Differential nitric oxide synthase activity, cofactor availability and cGMP accumulation in the central nervous system during anaesthesia.

We investigated the effects of anaesthesia on dynamic nitric oxide production, concentrations of tetrahydrobiopterin and the accumulation of cyclic GMP (cGMP) in the rat central nervous system (CNS). Rats were assigned to anaesthesia with halothane, isoflurane, pentobarbital, diazepam, ketamine or xenon (n=6 per group). After 30 min, [14C]L-arginine (i.v.) was given and, after a further 60 min of anaesthesia, rats were killed and exposed immediately to focused microwave radiation. After removal of the brain and spinal cord, nitric oxide production from radiolabelled arginine (and hence nitric oxide synthase activity during anaesthesia) was measured as [14C]L-citrulline by scintillation counting. cGMP was determined by enzyme immunoassay and tetrahydrobiopterin by fluorescence HPLC, in brain regions and the spinal cord. Nitric oxide synthase activity was similar in all brain regions but was lower in the spinal cord, and was unaffected by anaesthesia. cGMP was similar in all areas of the CNS and was significantly decreased in rats anaesthetized with halothane. Isoflurane produced similar effects. In contrast, ketamine and xenon anaesthesia increased cGMP in the spinal cord, brainstem and hippocampus. Diazepam and pentobarbital had no effect. Tetrahydrobiopterin concentrations were similar in all areas of the CNS and were increased in the cortex and hippocampus after anaesthesia. We have shown profound differential effects of anaesthesia on the nitric oxide pathway in the rat CNS.

Electrophysiological properties of electrical synapses between rat sympathetic preganglionic neurones in vitro.

1. The electrophysiological properties of electrical synaptic transmission between sympathetic preganglionic neurones (SPNs) in slices of rat spinal cord were investigated using simultaneous dual-electrode patch-clamp recordings. Electrotonic coupling was directly demonstrated between 21 pairs of SPNs. 2. Coupling coefficients determined from the steady-state response of both neurones to current steps injected into either neurone ranged from 0. 02 to 0.48 (0.18 +/- 0.02, mean +/- s.e.m.). Synapses were bidirectional and symmetrical for the majority of connections with coupling coefficients similar in either direction. Asymmetrical coupling between a minority of cell pairs was due to differences in passive neuronal properties rather than rectification of the synaptic conductances. 3. Action potentials were manifest in adjoining cells as biphasic electrical postsynaptic potentials (ePSPs), composed of a rapid depolarising component followed by a more prolonged hyperpolarisation with amplitudes of 1.2 +/- 0.2 and 2.1 +/- 0.6 mV, respectively. 4. Postsynaptic potentials resembled low-pass filtered presynaptic spikes with frequency dependence determined by the junctional conductance and postsynaptic membrane properties. Increases in presynaptic action potential frequency caused attenuation of the hyperpolarising component of the ePSP that was attributed to shorter duration presynaptic spikes being more markedly filtered. 5. Synchronisation of spontaneous action potentials between electrotonically coupled neurones was driven by subthreshold membrane potential activity resembling repetitive ePSPs. Synchronous spike firing in previously silent neurones could be driven by suprathreshold ePSPs induced by suprathreshold depolarisation of a single adjoining neurone. 6. These data characterise reliable communication of sub- and suprathreshold activity by electrical synapses enabling synchronised SPN firing which may contribute to generation of coherent sympathetic rhythms and promote summation of inputs to postganglionic neurones.

Metabotropic glutamate receptor-mediated excitation and inhibition of sympathetic preganglionic neurones.

