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1. Whole cell voltage-clamp recordings were made from dorsomedial nucleus tractus solitarii (dmNTS) neurons in coronal rat medullary slices. Synaptic activity was evoked by electrical stimulation in the region of the tractus solitarius (TS). Excitatory postsynaptic current/inhibitory postsynaptic current (EPSC/IPSC) complexes were recorded with K-gluconate-containing electrodes. Monosynaptic, gamma-aminobutyric acid-A (GABAA) receptor-mediated evoked IPSCs (evIPSCs) were recorded in the presence of D(-)2-amino-5-phosphonopentanoic acid (AP5, 50 microM) and 6,7-dinitroquinoxaline-2,3-dione (DNQX, 10 microM) with CsCl-containing electrodes. Control synaptic stimulation was applied at 0.1 Hz. 2. Tetanic stimulation (50 Hz, 2 s) produced an increase in the 10-90% rise time of the evIPSC component of mixed EPSC/IPSC complexes (assessed at Vhold = 0 mV) in three of eight recordings that remained significantly potentiated above control for > 15 min. This potentiation was further characterized with the use of Cs-containing electrodes. Tetanus similarly potentiated the amplitude of monosynaptic evIPSCs by 168.0 +/- 10.4% (mean +/- SE) control at 15 min posttetanus in 49 of 114 recordings. This tetanus-induced sustained potentiation of monosynaptic evIPSCs (TIP) was reproducible after recovery to control levels. 3. High Mg2+/low Ca2+ solutions reversibly blocked induction of TIP. TIP was reproducible in slices treated (> 7 min) with the N-type voltage-dependent Ca2+ channel (VDCC) antagonist omega-conotoxin-GVIA (1 microM) or L-type channel blocker nimodipine (10 microM), but not in those slices treated with either omega-agatoxin-IVA (200 nM, 20 min) or omega-conotoxin-MVIIC (2 microM). 4. The GABAB receptor antagonist CGP35348 (100 microM) reversibly blocked induction of TIP, reduced resting, and blocked tetanus-induced increases in spontaneous IPSC frequency. Spontaneous IPSC amplitude was unaffected by CGP35348. 5. These results suggest a presynaptic locus for TIP in dmNTS, which depends in part on Ca2+ influx through P/Q-type VDCCs.

We have previously demonstrated that the metabotropic glutamate receptor (mGluR) agonist (1S,3R)-1 aminocyclopentane-1,3-dicarboxylate (ACPD) presynaptically inhibits evoked glutamatergic EPSCs and GABAergic IPSCs in patch clamped rat nucleus tractus solitarius (NTS) neurons recorded in this slices. The present study investigated the pharmacology of the presynaptic mGluRs, the the voltage dependent Ca2+ channel (VDCC) subtypes supporting neurotransmitter release, and possible interactions between the two. Monosynaptic EPSCs or IPSCs were evoked by electrical stimulation in the region of the tractus solitarius (TS). The effects of the mGluR agonists ACPD, (2S,3S,4S)-alpha-(carboxycyclopropyl)glycine (L-CCG-I) and L-2-amino-4-phosphonobutyrate (AP4) were examined upon EPSCs. The effects of the above compounds and quisqualate (QUIS) were examined upon IPSCs. L-CCG-I proved the most potent inhibitor of EPSCs and IPSCs. The VDCC blockers omega-AGA-IVA (AGA), omega-conotoxin GVIA (GVIA), omega-conotoxin MVIIC (MVIIC) and nimodipine (NIM) were assessed for their ability to inhibit monosynaptic EPSCs and IPSCs. EPSCs were inhibited by GVIAAGAor = MVIIC. IPSCs were inhibited by AGAor = MVIICGVIA. NIM was without effect on the EPSC or IPSC. The potency of mGluR inhibition of evoked synaptic transmission was assessed in the absence and following treatment with VDCC blockers. mGluR agonists blocked a greater percentage of the EPSC or IPSC following treatment with GVIA, but not the other VDCC antagonists, than under control conditions. We have previously demonstrated that the postsynaptic inhibitory effects of mGluR activation upon GABAA mediated currents can be mimicked by cyclic guanosine monophosphate (cGMP) analogs. The cGMP-dependent protein kinase (PKG) inhibitors H8 and Rp-8-4-chlorophenylthio-guanosine-3',5'-cyclic monophosphorothioate (Rp-cG) blocked mGluR inhibition of GABAA mediated currents without blocking the ability of mGluR agonists to inhibit the IPSC. The effect of L-CCGI was enhanced following treatment with GVIA in the presence of Rp-cG, confirming a presynaptic locus of mGluR mediated inhibition of the IPSC. In contrast, cGMP analogues potentiate postsynaptic responses to glutamate agonists but depress the EPSC. As with the mGluR agonists, the inhibition of the EPSC by cGMP was potentiated following treatment with GVIA. These results suggest that presynaptic mGluR reduce both glutamate release from afferent fibers and GABA release from inhibitory interneurons following electrical stimulation in the region of the TS. Although different VDCCs support the majority of glutamate and GABA release and mGluR effects on release appear to utilize differing intracellular pathways, presynaptic GVIA-insensitive VDCCs are favorably targeted for inhibition by mGluR agonists.

