Inhibition Of Cell Firing Research Articles

Quantal Release of Dopamine and Action Potential Firing Detected in Midbrain Neurons by Multifunctional Diamond-Based Microarrays.

Micro-Graphitic Single Crystal Diamond Multi Electrode Arrays (μG-SCD-MEAs) have so far been used as amperometric sensors to detect catecholamines from chromaffin cells and adrenal gland slices. Besides having time resolution and sensitivity that are comparable with carbon fiber electrodes, that represent the gold standard for amperometry, μG-SCD-MEAs also have the advantages of simultaneous multisite detection, high biocompatibility and implementation of amperometric/potentiometric protocols, aimed at monitoring exocytotic events and neuronal excitability. In order to adapt diamond technology to record neuronal activity, the μG-SCD-MEAs in this work have been interfaced with cultured midbrain neurons to detect electrical activity as well as quantal release of dopamine (DA). μG-SCD-MEAs are based on graphitic sensing electrodes that are embedded into the diamond matrix and are fabricated using MeV ion beam lithography. Two geometries have been adopted, with 4 × 4 and 8 × 8 microelectrodes (20 μm × 3.5 μm exposed area, 200 μm spacing). In the amperometric configuration, the 4 × 4 μG-SCD-MEAs resolved quantal exocytosis from midbrain dopaminergic neurons. KCl-stimulated DA release occurred as amperometric spikes of 15 pA amplitude and 0.5 ms half-width, at a mean frequency of 0.4 Hz. When used as potentiometric multiarrays, the 8 × 8 μG-SCD-MEAs detected the spontaneous firing activity of midbrain neurons. Extracellularly recorded action potentials (APs) had mean amplitude of ∼-50 μV and occurred at a mean firing frequency of 0.7 Hz in 67% of neurons, while the remaining fired at 6.8 Hz. Comparable findings were observed using conventional MEAs (0.9 and 6.4 Hz, respectively). To test the reliability of potentiometric recordings with μG-SCD-MEAs, the D2-autoreceptor modulation of firing was investigated by applying levodopa (L-DOPA, 20 μM), and comparing μG-SCD-MEAs, conventional MEAs and current-clamp recordings. In all cases, L-DOPA reduced the spontaneous spiking activity in most neurons by 70%, while the D2-antagonist sulpiride reversed this effect. Cell firing inhibition was generally associated with increased APs amplitude. A minority of neurons was either insensitive to, or potentiated by L-DOPA, suggesting that AP recordings originate from different midbrain neuronal subpopulations and reveal different modulatory pathways. Our data demonstrate, for the first time, that μG-SCD-MEAs are multi-functional biosensors suitable to resolve real-time DA release and AP firing in in vitro neuronal networks.

Optogenetic Destabilization of the Memory Trace in CA1: Insights into Reconsolidation and Retrieval Processes.

Reactivation of memory can cause instability necessitating the reconsolidation of the trace. This process can be blocked by amnestic treatments administered after memory reactivation resulting in subsequent memory deficits. While the basolateral amygdala (BLA) is known to be crucial for reconsolidation, evidence for a contribution of the hippocampal CA1 region has only started to accumulate. Moreover, the effect of a reconsolidation blockade in CA1 has only been evaluated behaviorally, and it is unknown whether this manipulation has a long-term effect on neuronal activity. We combined optogenetic and high-resolution molecular imaging techniques to inhibit cell firing in CA1 following the reactivation of a fear memory in mice, evaluated memory performance and imaged neuronal activity the next day upon reexposure to the conditioning context. Blocking memory reconsolidation led to severe memory impairments that were associated with reduced neuronal activity not only in CA1 but also in CA3 and the BLA. Thus, our results indicate that CA1 is necessary for reconsolidation and suggest the involvement of a CA3-CA1-BLA network in the retrieval of contextual fear memory. Further investigations of this network might contribute to the validation of new brain targets for the treatment of pathologies such as posttraumatic stress disorders.

5-HT1A Receptor-Mediated Autoinhibition and the Control of Serotonergic Cell Firing.