The effects of metabotropic glutamate receptor (mGluR) subtype selective compounds on the excitability of sympathetic preganglionic neurones (SPNs) were investigated. Non-selective mGluR agonists (1S,3R)-aminocyclopentane-1,3-dicarboxylic acid and (2S,1'S,2'S)-2-(carboxycyclopropyl)glycine, induced dose-dependent depolarisations in 96 and 75% of SPNs, respectively and hyperpolarisations in 2 and 21% of SPNs. Both agonists could induce subthreshold membrane potential oscillations in previously non-oscillating SPNs and either increased or reduced the frequency of spontaneously occurring oscillations. A selective group I mGluR agonist, 3,5-dihydroxyphenylglycine, depolarised all SPNs tested, induced oscillations in membrane potential of otherwise non-oscillating SPNs and increased the frequency of spontaneous oscillations. Agonists with selectivity for group II mGluRs (1S,3S)-aminocyclopentane-1,3-dicarboxylic acid and (S)-4-carboxy-3-hydroxy-phenylglycine ((S)-4C3HPG) did not induce depolarising responses. However (S)-4C3HPG induced hyperpolarising responses associated with a reduction in the frequency of spontaneous oscillations in two of six SPNs tested. Depolarising and hyperpolarising responses were maintained in the presence of tetrodotoxin indicating a direct action of the agonists upon SPNs. In individual SPNs responses of opposite polarity could be induced from the same initial membrane potential using different agonists, indicating that the opposing responses involved different ionic mechanisms. The broad spectrum mGluR antagonist (S)-alpha-methyl-4-carboxyphenylglycine and the selective group I mGluR antagonist (S)-4-carboxyphenylglycine reversibly depressed mGluR agonist induced depolarisations. These results indicate that SPNs express two mGluR populations with opposing actions on neuronal excitability: group I mGluRs depolarise SPNs and can drive oscillatory membrane potential activity; a minority of SPNs express group II mGluRs which mediate membrane hyperpolarisations and reduce the frequency of membrane potential oscillations.

Bilaterally evoked monosynaptic EPSPs, NMDA receptors and potentiation in rat sympathetic preganglionic neurones in vitro.

1. Whole-cell patch clamp and intracellular recordings were obtained from 190 sympathetic preganglionic neurones (SPNs) in spinal cord slices of neonatal rats. Fifty-two of these SPNs were identified histologically as innervating the superior cervical ganglion (SCG) by the presence of Lucifer Yellow introduced from the patch pipette and the appearance of retrograde labelling following the injection of rhodamine-dextran-lysine into the SCG. 2. Electrical stimulation of the ipsilateral (n = 71) or contralateral (n = 32) lateral funiculi (iLF and cLF, respectively), contralateral intermediolateral nucleus (cIML, n = 41) or ipsilateral dorsal horn (DH, n = 34) evoked EPSPs or EPSCs that showed a constant latency and rise time, graded response to increased stimulus intensity, and no failures, suggesting a monosynaptic origin. 3. In all neurones tested (n = 60), fast rising and decaying components of EPSPs or EPSCs evoked from the iLF, cLF, cIML and DH in response to low-frequency stimulation (0.03-0.1 Hz) were sensitive to non-NMDA receptor antagonists. 4. In approximately 50 % of neurones tested (n = 29 of 60), EPSPs and EPSCs evoked from the iLF, cLF, cIML and DH during low-frequency stimulation were reduced by NMDA receptor antagonists. In the remaining neurones, an NMDA receptor antagonist-sensitive EPSP or EPSC was revealed only in magnesium-free bathing medium, or following high-frequency stimulation. 5. EPSPs evoked by stimulation of the iLF exhibited a sustained potentiation of the peak amplitude (25.3 +/- 11.4 %) in six of fourteen SPNs tested following a brief high-frequency stimulus (10-20 Hz, 0.1-2 s). 6. These results indicate that SPNs, including SPNs innervating the SCG, receive monosynaptic connections from both sides of the spinal cord. The neurotransmitter mediating transmission in some of the pathways activated by stimulation of iLF, cLF, cIML and DH is glutamate acting via both NMDA and non-NMDA receptors. Synaptic plasticity is a feature of glutamatergic transmission in some SPNs where EPSPs are potentiated following a brief high-frequency stimulus. Our data also suggest a differential expression of NMDA receptors by these neurones.

Leptin inhibits hypothalamic neurons by activation of ATP-sensitive potassium channels.

Leptin, the protein encoded by the obese (ob) gene, is secreted from adipose tissue and is thought to act in the central nervous system to regulate food intake and body weight. It has been proposed that leptin acts in the hypothalamus, the main control centre for satiety and energy expenditure. Mutations in leptin or the receptor isoform (Ob-R[L]) present in hypothalamic neurons result in profound obesity and symptoms of non-insulin-dependent diabetes. Here we show that leptin hyperpolarizes glucose-receptive hypothalamic neurons of lean Sprague-Dawley and Zucker rats, but is ineffective on neurons of obese Zucker (fa/fa) rats. This hyperpolarization is due to the activation of a potassium current, and is not easily recovered on removal of leptin, but is reversed by applying the sulphonylurea, tolbutamide. Single-channel recordings demonstrate that leptin activates an ATP-sensitive potassium (K[ATP]) channel. Our data indicate that the K(ATP) channel may function as the molecular end-point of the pathway following leptin activation of the Ob-R(L) receptor in hypothalamic neurons.