Whole-cell recordings were made in the nucleus tractus solitarii (NTS) in transverse brainstem slices from rats. Monosynaptic GABAA-receptor-mediated inhibitory postsynaptic currents (IPSCs) or potentials (IPSPs) were evoked (0.1-0.2 Hz) by electrical stimulation within and medial to the tractus solitarius in the presence of the ionotropic glutamate receptor antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10 microM) or 6,7-dinitroquinoxaline-2,3-dione (DNQX; 10 microM) and D-amino-5-phosphonopentanoic acid (APV; 50 microM). A brief period of tetanic stimulation (20 Hz, 2 s) resulted in posttetanic (5 min, 69 of 73 recordings) and sustained potentiation (15 min, 31 of 73 recordings) of the IPSP/Cs. Sustained potentiation was not due to alterations in the reversal potential of IPSP/Cs. Both pre- and post-tetanus IPSP/Cs were completely blocked by the GABAA antagonist bicuculline (10 microM). Postsynaptic responses to pressure ejection of the GABAA-receptor agonist muscimol were unaltered in cells displaying sustained potentiation. Sustained potentiation of IPSP/Cs could be induced by tetanus in the presence of either metabotropic glutamate receptor antagonists or bicuculline. However, sustained potentiation could not be induced in the presence of the GABAB-receptor antagonists 2-OH-saclofen (400 microM) or CGP35348 (3-amino-propyl-(diethoxymethyl)phosphinic acid, 100 microM), although a subsequent tetanus following washout induced sustained potentiation. Posttetanic potentiation was unaffected by GABAB-receptor antagonists. These data suggest that neuronal or terminal excitability of GABAergic interneurons in the NTS is enhanced following brief periods of increased frequency of activation in vitro. This novel phenomenon within the rat medulla may be involved in the temporal modulation of autonomic reflex sensitivity observed during certain behavioral states, such as the defense reaction.