The idea that serotonergic synaptic transmission plays an essential role in the control of mood and the pharmacotherapy of anxiety and depression is one of the cornerstones of modern biological psychiatry. As a result, there is intense interest in understanding the mechanisms controlling the activity of serotonin-synthesizing (serotonergic) neurons. One of the oldest and most durable ideas emerging from this work is that serotonergic neurons are capable of autonomously regulating their own basal firing rate. Serotonergic neurons express on their surface 5-HT1A receptors (autoreceptors) that, when activated, induce the opening of potassium channels that hyperpolarize and thereby inhibit cell firing. Activity-dependent release of serotonin within serotonergic nuclei is thought to activate these autoreceptors, thus completing an autoinhibitory feedback loop. This concept, which was originally proposed in the 1970s, has proven to be enormously fruitful and has guided the interpretation of a broad range of clinical and preclinical work. Yet, remarkably, electrophysiological studies seeking to directly demonstrate this phenomenon, especially in in vitro brain slices, have produced mixed results. Here, we critically review this work with a focus on electrophysiological studies, which directly assess neuronal activity. We also highlight recent work suggesting that 5-HT1A receptor-mediated autoinhibition may play other roles in the control of firing besides acting as a feedback regulator for the pacemaker-like firing rate of serotonergic neurons.

The neuroethics of non-invasive brain stimulation

Transcranial direct current stimulation (TDCS) is a brain stimulation tool that is portable, painless, inexpensive, apparently safe, and with potential long-term efficacy. Recent results obtained from TDCS experiments offer exciting possibilities for the enhancement and treatment of normal or impaired abilities, respectively. We discuss new neuroethical problems that have emerged from the usage of TDCS, and also focus on one of the most likely future applications of TDCS: enhancing learning and cognition in children with typical and atypical development.

Robust tonic GABA currents can inhibit cell firing in mouse newborn neocortical pyramidal cells

Within the hippocampus and neocortex, GABA is considered to be excitatory in early development due to a relatively depolarized Cl(-) reversal potential (E(Cl)). Although the depolarizing nature of synaptic GABAergic events has been well established, it is unknown whether cortical tonic currents mediated by extrasynaptically located GABA(A) receptors (GABA(A) Rs) are also excitatory. Here we examined the development of tonic currents in the neocortex and their effect on neuronal excitability. Mean tonic current, recorded from layer 5 (L5) pyramidal cells of the mouse somatosensory cortex, is robust in newborns [postnatal day (P)2-4] then decreases dramatically by the second postnatal week (P7-10 and P30-40). Pharmacological studies, in combination with Western blot analysis, show that neonatal tonic currents are partially mediated by the GABA(A) R α5 subunit, and probably the δ subunit. In newborns, the charge due to tonic current accounts for nearly 100% of the total GABA charge, a contribution that decreases to < 50% in mature tissue. Current clamp recordings show that tonic current contributes to large fluctuations in the membrane potential that may disrupt its stability. Bath application of 5 μM GABA, to induce tonic currents, markedly decreased cell firing frequency in most recorded cells while increasing it in others. Gramicidin perforated patch recordings show heterogeneity in E(Cl) recorded from P2-5 L5 pyramidal cells. Together, these findings demonstrate that tonic currents activated by low GABA concentrations can dominate GABAergic transmission in newborn neocortical pyramidal cells and that tonic currents can exert heterogeneous effects on neuronal excitability.

Modulation of nocioceptive transmission with calcitonin gene-related peptide receptor antagonists in the thalamus

Calcitonin gene-related peptide receptor antagonists are effective acute migraine treatments without the vascular contraindications associated with triptans. While it has been demonstrated that calcitonin gene-related peptide receptor antagonists act in the central nervous system, their effects in preclinical migraine models have been investigated in only the trigeminocervical complex. Migraine is a complex neurological disorder; sites in the brainstem and forebrain are clearly involved in its expression. We have performed electrophysiological recordings in thalamic neurons of rats responding to nocioceptive trigeminovascular inputs and tested the effect of olcegepant, a calcitonin gene-related peptide receptor antagonist (1 mg/kg, intravenously), on cell firing. We further tested the effect of microiontophoresed calcitonin gene-related peptide and the receptor antagonists calcitonin gene-related peptide 8-37 and olcegepant on thalamic cell firing, elicited by stimulation of the superior sagittal sinus or by microiontophoretic application of l-glutamate. Additionally, we used immunofluorescent staining to demonstrate the presence of functional calcitonin gene-related peptide receptors in the ventroposteromedial thalamic nucleus by specifically co-staining for the calcitonin gene-related peptide receptor subunits calcitonin receptor-like receptor and receptor activity modifying protein 1. Intravenously administered olcegepant significantly inhibited cell firing evoked by stimulation of the superior sagittal sinus as well as the background activity. Microiontophoresis of calcitonin gene-related peptide 8-37 also showed a significant inhibition of l-glutamate-evoked cell firing and firing evoked by stimulation of the superior sagittal sinus. Immunofluorescent staining confirmed the presence of the components of a functional calcitonin gene-related peptide receptor, the calcitonin receptor-like receptor and the receptor activity modifying protein 1, within the area of the ventroposteromedial thalamic nucleus. This is the first report on the efficacy of calcitonin gene-related peptide receptor antagonists at the level of third-order neurons in the migraine pathway, showing that the central effects of calcitonin gene-related peptide receptor antagonists extend beyond the trigeminocervical complex at least to the sensory thalamus.