Actions of the anaesthetic Saffan on rat sympathetic preganglionic neurones in vitro.

1. Whole-cell patch-clamp recordings were used to investigate the effects of the anaesthetic Saffan on the electrophysiological properties of sympathetic preganglionic neurones (SPNs) in rat spinal cord slices. 2. Saffan (1-54 microM) abolished or reduced the frequency of spontaneous action potential firing and abolished spontaneous, sub-threshold membrane potential oscillations. Saffan caused dose-dependent decreases in input resistance and depending upon the initial resting membrane potential, either a depolarization, a hyperpolarization or no change in membrane potential. 3. Responses to Saffan were blocked by the GABAA receptor antagonists bicuculline (5-20 microM) and picrotoxin (20 microM), but not by the glycine receptor antagonist strychnine (20 microM) indicating that they were mediated by GABAA receptors. 4. Changes in the properties of SPN action potentials were also observed. In the presence of Saffan the amplitude and duration of the action potential after-hyperpolarization were reduced and larger depolarizations were required in order to evoke trains of action potentials. 5. To examine the effects of Saffan on electrotonic coupling between SPNs, experiments were performed with the Na+ channel blocker QX-314 in the intracellular solution and antidromic oscillations were evoked by ventral root stimulation. Saffan failed to abolish antidromic oscillations, but reduced their amplitude and duration. This indicates that the abolition of spontaneous membrane potential oscillations was not a direct effect on the coupling between SPNs, but was a result of the abolition of spontaneous activity by Saffan. 6. The responses to Saffan occurred within the plasma concentration range of Saffan during anaesthesia, suggesting that the electrophysiological properties of SPNs may be altered during anaesthesia with Saffan. This would be expected to lead to changes in sympathetic tone and in the integration of sympathetic output.

Electrotonic coupling between rat sympathetic preganglionic neurones in vitro.

1. Using the whole-cell recording technique in rat spinal cord slices we have shown that 26% of sympathetic preganglionic neurones (SPNs) show spontaneous membrane potential oscillations. These oscillations consist of trains of biphasic waves, which we have termed spikelets because of their similarity to truncated action potentials. 2. The spikelets were inhibited by TTX and anaesthetics such as alpha-chloralose but not by the intracellular application of lidocaine N-ethyl bromide (QX-314). 3. By stimulating the ventral roots we have demonstrated the presence of short-latency depolarizations (SLDs) in oscillating neurones. These SLDs have a similar waveform to the spontaneous spikelets, and also show the ability to override the frequency of occurrence of the spontaneous spikelets. These observations suggest that the spikelets result from electrotonic coupling between the oscillating SPNs. 4. SLDs were also observed in a population of non-oscillating, electrotonically coupled, quiescent SPNs. It was possible to induce oscillations in these neurones by the injection of depolarizing current (in the presence of QX-314), suggesting that these neurones are also gap-junction coupled. 5. Simultaneous whole-cell recordings were obtained from twenty-three pairs of SPNs. Two pairs displayed both spontaneous, synchronized oscillations and action potentials. Electrotonic coupling was confirmed by the detection of membrane polarization in both neurones in response to current injected into one neurone. In a further two pairs of quiescent SPNs, injection of depolarizing current pulses into one neurone induced action potential discharge in that neurone and a depolarization and oscillations in the other neurone. 6. The ability of groups of electrotonically coupled SPNs to generate spontaneous discharges within the spinal cord provides a novel mechanism for the integration and synchronization of information within the sympathetic nervous system.

Excitation of rat sympathetic preganglionic neurones by selective activation of the NK1 receptor.