This study examined the electrophysiological consequences of selective activation of delta 1-, delta 2-, or mu-opioid receptors using whole- cell recordings made from visually identified lamina II neurons in thin transverse slices of young adult rat lumbar spinal cord. Excitatory postsynaptic currents (EPSCs) or potentials (EPSPs) were evoked electrically at the ipsilateral dorsal root entry zone after blocking inhibitory inputs with bicuculline and strychnine, and NMDA receptors with D-2-amino-5-phosphonopentanoic acid. Bath application of the mu receptor agonist [D-Ala2, N-MePhe4, Gly5-ol]enkephalin (DAMGO) or the delta 1 receptor agonist [D-Pen2, D-Pen5]enkephalin (DPDPE) produced a long-linear, concentration-dependent reduction in the amplitude of the evoked EPSP/EPSC. By comparison, the delta 2 receptor agonist [D- Ala2,Glu4]deltorphin (DELT) was unable to reduce the evoked EPSP/EPSC by more than 50% at 100 microM, the highest concentration tested. At concentrations that reduced evoked EPSP/EPSCs by 40–60%, neither DAMGO, DPDPE, nor DELT decreased the amplitude of the postsynaptic current produced by brief pressure ejection of (S)-alpha-amino-3-hydroxy-5- methyl-4-isoxazole-propionic acid, suggesting a presynaptic site of action of these opioid receptor agonists. Bath application of 200 nM naltriben (NTB), a delta 2 receptor antagonist, competitively increased the EC75 of DELT by 15.3-fold, but did not antagonize either DPDPE or DAMGO. The EC75 of DELT was further increased by 169.7-fold in the presence of 1 microM NTB. However, this high concentration of NTB also increased the EC50 of DPDPE by about threefold in a noncompetitive manner and antagonized DAMGO in a noncompetitive manner.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of the metabotropic glutamate receptor (mGluR) agonist (1S,3R)-1-aminocy-clopentane-1,3-dicarboxylic acid [(1S,3R)-ACPD] and a series of phenylglycine-derived putative mGluR antagonists were examined on electrophysiological responses mediated by glutamate and GABA receptors in the nucleus of the tractus solitarius (NTS) in transverse brainsten slices of the rat. Monosynaptic excitatory currents (EPSC's) evoked by electrical stimulation in the region of the tractus solitarius (TS) were reduced in the presence of (1S,3R)-ACPD in > 90% of neurons recorded in the dorsomedial subdivision of the NTS adjacent to the area postrema (AP). Monosynaptic evoked inhibitory currents (IPSC's) were similarly inhibited by (1S,3R)-ACPD. The inward current evoked by pressure application of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid ( I AMPS ) was potentiated in the presence of (1S,3R)-ACPD, whereas the outward current evoked by the γ-amino-butyric acid-A (GABA-A) receptor agonist muscimol ( I MUSC ) was inhibited. (1S,3R)-APCD also produced a postsynaptic inward current ( I K(ACPD) ) associated with a decrease in membrane conductance in approximately 50% of cells. The novel mGluR antagonists (S)-4-carboxy-3-hydroxy-phenylglycine (4C3H-PG), (R,S)-4-carboxy-phenylglycine (4C-PG) and (R,S)-α-methyl-4-carboxy-phenylglycine (αM4C-PG) reversibly antagonized the effects of (1S,3R)-ACPD on EPSC's IPSC's, I AMPA and I MUSC . The first two compounds also displayed weak agonist activity. However, none of the antagonists significantly inhibited I K(ACPD) concentrations which blocked (1S,3R)-ACPD effects on synaptic transmission. These results suggest that pharmacologically distinct mGluR's may be present in the NTS.

1. We have previously demonstrated that the metabotropic glutamate receptor (mGluR) agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylate [(1S;3R)-ACPD] presynaptically inhibits evoked glutamatergic excitatory postsynaptic currents (EPSCs) in patch-clamped rat nucleus tractus solitarius (NTS) neurons recorded in thin slices. The present study investigated the ability of endogenously released glutamate to modulate EPSCs in the NTS. 2. A low-frequency tetanus of the tractus solitarius (TS) resulted in either posttetanic potentiation (PTP) (8 of 21 cells) or depression (13 of 21 cells) of monosynaptic EPSCs recorded in the presence of D(-)2-amino-5-phosphonopentanoic acid (AP5) and bicuculline. 3. The amplitude of the EPSC was not significantly affected by the bath application of the mGluR antagonist (+) alpha-methyl-4-carboxyphenylglycine (MCPG). 4. In the presence of MCPG, a low-frequency tetanus resulted in PTP of the EPSC in all neurons. PTP was significantly enhanced in those cells previously exhibiting PTP. 5. The results suggest that presynaptic mGluRs on TS projections to the NTS may be activated by endogenously released glutamate at physiologically relevant stimulus frequencies and therefore play a role in the modulation of autonomic afferent transmission.

