on dopaminergic cells. In 13 of 21 electrophysiologically identified, presumably GABAergic neurons located in the pars compacta of the substantia nigra, excitatory responses were evoked concentration dependently by substance P and [Sar9,Met(O2)11]substance P, with EC50 values of 18.6 and 41.9 nM respectively. These responses were inhibited by SR 140333 (100 nM; n=3), but neither by its R-enantiomer SR 140603 nor by SR 142801 (both at 100 nM; n=3). Senktide and [Nle10]neurokinin A(4-10) (both at 30-100 nM; n=10) were without effect on these presumed GABAergic neurons. A small population (12%) of pars compacta neurons was insensitive to any of the three selective tachykinin agonists. In the nigral pars reticulata, 12 neurons were recorded which had an electrophysiological profile similar to that of presumed GABAergic neurons in the pars compacta. Of these 12 cells, seven did not respond to any of the selective tachykinin agonists tested, while five were excited by senktide in a concentration-dependent manner (EC50=98.5 nM). Although this value was significantly higher than that found for dopaminergic neurons in the pars compacta, senktide-evoked responses were inhibited by SR 142801 (100 nM; n=3). We conclude that, in the guinea-pig substantia nigra, tachykinins evoke excitatory responses in both dopaminergic and non-dopaminergic neurons; however, the sensitivity to tachykinin agonists (neurokinin-1 versus neurokinin-3) depends on both neuronal type and localization.

Mesencephalic trigeminal neuron responses to gamma-aminobutyric acid.

Mesencephalic trigeminal neurons are primary sensory neurons which have cell somata located within the brain stem. In spite of the presence of synaptic terminals on and around the cell somata, applications of a variety of neurotransmitter substances in earlier studies have failed to demonstrate responses. Using intracellular recording in a brain slice preparation, we have observed prominent depolarizations and decreases in input resistance in response to applications of gamma-aminobutyric acid (GABA) in most recorded mesencephalic trigeminal neurons. Those cells failing to respond were located deeply within the slice, and the low responsiveness was shown to be related to uptake of GABA in the slice. The responses were direct, since they remained during perfusion with a low calcium, high magnesium solution that blocks synaptic transmission. The responses were mimicked by the GABA(A) receptor agonist isoguvacine, and blocked by GABA(A) receptor antagonists. The GABA(B) receptor agonist baclofen evoked no changes in membrane potential or input resistance in neurons exhibiting depolarizations with GABA application. Tests of neuronal excitability during GABA applications indicated that the excitatory effects of the depolarization prevail over the depressant effects of the increase in membrane conductance. In situ hybridization histochemistry indicated that the GABA(A) receptors in Me5 cells are comprised of alpha2, beta2 and gamma2 subunits.

Modulation of GABAA receptor-mediated IPSCs by neuroactive steroids in a rat hypothalamo-hypophyseal coculture model.

1. We have used the whole-cell configuration of the patch-clamp technique to investigate the effects of neuroactive steroids on GABAA receptor-mediated synaptic transmission between rat hypothalamic neurones and pituitary intermediate lobe (IL) cells grown in coculture. In order to discriminate between possible pre- and postsynaptic sites of action, the effects of neurosteroids on GABAA receptor-mediated synaptic currents (IPSCs) were compared with those of GABAA currents (IGABA) triggered by local application of 50 or 500 microM GABA, which yielded approximately half-maximal and maximal responses, respectively. 2. In primary cultures of rat pituitary IL cells, allopregnanolone (5 alpha-pregnan-3 alpha-ol-20-one) reversibly potentiated IGABA in a dose-dependent manner with a threshold between 0.1 and 1 nM. At a concentration of 10 nM, allopregnanolone increased the response evoked by 50 microM GABA by +21.4 +/- 5.1% (n = 8), but had no effect on IGABA induced by 500 microM GABA. The beta-isomer of allopregnanolone, epipregnanolone (5 beta-pregnan-3 beta-ol-20-one, 10 nM), had no effect on IGABA at any concentration of GABA tested. 3. At concentrations lower than 10 microM, pregnenolone sulphate (5-pregnen-3 alpha-ol-20-one sulphate) did not significantly inhibit IGABA. However, at 10 microM, a systematic reduction of IGABA evoked by 50 and 500 microM GABA was observed, with mean values of -80 and -60%, respectively. This blocking effect was reversible and accompanied by a marked acceleration of decay of GABAA currents during the application of GABA. 4. In isolated pairs of synaptically connected hypothalamic neurones and IL cells, allopregnanolone (10 nM) augmented the mean amplitude of spontaneous IPSCs (sIPSCs) and electrically evoked IPSCs (eeIPSCs) by about 40% and increased the mean frequency of sIPSCs. Allopregnanolone (10 nM) also markedly increased the frequency of miniature IPSCs (mIPSCs) recorded in the presence of TTX (0.5 microM), but without modifying their mean amplitude. Epipregnanolone had no effect on the amplitude or frequency of sIPSCs. Neither epipregnanolone nor allopregnanolone modified the time to peak and decay time constants of GABAergic IPSCs. 5. Pentobarbitone (50 microM), a positive allosteric modulator of GABAA receptors, did not affect the amplitude of sIPSCs or eeIPSCs, but significantly increased the decay time constants of both types of IPSCs. Pentobarbitone had no effect on the frequency of sIPSCs. 6. Pregnenolone sulphate (10 microM) completely and reversibly blocked sIPSCs and eeIPSCs. Progressive block of IPSCs was correlated with a gradual decrease of the mean decay time constant. 7. Our results suggest that, under physiological conditions, allopregnanolone might be a potent modulator of GABAergic synaptic transmission, acting at both pre- and postsynaptic sites. The involvement of pregnenolone sulphate as a modulator of GABAergic IPSCs under physiological conditions is, however, more questionable. The mechanisms of action of both types of neurosteroids are discussed.