Whole-cell current clamp recordings were made from 32 identified sympathetic preganglionic neurones (SPN) in neonatal rat thoracolumbar spinal cord slices. Perfusion of substance P (SP; 0.1-10 microM) depolarised 7 out of 15 neurones tested and the selective NK1 receptor agonist GR73632 (0.075-5 microM) depolarised 14 out of 24 neurones. The response to GR73632 could be maintained in the presence of tetrodotoxin (0.25-0.5 microM; n = 3) and was irreversibly reduced or abolished by the selective NK1-receptor antagonist CP-99,994 (1-3 microM; n = 3). In 3 neurones, GR73632 evoked both a depolarising response and inhibitory postsynaptic potentials (IPSPs) and in the one neurone tested, the IPSPs were blocked by strychnine (20 microM), suggesting that NK1 receptors may also be found upon inhibitory glycinergic interneurones. The application of either GR73632 or SP to a subpopulation of previously quiescent SPN evoked rhythmical oscillations in membrane potential (n = 6). These results indicate that the NK1 receptor may play an important role in the regulation of both SPN and of inhibitory interneurones presynaptic to SPN.

Excitation of sympathetic preganglionic neurons via metabotropic excitatory amino acid receptors.

The role of excitatory amino acid metabotropic receptors in the regulation of excitability of sympathetic preganglionic neurons was investigated. This study used both conventional intracellular and whole-cell patch clamp techniques to record from sympathetic preganglionic neurons in transverse spinal cord slices of the rat (9-21 days old). The metabotropic receptor agonists (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) (10-200 microM, superfused for 2-60 s) and quisqualate (1-50 microM, superfused for 2-60 s) induced concentration-dependent depolarizing responses which did not desensitize. These responses were unaffected by the glutamate ionotropic receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX, 10-50 microM), 6,7-dinitroquinoxaline-2,3-dione (DNQX, 10 microM), dizocilpine (MK-801, 10-40 microM), 3-[(R)-2-carboxy-piperazin-4-yl]-propyl-1-phosphonic acid (D-CPP, 10-50 microM) and DL-2-amino-5-phosphonovaleric acid (DL-AP5, 20-100 microM). Depolarizing responses to 1S,3R-ACPD and quisqualate were unaffected by L-2-amino-3-phosphonopropionic acid (L-AP3, 30 microM-1mM) and L-2-amino-4-phosphonobutanoic acid (L-AP4, 100 microM-1 mM)). The responses to 1S,3R-ACPD and quisqualate were reduced by including the G-protein blocker GDP-beta-S (400 microM) in the patch pipette solution by 77 +/- 2% (mean +/- S.E) of control (n = 3), suggesting that these agonists activate a G-protein-coupled receptor. Metabotropic receptor-mediated responses were maintained in the presence of tetrodotoxin (500 nM), progressively reduced with increased membrane hyperpolarization to around -95 mV and associated with either an increase of 16.5 +/- 2.8% (data from four neurons) in the majority of neurons (n = 22 of 34) or no measurable change (n = 12) in neuronal input resistance. These data suggest that the agonists exert a direct action on 1S,3R-ACPD and quisqualate had several effects on sympathetic preganglionic neuron membrane properties including: inhibition of a slow apamin-insensitive component of the afterhyperpolarization; a reduction in spike frequency adaptation leading to increases in firing frequency from 6.4 +/- 2.8 Hz in control experiments up to 14.7 +/- 3.0 Hz (n = 6 neurons) in the presence of a metabotropic receptor agonist: a broadening of the action potential by 37.5 +/- 6.4% (n = 6 neurons) of control. These observations suggest that the metabotropic receptor-mediated depolarization is due, at least in part, to the reduction of potassium conductances involved in the spike afterhyperpolarisation potential.(ABSTRACT TRUNCATED AT 400 WORDS)

Effects of muscarinic receptor stimulation of sympathetic preganglionic neurones of neonatal rat spinal cord in vitro.

Whole cell current-clamp recordings were made from 85 sympathetic preganglionic neurones (SPN) of the neonatal spinal cord in vitro. Superfusion of up to 500 microM acetylcholine (2-30 sec) gave weak responses. Carbachol (CChol; 5-50 microM) superfused for 1-20 sec, hyperpolarized SPN. This response was associated with a mean reduction in input resistance of 22%. Ion substitution studies suggested that potassium is the likely carrier of at least part of the current underlying the CChol-induced hyperpolarization. Some SPN display spontaneous rhythmic oscillations in their membrane potentials which may be due to action potential discharge in electrically-coupled neurones. CChol also acts to inhibit these oscillations concomitant with the hyperpolarization. Responses to CChol were unaffected by addition of 500 nM TTX to the bathing medium suggesting that CChol acts directly upon SPN. Carbachol-induced hyperpolarizing responses were totally abolished by the non-specific muscarinic receptor antagonist atropine (20-50 microM). Pirenzepine, at concentrations over 5 microM reversibly reduced the responses to CChol. Gallamine, an M2 receptor antagonist applied at 25 microM also reversibly abolished the CChol responses. These results suggest that CChol-mediated hyperpolarizations may be due to M2 receptor activation.