Whole-cell patch clamp recordings were made from neurons in the rat nucleus tractus solitarius (NTS) in transverse brainstem slices. 5-Hydroxytryptamine (5-HT, 100 microM) and the selective 5-HT3 receptor agonist 2-methyl-5-HT (2-CH3-5-HT, 100 microM) depolarized 86% of NTS neurons at resting membrane potential (Vm). This response was resistant to tetrodotoxin (TTX) and Co2+ application. In addition, 2-CH3-5-HT (500 nM-100 microM) increased the amplitude and frequency of both excitatory and inhibitory spontaneous synaptic potentials. This effect was also TTX-resistant, but was abolished by Co2+. The effects of 2-CH3-5-HT on EPSPs and IPSPs evoked by electrical stimulation of the tractus solitarius (TS) were analyzed separately in the presence of bicuculline or 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), respectively. Concentrations of 2-CH3-5-HT between 500 nM and 1 microM decreased the amplitude of evoked EPSPs and IPSPs with similar potency. The selective 5-HT3 receptor antagonists ICS 205-930 (10 nM) and MDL 72222 (10 microM) reversibly blocked the effects of 2-CH3-5-HT at all doses examined. It is concluded that 5-HT3 receptors can mediate both pre- and postsynaptic responses in the NTS.

The release of pituitary GH appears to be critically dependent on alterations in the free intracellular Ca2+ concentration ([Ca2+]i). However, little is known about the nature of Ca2+ signalling within normal pituitary cells. We, therefore, examined [Ca2+]i patterns in individual cultured pituicytes of adult male rats under basal conditions and in response to GH regulatory agents, using the calcium-sensitive dye fura-2 together with digital imaging microscopy. Perfusion of cultured anterior pituitary cells with GH-releasing factor (GHRF) resulted in a marked increase in [Ca2+]i in specific pituitary cells. These cells did not respond to other hypothalamic secretagogues (GnRH, TRH, or CRF), and there was no evidence of desensitization on repetitive administration of GHRF. Somatotrophs (n = 134) exhibited spontaneous oscillations of [Ca2+]i in the basal state, with considerable heterogeneity of oscillatory patterns among cells. After application of a near-maximal stimulatory dose of GHRF (1 nM), there was a striking 2.2-fold increase in the amplitude of [Ca2+]i oscillations and only a modest increase in their frequency. Forskolin (1 microM) augmented somatotroph [Ca2+]i in patterns similar to those of GHRF. Somatostatin (10 nM) abolished the [Ca2+]i response to GHRF (n = 26); this reflected a marked reduction in the amplitude of [Ca2+]i oscillations and a slight reduction in their frequency. Ca(2+)-free medium or the Ca2+ channel antagonist nimodipine (0.1-1 microM) suppressed the Ca2+ stimulatory effect of GHRF. Conversely, the Ca2+ channel agonist BAY K8644 (1 microM) strikingly augmented the GHRF-induced rise in [Ca2+]i, with a major stimulatory effect on the amplitude of [Ca2+]i oscillations and no observed effect on their frequency. In summary, GHRF and other hypothalamic secretagogues increase [Ca2+]i in pituitary cells in a highly specific manner, consistent with the known specificity of their effects on hormone release. Somatotrophs exhibit spontaneous rhythmic oscillation of [Ca2+]i in the basal state. Known regulators of GH release markedly alter the [Ca2+]i oscillatory pattern in characteristic manners, exerting predominant effects on the amplitude of [Ca2+]i pulses and lesser effects on their frequency. These striking effects of GH regulatory agents on pituitary Ca2+ signalling are consistent with the concept that modulation of [Ca2+]i is a critical mediator of somatotroph function.

We carried out experiments designed to investigate the effects of sarafotoxin-6B (SFTx) on [Ca2+]i in cerebellar astrocytes using the Ca2+ indicator fura-2. Both endothelin-1 and sarafotoxin-6B increased [Ca2+]i in individual cerebellar astrocytes in cell culture. The shape of the response was variable but usually consisted of an initial peak of [Ca2+]i followed by an extended plateau increase in [Ca2+]i. In Ca(2+)-free medium only the initial peak was observed. If Ca2+ was subsequently readmitted to the external medium a plateau was now formed. When external Ca2+ was removed during a plateau, [Ca2+]i rapidly declined; replacing the external Ca2+ reversed this decline. The plateau was also reversibly reduced by addition of Ni2+ (5 mM) to the external medium. Addition of 50 mM K+ produced a small increase in [Ca2+]i in most cells. This response was blocked by nimodipine. However, nimodipine only slightly blocked the plateau increase in [Ca2+]i that was formed following activation of endothelin receptors. Furthermore, perfusion of cells with 50 mM K+ during the plateau portion of a response to SFTx reduced [Ca2+]i. In some cells addition of a phorbol ester produced a sustained increase in [Ca2+]i that was blocked by nimodipine. In conclusion, activation of endothelin receptors by SFTx in cerebellar astrocytes produces both Ca2+ mobilization and Ca2+ influx. The pathway for Ca2+ influx is predominantly a non-voltage-dependent one, although some entry through a dihydropyridine-sensitive pathway also appears to occur. Furthermore, activation of protein kinase C in cerebellar astrocytes activates voltage-sensitive Ca2+ channels.