Adrenergic responses in silent and putative inhibitory pacemaker-like neurons of the rat rostral ventrolateral medulla in vitro.

Noradrenaline and adrenergic agonists were tested on pacemaker-like and silent neurons of the rat rostral ventrolateral medulla using intracellular recording in coronal brainstem slices as well as in punches containing only the rostral ventrolateral medullary region. Noradrenaline (1-100 microM) depolarized or increased the frequency of discharge of all cells tested in a dose-dependent manner. The noradrenaline-induced depolarization was associated with an apparent increase in cell input resistance at low concentrations and a decrease or no significant change at higher concentrations. Moreover, it was voltage dependent and its amplitude decreased with membrane potential hyperpolarization. Noradrenaline caused a dose-related increase in the frequency and amplitude of spontaneous inhibitory postsynaptic potentials. The alpha 1-adrenoceptor antagonist prazosin (0.5 microM) abolished the noradrenaline depolarizing response as well as-the noradrenaline-evoked increase in synaptic activity and unmasked an underlying noradrenaline dose-dependent hyperpolarizing response associated with a decrease in cell input resistance and sensitive to the alpha 2-adrenoceptor/antagonist yohimbine (0.5 microM). The alpha 1-adrenoceptor agonist phenylephrine (10 microM) mimicked the noradrenaline depolarizing response associated with an increase in membrane resistance as well as the noradrenaline-induced increase in synaptic activity. The alpha 2-adrenoceptor agonists UK-14,304 (1-3 microM) and clonidine (10-30 microM) produced only a small hyperpolarizing response, whereas the beta-adrenoceptor agonist isoproterenol (10-30 microM) had no effect. Baseline spontaneous postsynaptic potentials were abolished by strychnine (1 microM), bicuculline (30 microM) or both. However, only the strychnine-sensitive postsynaptic potentials had their frequency increased by noradrenaline or phenylephrine and they usually occurred with a regular pattern. Tetrodotoxin (1 microM) eliminated 80-95% of baseline spontaneous postsynaptic potentials and prevented the increase in synaptic activity evoked by noradrenaline and phenylephrine. Similar results were obtained in rostral ventrolateral medulla neurons impaled in both coronal slices and punches of the rostral ventrolateral medulla. It is concluded that noradrenaline could play an important inhibitory role in the rostral ventrolateral medulla via at least two mechanisms: an alpha 2-adrenoceptor-mediated hyperpolarization and an enhancement of inhibitory synaptic transmission through activation of alpha 1-adrenoceptors located on the somatic membrane of glycinergic interneurons. Some of these interneurons exhibit a regular discharge similar to the pacemaker-like neurons and might, at least in part, constitute a central inhibitory link in the baroreceptor-vasomotor reflex pathway.

Evidence that GABAA receptor subunit mRNA expression during development is regulated by GABAA receptor stimulation.