5-Hydoxytryptamine evokes depolarizations and membrane potential oscillations in rat sympathetic preganglionic neurones.

1. Whole-cell recordings were made from seventy-seven identified rat sympathetic preganglionic neurones (SPN) in spinal cord slices. Perfusion of 5-HT (0.5-30 microM) strongly depolarized 90% of neurones. The response was slow in onset, could last over 10 min and was associated with an increase in input resistance. 5-HT could also evoke rhythmical membrane potential oscillations in a population of previously quiescent neurones. 2. The 5-HT response persisted in TTX and also in low-Ca(2+)-high-Mg2+ artificial cerebrospinal fluid (ACSF), suggesting that the receptors are on SPN. The 5-HT uptake inhibitor 6-nitroquipazine potentiated the 5-HT-induced depolarization. 3. The 5-HT-induced depolarization was reduced and then abolished by membrane hyperpolarization to potentials of about -100 mV, but was not reversed in sign by further hyperpolarization. In voltage clamp, 5-HT evoked inward currents associated with the reduction of an outwardly rectifying potassium conductance. 4. The 5-HT2 receptor agonist alpha-methyl-5-HT mimicked the 5-HT response on all neurones, as did the 5-HT1 receptor agonist 5-carboxamidotryptamine (5-CT) on 71% of SPN. The responses to 5-HT, alpha-methyl-5-HT and 5-CT were inhibited by the 5-HT2 antagonists ketanserin and ritanserin. 5. Pressure ejection of 5-HT over the central canal region could evoke a biphasic inhibitory-excitatory response. This response persisted in TTX, suggesting that an inhibitory 5-HT receptor may be located on the medial dendrites. 6. SPN are powerfully depolarized by 5-HT acting at 5-HT2 receptors, via the closure of an outwardly rectifying potassium conductance. The long duration of the response and the ability of 5-HT to induce rhythmical oscillations suggest that 5-HT may have an important role in regulating SPN excitability.

Inhibition of sympathetic preganglionic neurons by spinal glycinergic interneurons.

Intracellular and whole-cell patch-clamp recordings were obtained from sympathetic preganglionic neurons in rat spinal cord slices. Perfusion of selective ionotropic and metabotropic excitatory amino acid agonists induced depolarizing responses in all neurons. In approximately 20% of neurons the application of these agonists also evoked inhibitory postsynaptic potentials. The application of the ionotropic receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (5-40 microM) blocked the inhibitory postsynaptic potential discharges induced by (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (0.5-50 microM) and N-methyl-D-aspartate (0.5-50 microM), but failed to block the inhibitory postsynaptic potentials induced by quisqualate (0.5-50 microM) and (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (10-200 microM). Similar inhibitory postsynaptic potentials were seen to occur spontaneously or could be evoked by electrical stimulation of the dorsal horn. The application of tetrodotoxin blocked the spontaneous and evoked inhibitory postsynaptic potential, indicating that they result from activity-dependent release of neurotransmitter. Strychnine antagonized all inhibitory postsynaptic potentials suggesting that they were mediated via glycine receptors. The reversal potential of the inhibitory postsynaptic potentials was -65 mV for intracellular and -55 mV for whole-cell recordings. This latter value is close to the reversal potential for chloride, suggesting that the inhibitory postsynaptic potentials were mediated by a chloride conductance. Perfusion of glycine (0.1-1 mM) induced inhibitory hyperpolarizing responses in the majority of neurons. This hyperpolarizing response was associated with a reduction in neuronal input resistance, persisted in the presence of tetrodotoxin, was blocked by strychnine and reversed at -55 mV. In some neurons, glycine induced a membrane depolarization and increased the rate of spontaneous action potential firing. This excitatory effect of glycine was blocked by tetrodotoxin, showed voltage dependency and was less sensitive to strychnine than the glycine-induced inhibitory response. We conclude from these data that spinal interneurons which synapse with sympathetic preganglionic neurons can be activated through multiple subtypes of excitatory amino acid receptor, including both ionotropic and metabotropic receptors. These interneurons release glycine to evoke inhibitory postsynaptic potentials which are mediated via a strychnine-sensitive glycine receptor coupled to a chloride conductance.