Nociceptin/orphanin FQ and the regulation of neuronal excitability in the rat bed nucleus of the stria terminalis: Interaction with glucocorticoids

Nociceptin (N/OFQ) peptide has regulatory roles in neuroendocrine responses to stress. We sought to clarify the roles of nociceptin and its receptors (NOP receptors) in the regulation of rat bed nucleus of the stria terminalis (BNST) neurones in vitro. The effect of nociceptin (75 nM) across subregions of the anterior BNST was determined using extracellular single-unit recordings in rat brain slices. Firing patterns of the neurones were recorded in the presence of N-methyl-D-aspartate (NMDA, 10 μm) for the classification of putative cell types. Based on the firing patterns, four cell types were identified. The distribution of cell types differed between the dorsal and ventral BNST. Nociceptin inhibited the activity of 53.2% of all the neurones tested (n = 47), regardless of the cell type or subregion. The duration of nociceptin-mediated inhibition of cell firing was significantly attenuated by pre-treatment with the NOP receptor antagonist, UFP-101 (750 nM), indicating that nociceptin-induced suppression of firing rate involves NOP receptor activation in the BNST. Pre-treatment of slices with 100-nM corticosterone (CORT) vs. dimethylsulphoxide (DMSO) for 20 min significantly abolished the nociceptin-induced inhibition of firing rate (P < 0.001) when tested 2 h later. We did not, however, observe a significant effect of CORT on baseline firing rate or pattern in BNST neurones. We suggest that the interaction between nociceptin and glucocorticoids in the BNST may be essential for normal adaptive stress responses.

Differential long-term effects of MDMA on the serotoninergic system and hippocampal cell proliferation in 5-HTT knock-out vs. wild-type mice

Although numerous studies investigated the mechanisms underlying 3,4-methylenedioxymethamphetamine (MDMA)-induced neurotoxicity, little is known about its long-term functional consequences on 5-HT neurotransmission in mice. This led us to evaluate the delayed effects of MDMA exposure on the 5-HT system, using in-vitro and in-vivo approaches in both 5-HTT wild-type and knock-out mice. Acute MDMA in-vitro application on slices of the dorsal raphe nucleus (DRN) induced concentration-dependent 5-HT release and 5-HT cell firing inhibition. Four weeks after MDMA administration (20 mg/kg b.i.d for 4 d), a 2-fold increase in the potency of the 5-HT1A receptor agonist ipsapirone to inhibit the discharge of DRN 5-HT neurons and a larger hypothermic response to 8-OH-DPAT were observed in MDMA- compared to saline-treated mice. This adaptive 5-HT1A autoreceptor supersensitivity was associated with decreases in 5-HT levels but no changes of [3H]citalopram binding in brain. Long-term MDMA treatment also induced a 30% decrease in BrdU labelling of proliferating hippocampal cells and an increased immobility duration in the forced swim test suggesting a depressive-like behaviour induced by MDMA treatment. All these effects were abolished in 5-HTT-/- knock-out mice. These data indicated that, in mice, MDMA administration induced a delayed adaptive supersensitivity of 5-HT1A autoreceptors in the DRN, a deficit in hippocampal cell proliferation and a depressive-like behaviour. These 5-HTT-dependent effects, opposite to those of antidepressants, might contribute to MDMA-induced mood disorders.

Sustained pharmacological blockade of NK1 substance P receptors causes functional desensitization of dorsal raphe 5‐HT1A autoreceptors in mice

Antagonists at NK1 substance P receptors have demonstrated similar antidepressant properties in both animal paradigms and in human as selective serotonin reuptake inhibitors (SSRIs) that induce desensitization of 5-HT 1A autoreceptors within the dorsal raphe nucleus (DRN). We investigated whether this receptor adaptation also occurs upon NK1 receptor blockade. C57B/L6J mice were treated for 21 days with the selective NK1 receptor antagonist GR 205171 (10 mg/kg daily) through subcutaneously implanted osmotic mini pumps, and DRN 5-HT 1A autoreceptor functioning was assessed using various approaches. Recording of DRN serotonergic neurons in brainstem slices showed that GR 205171 treatment reduced (by approximately 1.5 fold) the potency of the 5-HT 1A receptor agonist, ipsapirone, to inhibit cell firing. In parallel, the 5-HT 1A autoreceptor-mediated [35S]GTP-gamma-S binding induced by 5-carboxamidotryptamine onto the DRN in brainstem sections was significantly decreased in GR 205171-treated mice. In vivo microdialysis showed that the cortical 5-HT overflow caused by acute injection of the SSRI paroxetine (1 mg/kg) was twice as high in GR 205171-treated as in vehicle-treated controls. In the DRN, basal 5-HT outflow was significantly enhanced by GR 205171 treatment. These data supported the hypothesis that chronic NK1 receptor blockade induces a functional desensitization of 5-HT 1A autoreceptors similar to that observed with SSRIs.