The expression of six mRNA species (alpha 2, alpha 3, alpha 5, beta 2, beta 3, and gamma 2) encoding for GABAA receptor subunits was followed in cultured early postnatal cortical neurons by in situ hybridization histochemistry. In untreated control cultures it was found that these subunit mRNA expression profiles closely follow those seen during development in vivo. alpha 3, alpha 5, and beta 3 subunit expression declined, alpha 2 expression increased, whereas beta 2 and gamma 2 subunit mRNA expression remained relatively constant. To test the hypothesis that GABAA receptor stimulation regulates these expression profiles, we tested the effect of a GABAA receptor positive modulator, allopregnanolone, and a GABAA receptor noncompetitive antagonist, tert-butylbicyclophosphorothionate (TBPS). It was found that allopregnanolone augmented the rate at which the alpha 3, alpha 5, or beta 3 subunit mRNA expression declined and prevented the increase in alpha 2 subunit mRNA expression. As well, allopregnanolone down-regulated beta 2 subunit mRNA expression. TBPS, on the other hand, up-regulated alpha 3, alpha 5, beta 2, and beta 3 subunit mRNA expression. It also down-regulated the expression of alpha 2 subunit mRNA. Both allopregnanolone and TBPS had no effect on gamma 2 subunit mRNA expression. These results imply that the developmental switchover of GABA receptor subunit mRNA expression is regulated by GABAA receptor activity.

Recombinant GABAA receptor desensitization: the role of the gamma 2 subunit and its physiological significance.

1. The purpose of these investigations was to examine the role that the gamma 2 subunit plays in human GABAA receptor desensitization. Two different recombinant GABAA receptors (alpha 1 beta 3 and alpha 1 beta 3 gamma 2) were compared by measuring the relaxation of whole-cell currents during the application of GABA, isoguvacine or taurine. 2. At concentrations which trigger a maximum response (100-500 microM GABA) the current relaxation usually fitted the sum of two exponentials. For alpha 1 beta 3 subunit receptors these values were tau 1 = 145 +/- 12 ms and tau 2 = 6.3 +/- 2.1 s (means +/- S.E.M.). Receptors consisting of alpha 1 beta 3 gamma 2 subunits desensitized faster: tau 1 = 41.6 +/- 8.3 ms and tau 2 = 2.4 +/- 0.6 s. 3. The Hill slope, determined for each receptor subunit combination, was the same and greater than 1.0, implying two binding steps in the activation of both receptor subunit combinations. 4. For alpha 1 beta 3 subunit receptors the fast desensitization rates were unaltered by reducing the GABA concentration from the EC100 (100 microM) to the approximate EC50 values (10-20 microM), whereas for alpha 1 beta 3 gamma 2 subunit receptors a significant slowing was observed. The fast desensitization disappeared at agonist concentrations below the EC50 for both subunit combinations. In contrast, the slow desensitization appeared at agonist concentrations near the EC20. This rate was dependent on agonist concentration reaching a maximum near the EC60 value of GABA. 5. The fast desensitization rates were unaltered by changing the holding potential of the cell during agonist application. However, for alpha 1 beta 3 gamma 2 subunit receptors the slow desensitization rate increased by approximately 15- to 20-fold over the range of voltages of -60 to +40 mV. This indicates that the gamma 2 subunit makes GABAA receptor desensitization voltage dependent. 6. Recovery from desensitization was also biphasic. The first recovery phase was faster for alpha 1 beta 3 gamma 2 than for alpha 1 beta 3 subunit receptors (0.13 vs. 0.03 s-1, respectively). The second phase of recovery for the two receptors were the same (approximately 0.003 s-1). 7. There was only a poor correlation between agonist potency and the degree or time course of desensitization. Isoguvacine (EC50 approximately to 10 microM) induced biphasic relaxation for both alpha 1 beta 3 and alpha 1 beta 3 gamma 2 subunit receptors (tau 1 = 288.6 +/- 43.3 and 167 +/- 15 ms, and tau 2 = 8.0 +/- 1.9 and 4.4 +/- 0.4 S, respectively, for each subunit combination). Taurine (EC50 approximately 7 mM) usually induced monophasic relaxation for both subunit combinations (tau 2 = 7.1 +/- 1.6 and 23.0 +/- 6.6 s, respectively). 8. A computer model was developed to examine the effect of the gamma 2 subunit on the time course of a synaptic potential. It was found that the gamma 2 subunit theoretically prolongs the time course of a synaptic potential by inducing desensitization more rapidly. The subsequent relaxation of the desensitized receptors through the open state increases Popen (the probability that the GABAA receptor is in an open conducting state) altering the time course of the modelled potential. alpha 1 beta 3 subunit receptors do not desensitize sufficiently rapidly to induce this desensitized state and, therefore, are shorter in time course. These data imply that the physiological role of the gamma 2 subunit is to increase synaptic efficacy by prolonging Popen.