The actions of phorbol esters upon isolated calcium currents of Helix aspersa neurones.

1. Voltage-clamp recordings have been made from identified neurones in the suboesophageal ganglia of Helix aspersa. 2. Calcium currents were isolated pharmacologically and studied under two-electrode voltage-clamp. 3. PdBu but not PMA caused a transient enhancement followed by an irreversible inhibition of the calcium current. 4. It is concluded that activation of protein kinase C within these neurones may show selectivity with respect to the exogenous activator used.

Whole-cell recordings from sympathetic preganglionic neurons in rat spinal cord slices.

Whole-cell patch-clamp recordings (WCR) were made from sympathetic preganglionic neurons (SPN) in neonate rat spinal cord slices. SPN were identified histologically by filling them with the fluorescent dye Lucifer Yellow contained within the patch pipette solution. Current clamp recordings were obtained from SPN with a potassium based pipette solution. The cells exhibited many of the characteristic properties of SPN seen previously with intracellular recordings in both the rat and the cat. However, we found an order of magnitude increase in both cell input resistance (950 M omega) and time constant (118 ms) over those seen with conventional recordings. We believe these values approximate better the situation in intact cells, and will have a vital bearing upon how SPN integrate inputs. We conclude that WCR in spinal cord slices provides a powerful tool for investigating the cellular properties of SPN.

Studies on the mechanisms of signalling and inhibition by pertussis toxin of fibroblast growth factor-stimulated mitogenesis in Balb/c 3T3 cells.

Basic fibroblast growth factor (bFGF) stimulates mitogenesis of Balb/c 3T3 fibroblast cells. This stimulation may be mediated by multiple signal pathways as it is accompanied by the formation of inositol phosphates, activation of PKC (protein kinase C) and a decrease in intracellular cAMP levels. The multiple positive and negative pathways implicated for FGF-induced mitogenesis may interact and each may contribute in varying degrees to the final cellular response. At least two types of G-proteins may be involved in the intracellular signalling pathways of FGF. Pertussis toxin blocks FGF and TPA (12-O-tetradecanoylphorbol-13-acetate) induced. PKC-mediated mitogenesis and also the associated fall in intracellular cAMP levels. However, pertussis toxin has no effect upon FGF-induced inositol phosphates formation. Thus, inhibition of mitogenesis by pertussis toxin may involve pertussis toxin sensitive G-proteins which may affect at least two separate putative signal pathways involving adenylate cyclase and protein kinase C. Pertussis toxin insensitive G-proteins may also be involved in coupling the FGF receptor to phosphoinositidase C.

Sympathetic preganglionic neurones in neonatal rat spinal cord in vitro: electrophysiological characteristics and the effects of selective excitatory amino acid receptor agonists.

Intracellular recordings were made from 52 lateral horn neurones in thin slices of neonatal rat thoracolumbar spinal cord. Of these neurones 12 were spontaneously active and the remainder silent. A number of these cells could be activated antidromically by stimulation of ventral roots. The conduction velocity of the antidromic potential was estimated to be 0.9-2 m/s which is within the range reported for axons of sympathetic preganglionic neurones (SPNs). The membrane properties of antidromically identified SPNs were similar to other lateral horn neurones included in this study and comparable to those reported for SPNs by others. Spontaneous burst firing was recorded in 3 neurones and activity in a further 5 neurones was characterized by the discharge of an action potential followed by an afterhyperpolarization potential (AHP) of peak amplitude 3-13 mV and duration 0.5-4 s. The AHP had an initial fast component (fAHP) which was sensitive to the potassium channel blocker tetraethylammonium (TEA), and a second slower component (sAHP) which was both sensitive to extracellular calcium and TEA. The effects of the selective excitatory amino acid receptor agonists N-methyl-D-aspartate (NMDA), kainate and quisqualate were investigated by superfusion of the agonists, at known concentrations (100 nM to 100 microM). These agonists induced concentration-dependent depolarizations which were primarily associated with a reduction in neuronal input resistance. NMDA-induced depolarizations were potentiated in the absence of magnesium.(ABSTRACT TRUNCATED AT 250 WORDS)

Spontaneous rhythmic activity in the intermediolateral cell nucleus of the neonate rat thoracolumbar spinal cord in vitro.