1. Neuropeptide Y (NPY) and peptide YY (PYY) act at receptors referred to as Y1 and Y2, while the Y3 receptor is specific to NPY and does not recognize PYY. The effects of NPY, its related peptides and a series of newly constructed chimeric NPY-PYY peptides were examined on excitatory and inhibitory postsynaptic currents (e.p.s.cs and i.p.s.cs, respectively) in rat dorsomedial nucleus tractus solitarius (NTS) neurones recorded in coronal brainstem slices. Monosynaptic activity was evoked by electrical stimulation in the region of the tractus solitarius. 2. NPY (5-500 nM) inhibited e.p.s.cs and i.p.s.cs in a concentration-dependent manner. In contrast, PYY (500 nM) failed to affect either e.p.s.cs or i.p.s.cs. The N- and C-terminal parts of a series of chimeric NPY-PYY peptides were joined at positions where NPY and PYY sequences differ. In binding experiments the chimeric peptides were all about equipotent with NPY and PYY in displacing [125I]-PYY from Y1 and Y2 binding sites on SK-N-MC cells and rat hippocampus respectively. 3. In the whole cell voltage clamp recordings of NTS neurones, NPY(1-23)-PYY(24-36) and NPY(1-14)-PYY(15-36) evoked a concentration-dependent inhibition of e.p.s.cs and i.p.s.cs, while NPY(1-7)-PYY(8-36) and NPY(1-3)-PYY(4-36) were inactive. The only differences in amino acid residues between NPY(1-14)-PYY(15-36) and NPY(1-7)-PYY(8-36) reside in positions 13 and 14. 4. Furthermore, [Pro34]NPY (500 nM) was equivalent in potency to NPY itself at inhibiting monosynaptic transmission in NTS, while [Leu31,Pro34]NPY and pancreatic polypeptide (both at 500 nM) failed to affect synaptic transmission. 5. The present study has shown that NPY acts at Y3 receptors to suppress both excitatory and inhibitory currents in the NTS. The different efficacy of the chimeric NPY-PYY peptides suggests that positions 13 and 14 are of great importance for Y3 receptor recognition. Finally, this receptor type readily recognizes [Pro34]NPY, but not [Leu31,Pro34]NPY.

Publisher Summary This chapter describes the use of whole-cell patch-clamp recording methods in brain slices combined with the photometric method to monitor neurons dialyzed with fura-2 and imaging methods using fluo-3. The primary advantage of examining intracellular Ca 2+ concentration ([Ca 2+ ] i ) in brain slices by the photometric method is that it provides a rapid temporal means of resolving changes in [Ca 2+ ] i . The method maintains the advantages of the slice preparation, such as the avoidance of culture or dissociation artifacts, the preservation of a degree of endogenous connectivity, and control of the extracellular milieu. The photometric method provides a high temporal means of simultaneously monitoring the fluorescence of intracellular ion-sensitive dyes and the transmembrane voltage or current in neurons in slices. The key advantage of the method is speed. The chief disadvantage is the limited region of the cell that can be accurately examined and the absence of spatial resolution. Imaging methods are needed where the spatial resolution of the calcium signal is of interest, and speed can be compromised to some extent.