Nicotinic actions on neurones of the central autonomic area in rat spinal cord slices.

1. Nicotinic responses and actions on excitatory synaptic activity were studied in eighty-four neurones in the region dorsal to the central canal (lamina X) in transverse thoracolumbar spinal cord slices of neonate (P2-P10) rats by using the whole-cell patch-clamp technique. 2. Neurones (n = 15) labelled with Lucifer Yellow, showed the typical morphology of sympathetic preganglionic neurones (SPNs) in the central autonomic area (CA). Unlabelled neurones of comparable morphology were visually identified and recorded. 3. All neurones recorded responded to the nicotinic acetylcholine receptor (nAChR) agonist, DMPP. Under current-clamp conditions, pressure ejections of DMPP depolarized cells and induced the discharge of action potentials. Tetrodotoxin suppressed action potentials but not DMPP-induced depolarization. 4. Under voltage-clamp conditions at a holding potential (Vh) of -50 mV, DMPP induced a transient inward current (which reversed around 0 mV) and an increase in membrane current noise in 50% of the recorded neurones. In the others, DMPP increased membrane current noise without measurable inward current. The current-voltage relationship showed strong inward rectification at holding potentials more positive than 0 mV. 5. In neurones displaying a detectable current response to DMPP, the following agonist rank order potency could be established: DMPP = nicotine > cytisine > ACh. The DMPP response could be blocked by mecamylamine but was insensitive to methyllycaconite. 6. Pressure application of glutamate induced inward currents in all cells tested at a Vh of -50 mV. This response reversed at 10 mV, displayed a region of negative slope conductance at Vh more negative than -30 mV and was partially blocked by CNQX. Pressure application of DMPP transiently increased the amplitude of the glutamate-induced current in six out of nine cells tested. This potentiation persisted in the presence of tetrodotoxin. 7. Forty per cent of the recorded neurones displayed spontaneous excitatory postsynaptic currents (sEPSCs). At a Vh of -50 mV the sEPSCs had a mean amplitude of -19.3 pA and occurred at a frequency below 0.5 Hz. sEPSCs were blocked by CNQX and inverted around 0 mV. Brief application of DMPP increased the discharge frequency of sEPSCs without affecting their kinetics. Additionally, in some cells DMPP increased mean sEPSC amplitude. 8. Focal electrically evoked EPSCs reversed close to 10 mV and were sensitive to CNQX. They occurred with a constant latency, rise time and a mono-exponential decay time. Application of DMPP decreased the percentage of stimulation failures and increased the amplitude of evoked EPSCs, in all cells tested. 9. It is concluded that neurones in the CA, presumed to be SPNs, have functional nAChRs with activation having two distinct effects: firstly, a direct depolarization of the postsynaptic membrane; and secondly, a facilitation of the excitatory transmission onto these cells. This second effect is achieved by an increase of the size of the glutamate-induced current at the postsynaptic level as well as by an enhancement of the presynaptic release of glutamate.

Electrophysiological characterization of non-NMDA glutamate receptors on cultured intermediate lobe cells of the rat pituitary.