Intracellular recordings from the intermediolateral cell nucleus of the neonate rat thoracolumbar spinal cord slice preparation revealed a population of neurons which displayed three types of spontaneous rhythmic activity: burst firing, tonic beating and membrane oscillations. Most neurons displayed more than one of these types of activity. Neurons had mean resting potentials of -59 mV and input resistances ranging from 10 to 48 m omega. Spontaneous oscillations which were observed either independently or following hyperpolarization of neurons displaying tonic beating or bursting behaviour had a mean peak amplitude and frequency of approximately 14 mV and 1 Hz respectively. Oscillations were not obviously reversible as they were still apparent at potentials as negative as -120 to -140 mV. This suggests that the oscillations had a site of generation distant to the recording electrode. Neurons displaying tonic beating activity were characterized by low frequency firing activated at the peak of the depolarizing phase of the underlying oscillation and these neurons could be induced to exhibit burst behaviour by membrane depolarization. The frequency of firing in tonic beating neurons ranged from 0.1 to 8.8 Hz. Burst firing was characterized by: bursts of 3-17 action potentials; burst cycle frequency of approximately 1 Hz; an afterdepolarization potential mainly observed at the termination of a burst. Burst firing was abolished by cobalt and membrane hyperpolarization but not by barium, low calcium or tetraethylammonium chloride. The switch from tonic beating to burst firing may, in part, involve activation of a voltage- and calcium-dependent afterdepolarization potential. We conclude that a population of neurons in the lateral horn of the spinal cord are capable of rhythmic activity with underlying spontaneous pacemaker-like oscillations.

Production of inositol phosphates in BALB/C 3T3 cells stimulated with basic fibroblast growth factor.

When quiescent 3T3 fibroblast cells were pre-labelled with [3H]inositol and stimulated with basic fibroblast growth factor there was a stimulation of the hydrolysis of membrane lipids and the rapid production of [3H]inositol polyphosphates. Rapid and transient peaks of isomers of inositol phosphates with the chromatographic properties of inositol trisphosphates and inositol tetrakisphosphates were detectable by anion-exchange HPLC between 5 and 10 s after stimulation. These data suggest that upon stimulation the receptor for fibroblast growth factor is coupled to a phosphoinositidase C and that one of its signal-transducing pathways involves hydrolysis of inositol lipids and the production of inositol polyphosphates, some of which may act as intracellular signals mediating the cellular response. Chronic stimulation with basic fibroblast growth factor is associated with desensitization of the inositol lipid signaling pathway.

Inhibition of agonist-stimulated inositol lipid metabolism by the anticonvulsant carbamazepine in rat hippocampus.

1. The effect of the anticonvulsant, anti-manic drug carbamazepine was examined on inositol lipid signalling in rat hippocampus in vitro. 2. Hippocampal miniprisms were labelled with [3H]-inositol before stimulation with a variety of neuroactive agents that increase phosphoinositide turnover. 3. The presence of carbamazepine (0.1-100 microM) during labelling caused a dose-related reduction of basal and carbachol-evoked [3H]-inositol phosphate accumulations. The effect of the drug on basal inositol phosphate levels was lost when slices were labelled with [3H]-inositol before incubation with carbamazepine. 4. Incubation of slices with carbamazepine after labelling with [3H]-inositol and before stimulation showed the inhibitory effect of the drug to be selective according to the agonist used. Responses to carbachol, histamine and the sodium-channel agent veratrin were reduced by carbamazepine whilst the responses to 5-hydroxytryptamine, noradrenaline and substance P were unaffected. 5. Inhibition of carbachol, histamine and veratrin-induced stimulation by carbamazepine share a similar dependence on length of pre-incubation time with the drug. However, the effect of carbamazepine (100 microM) on the respective dose-response curves suggests that the mechanism of inhibition of the carbachol response differs from the inhibition of the histamine and veratrin responses. These effects may be significant in the mechanism of action of carbamazepine as an anticonvulsant and in its effectiveness against manic depression.