Two principal classes of glutamate receptors have been identified: (1) ligand-gated ion channels and (2) G-protein-coupled “metabotropic” receptors (Sugiyama et al., 1989). Activation of ionotropic α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA), kainate (KA), and N-methyl-d-aspartate (NMDA) receptors represents the principal route of fast excitatory transmission in the CNS. However, it is becoming increasingly clear that synaptic transmission also appears to be influenced by the actions of glutamate on metabotropic glutamate receptors (mGluRs) at both pre- and postsynaptic sites. At least seven mGluR subtypes (mGluR1–7) plus several splice varients have been identified by molecular biological methods (see Chapter 1). Expression of these receptors in a variety of cell types has shown that they are capable of interacting with most of the commonly recognized second-messenger systems. As detailed elsewhere in this volume, each expressed mGluR subtype also displays unique pharmacological specificity and shows a particular preference for one of the effector systems (Nakajima et al., 1993; Tanabe et al., 1993). However, which mGluRs mediate the various acute effects of mGluR activation on synaptic transmission and the underlying mechanisms are still poorly understood. In this chapter, we will review the current understanding regarding acute regulation of synaptic transmission by mGluRs and examine possible mechanisms of this regulation.

Whole-cell voltage-clamp recordings were made in thin transverse slices from neurons of the dorsomedial subdivision of the nucleus of the tractus solitarius (NTS) of the rat. Cells were exposed to either the ionotropic glutamate receptor agonist (R, S)-alpha-amino-3-hydroxy-5- methylisoxazole-4-propionate (AMPA) or the GABAA receptor agonist muscimol via pressure ejection directed at the cell soma. The metabotropic glutamate receptor agonist 1S,3R-1-aminocyclopentane-1,3- dicarboxylate (1S,3R-ACPD; 2–100 microM) reversibly depressed muscimol- evoked currents. Conversely, 1S,3R-ACPD reversibly potentiated AMPA- evoked currents. High-frequency stimulation of the tractus solitarius in the presence of 6,7-dinitroquinoxaline-2,3-dione and D-2-amino-5- phosphonopentanoic acid also produced a reversible depression of muscimol-evoked currents that was occluded in the presence of 100 microM 1S,3R-ACPD. 8-Br-cGMP or brain-derived natriuretic peptide mimicked the effects of 1S,3R-ACPD on AMPA and muscimol currents. However, agents that mimicked the actions of cAMP or diacylglycerol did not. These findings indicate that metabotropic glutamate receptors may mediate multiple components of excitatory transmission in the NTS including modulation of glutamate and GABA-activated ion channels.

Following microinjection into the nucleus tractus solitarius (NTS), the effects of glutamate on the baroreceptor reflex are poorly antagonized by kynurenic acid and DL-2-amino-5-phosphonovaleric acid, suggesting the possible involvement of metabotropic glutamate receptors in this response. The metabotropic glutamate receptor agonist 1S,3R-1- aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD) depolarized neurons located medial to the tractus solitarius (TS) at the level of the area postrema in coronal sections of the rat NTS. This effect was mimicked by glutamate and was not blocked by antagonists at alpha-amino- 3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA)/kainate or NMDA receptors. 1S,3R-ACPD also produced an inward current under voltage clamp that was not accompanied by a rise in [Ca2+]i, monitored with the Ca(2+)-sensitive dye fura-2. Conversely, the muscarinic agonist carbachol produced an outward current and a rise in [Ca2+]i. 1S,3R-ACPD reduced both the excitatory and the inhibitory postsynaptic current resulting from single electrical stimuli in the region of the TS. High- frequency stimulation of the TS produced an inward current in the presence of AMPA/kainate and NMDA receptor blockers. This current had similar properties to that produced by 1S,3R-ACPD. Thus, metabotropic glutamate receptors may mediate a component of excitatory transmission in the NTS.

We measured changes in the molar concentration of cytosolic Ca2+ ([Ca2+]i) in individual astrocytes in culture produced by the glutamate analog quisqualate (QA) and related substances by using fura-2 digital fluorescence microscopy. In cells cultured from the cortex, hippocampus, and cerebellum, the QA analog alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA; 10 microM) produced a slow increase in [Ca2+]i that was modest in amplitude (approximately 200 nM). These effects were completely abolished by 10 microM 6-nitro-7-cyano-quinoxaline-2,3-dione (CNQX). In cerebellar astrocytes, similar effects were produced by QA. However, in cortical and hippocampal astrocytes, the response to QA was much more complex. In these cells, QA produced an initial [Ca2+]i spike that was followed by a sustained influx of Ca2+ ("plateau"). In the absence of extracellular Ca2+, this plateau was abolished but the spike remained. CNQX did not block the spike and only slightly reduced the size of the plateau in some cells. Ni2+ (10 microM) but not nimodipine (10 microM) reduced the amplitude of the plateau. Pretreatment with 100 nM phorbol 12-myristate 13-acetate for 15 min abolished the spike but not the plateau portion of the QA response. Treatment with pertussis toxin at 250 ng/ml for 12-16 hr failed to alter the response. In some instances, the latency of the QA response differed considerably for individual cells in a group. It appeared that the response began in one cell and then spread to neighboring cells. Thus, QA appears to trigger a complex response in some astrocytes consisting of Ca2+ mobilization from intracellular stores and also Ca2+ influx resulting from the activation of AMPA-sensitive and -insensitive pathways.