Glutamate is the major excitatory neurotransmitter in the central nervous system, yet little is known about its actions on endocrine cells. We have investigated the membrane effects of glutamate in cultured neonatal rat pituitary intermediate lobe (IL) cells using the whole-cell configuration of the patch-clamp technique. In a standard Na(+)-based extracellular solution, glutamate failed to induce a detectable membrane current at a holding potential (HP) of -60 mV (n = 40). However, when cyclothiazide (50 microM), a benzothiazide that blocks desensitization of alpha-amino-2,3-dihydro-5-methyl-3-oxo-4-isoxazole-propanoic acid (AMPA)-type receptors, was added to the extracellular solution, glutamate (0.5-1 mM) induced an inward current at a HP of -60 mV in 65% of the cells tested (n = 72). This response was usually small in amplitude (mean amplitude: 28.6 +/- 37.5 pA, n = 47). The glutamate-induced current reversed polarity close to 0 mV and was reversibly blocked when extracellular Na+ was replaced by the impermeant cation N-methyl-D-glucamine, suggesting that this current was a nonselective cation current. The response to glutamate (1 mM) was reproduced by AMPA (50 microM), kainate (200 microM), and quisqualate (200 microM). N-Methyl-D-aspartate (NMDA, 100 microM) in the presence of 10 microM glycine did not induce any membrane current in cells responding to glutamate (n = 8). The non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (10 microM) reversibly inhibited the response to glutamate (0.5 mM) by 85 +/- 14% (n = 7), whereas D(-)-2-amino-5-phosphonopentanoic acid (20 microM), an antagonist of NMDA receptors, had no effect on the glutamate-induced current (n = 3). Moreover, we show that although the amplitude of the glutamate currents was small, the latter induced large (30-mV) membrane depolarizations and triggered the firing of action potentials. Taken together, our results indicate that neonatal rat IL cells possess AMPA-type glutamate receptors that could possibly underlie a fast excitatory glutamatergic synaptic input to these cells.

Sumatriptan inhibits the release of CGRP and substance P from the rat spinal cord.

The possible presynaptic action of the anti-migraine drug sumatriptan on primary afferent fibres containing substance P and/or calcitonin gene-related peptide was investigated on superfused rat horizontal spinal cord slices with attached dorsal roots. Electrical stimulation of dorsal roots triggered a significant overflow of both peptides; this could be reduced by sumatriptan in a concentration-dependent manner. As expected from the involvement of 5-HT1B/1.D beta receptors, methiothepin, (-)tertatolol and GR 127,935, but not WAY 100,635, prevented the inhibitors effect of sumatriptan. These data support the idea that the anti-migraine action of sumatriptan may involve, at least in part, a presynaptic inhibitory control of nociceptive (trigeminovascular) substance P- and/or calcitonin gene-related peptide-containing sensory fibres.

Characterization of functional GABAergic synapses formed between rat hypothalamic neurons and pituitary intermediate lobe cells in coculture: Ca2+ dependence of spontaneous IPSCs.

Rat hypothalamic neurons and endocrine cells from the intermediate lobe of the pituitary were grown in dissociated coculture. Neurons positively stained with an antibody against glutamate decarboxylase established apparent contacts with the alpha-melanocyte-stimulating hormone-positive endocrine cells. These sites of contact were intensely labeled with an antibody against the synaptic protein synapsin I and displayed ultrastructural features characteristic of synapses. Using patch-clamp recordings, we have demonstrated that these contacts correspond to functional GABAergic synapses. The synaptic currents were blocked reversibly by bicuculline (5 microM) and SR95531 (5 microM), two competitive antagonists of the GABAA receptor. At a holding potential of -60 mV, spontaneously occurring IPSCs (s-IPSCs) had small amplitudes (10-100 pA), whereas electrically evoked IPSCs (ee-IPSCs) had amplitudes up to 1 nA. The rise times of both types of IPSCs were fast ( < or = 1 msec), and their decaying phases were fitted in most cases with a single exponential function (time constant 50 msec). The amplitude distribution of s-IPSCs did not reveal clear, equally spaced peaks and was little affected by tetrodotoxin, suggesting that most s-IPSCs were miniature IPSCs. Reduction of extracellular calcium concentration to 0.3 mM induced a marked decrease in s-IPSC frequency and revealed a single amplitude peak at 10 pA, suggesting that a single quantum of GABA activates 8-10 GABAA channels. Thus, our preparation might be an interesting model to study different aspects of synapse formation between a central neuron and its target as well as the fundamental mechanisms of synaptic transmission at central synapses.

Selective inhibition of high voltage-activated L-type and Q-type Ca2+ currents by serotonin in rat melanotrophs.

1. Whole-cell Ca2+ currents (ICa) from cultured rat melanotrophs were identified by their sensitivity to Ca2+ channel blockers, and their modulation by serotonin (5-HT) was studied. All cells displayed high voltage-activated (HVA; > -30 mV) Ca2+ currents. A low voltage-activated (LVA; > -60 mV) Ca2+ current was detected in 92% of the cells. 2. The whole-cell ICa was insensitive to omega-conotoxin GVIA (0.5-1 microM) indicating the absence of N-type Ca2+ channels. 3. At a holding potential (Vh) of -70 mV, the L-type channel blocker nifedipine reduced ICa in a dose-dependent manner with a half-maximal effective concentration (IC50) of 28 nM. The L-type current represented 39% of the total ICa. 4. omega-Agatoxin IVA (omega-Aga IVA) produced a biphasic dose-dependent inhibition of ICa, with IC50 values of 0.4 and 91 nM, indicating the presence of P-type and Q-type Ca2+ channels, which accounted respectively for 16 and 45% of the total ICa. The P-type current was also blocked by synthetic funnel-web spider toxin (sFTX 3.3; 1-10 microM) and was present only in a subpopulation (60-70%) of cells. 5. All cells possessed a Ca2+ current which was resistant to nifedipine (10 microM) and omega-Aga IVA (50 nM). This current was not affected by Ni2+ (40 microM) but was abolished by a low concentration of Cd2+ (10 microM) and by omega-conotoxin MVIIC (1 microM) indicating that it was a Q-type Ca2+ current. 6. 5-HT (10 microM) inhibited the whole-cell ICa in 70% of the cells tested (n = 120) by activating 5-HT1A and 5-HT2C receptors. 5-HT produced either a kinetic slowing of the activation phase (37% of the cells) or a scaling down (14% of the cells) of ICa. In the majority of cells (49%) both types of inhibition were found to coexist. 7. The effects of 5-HT were voltage dependent, rendered irreversible when GTP-gamma-S (30 microM) was present in the pipette solution and abolished by pretreatment of the cells with pertussis toxin (PTX; 150 ng ml-1, 18 h). 8. Low concentrations of omega-Aga IVA (20 nM), which blocked mainly P-type channels, did not reduce the effect of 5-HT on ICa. The scaling down effect of 5-HT on ICa was eliminated in the presence of nifedipine (10 microM) and the kinetic slowing effect of 5-HT persisted after blockade of L- and P-type channels but was abolished by omega-conotoxin MVIIC (1 microM). 9. We conclude that rat melanotrophs possess functional L-, P- and Q-type Ca2+ channels and that 5-HT inhibits selectively L-type and Q-type Ca2+ currents with different modalities. These effects are voltage dependent and mediated by a PTX-sensitive G-protein.

Patch-clamp characterization of nicotinic receptors in a subpopulation of lamina X neurones in rat spinal cord slices.

1. Nicotinic acetylcholine receptors (nAChRs) on lamina X neurones in neonate (P1-P12) rat transverse thoracolumbar spinal cord slices were studied using the whole-cell patch-clamp technique. These visually selected neurones are located dorsal to the central canal, mainly in the ventral half of the dorsal commissure. 2. Pressure application of the nicotinic agonist 1,1-dimethyl-4-phenyl-piperazinium (DMPP) (1 mM) induced a rapid depolarization on which action potentials are superimposed. 3. At -50 mV, DMPP (1 mM), pressure ejected for 100 ms, induced a fast inward current with a mean amplitude of -280 pA (n = 28) in 90% of the neurones recorded. Superfusion of tetrodotoxin (TTX), a solution containing 0 Ca(2+)-high Mg2+, CdCl2 or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) did not abolish the DMPP-induced current, which confirmed a direct postsynaptic effect of DMPP on recorded neurones. 3. The current-voltage (I-V) relationship for DMPP-induced current exhibited a reversal potential of 0 mV (NaCl outside, potassium gluconate inside) and a strong inward rectification. 4. The DMPP-induced responses were blocked by mecamylamine, hexamethonium and d-tubocurarine (dTC) but were insensitive to alpha-bungarotoxin and methyllycaconitine (MLA). 5. We conclude that lamina X neurones located dorsally to the central canal possess nicotinic acetylcholine receptors. Activation of these nicotinic receptors results in depolarization and generation of action potentials. These receptors may be involved in the modulation of the somato- and viscerosensory transmission.

GABA-induced responses in electrophysiologically characterized neurons within the rat rostro-ventrolateral medulla in vitro.