The selective 5-HT3 receptor agonist 2-methyl-serotonin (2-Me-5-HT) mimicked the antinociceptive activity of 5-HT when intrathecally administered to rats. Two hundred micrograms (i.t.) doses of these agonists produced similar increases in tail flick latency. However, equal doses of 2-Me-5-HT and 5-HT doubled and tripled, respectively, the mean response latency as measured by the hot plate test. The potent and selective 5-HT3 receptor antagonists ICS 205-930 (3-tropanyl-indole-3-carboxylate) and MDL 72222 (3-tropanyl-3,5-dichlorobenzoate) antagonized the antinociceptive effects of both 5-HT and 2-Me-5-HT. However, there were differences in the efficacy of these antagonists. Thus, intrathecal pretreatment with ICS 205-930 (0.05 micrograms) or MDL 72222 (0.1 micrograms) blocked the antinociceptive effects of 5-HT (200 micrograms, i.t.) as measured by the tail flick test, however, higher doses (0.1 and 1.0 micrograms, respectively) were required in the hot plate test. Pretreatment with ICS 205-930 (0.1 microgram) or MDL 72222 (0.1 microgram) blocked the effects of 2-Me-5-HT (200 micrograms, i.t.) in both analgesiometric tests. It is concluded that 5-HT3 receptors are intimately involved in the modulation of spinal nociceptive responses.

The ability of the highly selective 5-HT3 receptor antagonist ICS 205-930 (3 alpha-tropanyl-1H-indole-3-carboxylic acid ester) to block the increase in tail flick (TFL) and hot plate latencies (HPL) produced by intrathecally (i.t.) administered serotonin (5-HT) was examined in pargyline pretreated rats. ICS 205-930 (0.1 microgram, i.t.) blocked the ability of 5-HT (200 micrograms) to increase TFL and HPL. Significant hyperalgesia, as measured by a decrease in TFL and HPL compared to saline controls, also resulted from either the coadministration of ICS 205-930 (10 micrograms) and 5-HT (200 micrograms) or from ICS 205-930 (100 micrograms) alone. These data suggest an important role for 5-HT3 receptors in modulating spinal nociceptive responses.

Caffeine at doses of 0.125, 1.25, 12.5 and 25 mg/kg was administered to rats and the subsequent effects on the sleep-wake cycle were measured. The 12.5 and 25 mg/kg doses of caffeine increased wakefulness, and decreased slow wave sleep-1 (SWS1), SWS2, rapid eye movement (REM) sleep and total sleep time (P less than or equal to 0.05). The 0.125 and 1.25 mg/kg doses of caffeine increased SWS1 at the expense of SWS2 (P less than or equal to 0.05), and did not affect total sleep time in any time period measured. Adenosine or adenosine agonists have been shown to increase SWS2 at the expense of waking or SWS1 with an increase in total sleep time. The effects of caffeine on sleep reported in this study suggest that caffeine administration not only antagonizes the effects of adenosine at the receptor level, but also at the behavioral level.

The 5-HT 3 receptor antagonists, ICS 205-930 and MDL 72222, displace 47–55% of the specific [ 3 H]serotonin (100 nM) binding to synaptosomal membranes derived from the dorsal, but not ventral, spinal cord of rats with IC 50 s less than 1.0 nM. Methiothepin (10 μM) increased displacement to 86–94% without shifting these IC 50 s. Scatchard plots of [ 3 H]5-HT binding in the presence of methiothepin (10 μM) reveal a single population of sites (K D = 11.5 nM, B max = 282 fmol mg −1 protein). These results indicate the presence of 5-HT 3 binding sites in dorsal spinal cord.