Rostro-ventrolateral medulla (RVL) neurons were recorded using conventional intracellular recording techniques in brain slices maintained in vitro at 32 degrees C and classified into 3 major groups. The first group included neurons having endogenous pacemaker-like (PL) activity with regular firing frequency (mean 8 Hz) and a linear current-voltage relationship (I-V). The second group of neurons were slowly and irregularly firing (IF) or quiescent, presenting membrane potential oscillations and their I-V usually displayed an inward rectification. These neurons had a relatively longer action potential duration. The third group included silent neurons (S) with no apparent membrane oscillations and they differed from the first two groups by having relatively shorter action potential duration and amplitude and lower cell input resistance. When recorded with KCl-filled electrodes, the majority of silent neurons displayed a time-dependent inward rectification. With KAc-filled electrodes, irregular slow hyperpolarizing and depolarizing spontaneous potentials could be recorded primarily on PL and IF neurons, respectively. Moreover, fast spontaneous inhibitory postsynaptic potentials (PSPs) were detected in about 15% of PL and S neurons. They generally exhibited a regular pattern and were depolarizing when KCl-filled electrodes were used for recording. The amplitude of these inhibitory PSPS was reversibly reduced by the GABA A antagonists bicuculline, SR 95531 and picrotoxin. With KAc-filled electrodes, pressure-applied GABA (20 mM) evoked complex responses. In PL neurons, it consisted of a fast hyperpolarization followed by a slower depolarization that were both sensitive to SR 95531 and picrotoxin. The response was terminated by a long-lasting hyperpolarization that was reduced, but not abolished, by the GABA B antagonist CGP 35348. In IF and S neurons, GABA application usually produced a fast followed by a slow monophasic hyperpolarization and depolarization, respectively. The fast component of these responses was sensitive to the GABA A antagonists. Pressure application of isoguvacine (10 mM) always induced monophasic responses in all types of neurons recorded. Baclofen (1-30 mu M) reduced the firing frequency and hyperpolarized PL and IF neurons, an effect that was antagonized by CGP 35348 (50-100 mu M); however, it had little effect on silent neurons. It is concluded that RVL neurons have heterogeneous electrophysiological characteristics. Their predominant synaptic input and GABA responsiveness might be additional criteria to identify the excitatory and inhibitory elements in the RVL circuitry. All neuronal types seem to have functional GABA A and GABA B receptors; however, only a subpopulation is under tonic inhibitory control in vitro, probably from local GABAergic pacemaker interneurons. Our results further emphasize the role of GABA as an important neurotransmitter in the RVL network.

GABAB receptors negatively regulate transcription in cerebellar granular neurons through cyclic AMP responsive element binding protein-dependent mechanisms.

GABAB receptors affect short-term signalling in various cell types. However, nothing is known about possible long-term effects on transcription. To analyse such effects in the CNS, we studied GABAB receptor-mediated gene regulation in primary cultures of cerebellar granule neurons. Transcription was followed using a chloramphenicol acetyl transferase reporter gene driven by the minimal cyclic AMP-responsive element (TGACGTCA). Transcription was stimulated by activation of both the cyclic AMP (forskolin: 5 x 10(-6) M) and the Ca2+ dependent (KCl: 30 mM) pathways (-)-Baclofen (10(-6) M to 10(-4) M), a specific GABAB receptor agonist, reduced by 50-70% the transcriptional stimulation evoked by both forskolin and KCl, whereas isoguvacine, a GABAA receptor agonist, was without effect. Moreover, the GABAB antagonist CGP 35348 abrogated the inhibitory effects of both GABA and baclofen, indicating that GABAB receptors were specifically implicated in this response. Measurements of cyclic AMP levels suggested that (-) baclofen inhibits forskolin-initiated transcription by reducing cyclic AMP production. Direct transcriptional activation, via the cyclic AMP pathway, by overexpression of the catalytic subunit of the cyclic AMP-dependent protein kinase, was not significantly altered by (-) baclofen. This indicates again that (-) baclofen-dependent inhibitory mechanisms operate upstream of cyclic AMP-dependent protein kinase at the level of second messenger formation. Further, we used a yeast transcriptional activator GAL4-cyclic AMP-responsive element binding protein to analyse whether GABAB receptor-mediated inhibition of cyclic AMP-responsive element transcription implicated the transacting factor cyclic AMP-responsive element binding protein. We show that the negative effects of (-) baclofen implicate this transcription factor and this holds good for both the forskolin and KCl-stimulated pathways. The results indicate that GABAB receptors negatively regulate cyclic AMP-responsive element binding protein-mediated transcription in the CNS.

Kinetics of A-currents in sympathetic preganglionic neurones and glial cells.

Transient outward currents (A-currents; IA) were recorded in sympathetic preganglionic neurones (SPNs) and glial cells of the intermediolateral cell column (IML) by whole-cell recordings in rat spinal cord slices. In both cell types IA activated at around -45 mV and the time-course of decay was monoexponential, but faster in glial cells than in neurones. In both cases decay time constants displayed the same increase with depolarization above -30 mV. In neurones, the activation curve was shifted to more negative value along the voltage axis and was steeper than the activation curve for glial cells whereas inactivation curves were similar. Recovery from inactivation followed a double and monoexponential decay in neurones and glial cells, respectively.

Electrophysiological evidence for oxytocin receptors on sympathetic preganglionic neurones--an in vitro study on the neonatal rat.

The action of oxytocin (0.01-1 microM) on sympathetic preganglionic neurones was studied by intracellular recording in slices of neonatal rat thoracic spinal cord. In 85% of the cells superfusion induced a slow tetrodotoxin-insensitive depolarization accompanied by the appearance or increase in frequency of repetitive discharges. Oxytocin also caused some cells to switch from silent neurones to spontaneously active ones. These effects were reversibly blocked by a specific oxytocin antagonist.

Excitatory postsynaptic currents and glutamate receptors in neonatal rat sympathetic preganglionic neurons in vitro.

1. We obtained whole cell patch-clamp recordings from visually identified sympathetic preganglionic neurons (SPNs) in thin (200-300 microns) transverse spinal cord slices of neonatal rats (1-14 days postnatal). Exogenous application of glutamate (100 microM), N-methyl-D-aspartate (NMDA; 100 microM), kainate (100 microM), quisqualate (1 microM), and alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA; 50 microM) induced inward currents at a holding potential of -30 mV. 2. Excitatory postsynaptic currents (EPSCs) were evoked by electrical stimulation either in the dorsal horn or the lateral funiculus. They reversed at 1.2 +/- 4.6 (SD) mV and could in most cases (49 of 51) be separated into two components. 3. In the presence of DL-2-amino-5-phosphonovalerate (10-40 microM) the current-voltage (I-V) relationship of the remaining EPSC was linear. When stimulated in the lateral funiculus, its rise time (10-90%) and the time constant of the monoexponential decay were 1.6 +/- 1.0 and 5.5 +/- 2.7 ms, respectively. By contrast, when stimulated in the dorsal horn, this component had a rise time (10-90%) of 3.0 +/- 0.8 ms and a decay time constant of 13.7 +/- 7.6 ms. 4. We studied the NMDA receptor-mediated component of the EPSCs after superfusion of 6-cyano-7-nitroquinoxaline-2,3-dione (5 microM). The I-V relationship of this component had a region of negative slope conductance between -30 and -80 mV, which was abolished in Mg(2+)-free saline. The rise time (10-90%) ranged from 3.3 to 9.5 ms and the decay was biexponential. Both decay time constants increased with depolarization. Mg(2+)-free saline reduced this voltage sensitivity. 5. At a membrane potential of -80 mV and in 1 mM extracellular Mg2+, the NMDA receptor-mediated component represented 74.8 +/- 11.2% of the total charge carried by the EPSCs evoked by stimulation in the dorsal horn. In contrast, when stimulated from the lateral funiculus, 28.9 +/- 18.9% of the total charge carried during the EPSC was mediated by the NMDA receptor-mediated component. The contribution of the NMDA receptor-mediated component increased in both cases with depolarization. In addition, in 2 of 18 SPNs the EPSC evoked in the dorsal horn was exclusively carried by NMDA receptors. 6. We conclude that L-glutamate or a related substance mediates the fast excitatory input onto SPNs. Viscerosomatic and supraspinal inputs form synapses with different topographical locations on the SPN.(ABSTRACT TRUNCATED AT 400 WORDS)

Postnatal change of glycinergic IPSC decay in sympathetic preganglionic neurons.

The characteristics of glycinergic inhibitory postsynaptic currents (IPSCs) in sympathetic preganglionic neurons (SPNs) of neonatal rats were studied by whole-cell recordings in transverse spinal cord slices. In relation to postnatal age, the decay time constants of these currents decreased without a comparable effect on their rise time. This may result from alpha-subunit switching of the glycine receptor and/or increased glycine uptake during this period of postnatal life. The kinetics of glycinergic IPSCs were also temperature- and voltage-dependent. Whereas, compared with room temperature, rise and decay of the events were faster at more physiological temperature, only the decay increased upon depolarization. Visual identification of SPNs was confirmed by intracellular staining and comparison with retrogradely labeled SPNs.