Differential Effects of the 5-Hydroxytryptamine (5-HT)1AReceptor Inverse Agonists Rec 27/0224 and Rec 27/0074 on Electrophysiological Responses to 5-HT1AReceptor Activation in Rat Dorsal Raphe Nucleus and Hippocampus in Vitro

The pharmacological properties of cyclohexanecarboxylic acid, {2-[4-(2-bromo-5-methoxybenzyl)piperazin-1-yl]ethyl}-(2-trifluoromethoxyphenyl)amide (Rec 27/0224), and cyclohexanecarboxylic acid, (2-methoxy-phenyl)-{2-[4-(2-methoxyphenyl)-piperazin-1-yl]ethyl}amide (Rec 27/0074), were characterized using radioligand displacement and guanosine 5'-O-(3-[35S]thiotriphosphate) ([35S]GTPgammaS) binding assays, as well as electrophysiological experiments, in rat hippocampal and dorsal raphe nucleus (DRN) slices. Both compounds showed a high affinity (Ki, approximately 1 nM) and selectivity (>70-fold) at human 5-hydroxytryptamine (5-HT)1A receptors versus other 5-HT receptors. In [35S]GTPgammaS binding assays on HeLa cells stably expressing human 5-HT1A receptors, Rec 27/0224 and Rec 27/0074 inhibited basal [35S]GTPgammaS binding by 44.8 +/- 1.7% (pEC50 = 8.58) and 25 +/- 2.5% (pEC50 = 8.86), respectively. In intracellularly recorded CA1 pyramidal cells, 5-HT1A (hetero)receptor-mediated hyperpolarization, elicited by 100 nM 5-carboxamidoytryptamine (5-CT), was partially antagonized by Rec 27/0224 (approximately 50%; IC50 = 18.0 nM) and Rec 27/0074 (74%; IC50 = 0.8 nM). In extracellularly recorded DRN serotonergic neurons, Rec 27/0224 and Rec 27/0074 fully antagonized the inhibition of firing caused by the activation of 5-HT1A (auto)receptors by 30 nM 5-CT with an IC50 of 34.9 nM and 16.5 nM, respectively. The antagonism had a slow time course, reaching a steady state within 60 min. Both compounds also antagonized the citalopram-elicited, endogenous 5-HT-mediated inhibition of cell firing. In conclusion, Rec 27/0224 and Rec 27/0074 exhibited inverse agonism in [35S]GTPgammaS binding assays and differential antagonistic properties on 5-HT1A receptor-mediated responses in the hippocampus but not in the DRN. Whether this differential effect is causally related to inverse agonist activity is unclear. The qualitatively different nature of the antagonism in the hippocampus versus the DRN clearly distinguishes the compounds from neutral antagonists, such as N-{2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl}-N-2-pyridinylcyclo-hexanecarboxamide (WAY-100635).

Molecular modeling and docking simulations of scorpion toxins and related analogs on human SKCa2 and SKCa3 channels

The small-conductance Ca 2+-activated K + (SKCa) channels modulate cytosolic Ca 2+ concentration in excitable and non-excitable tissues by regulating the membrane potential and are responsible of slow action potential after hyperpolarization that inhibits cell firing. Among these, human SKCa2 and SKCa3 channels differ in the pore region by only two residues: Ala331 and Asn367 (human small-conductance calcium-activated potassium channel, hSKCa2) instead of Val485 and His521 (hSKCa3). To design highly selective blockers of hSKCa channels, a number of known hSKCa2 and/or hSKCa3-active peptides (i.e. scorpion toxins and analogs thereof) were analyzed for their interactions and selectivities toward these channels. Molecular models of hSKCa2 and hSKCa3 channels (S5-H5-S6 portion) were generated, and scorpion toxins/peptides of unsolved three-dimensional (3D) structures were modeled. Models of toxin–channel complexes were generated by the bimolecular complex generation with global evaluation, and ranking (BiGGER) docking software and selected by using a screening method of the docking solutions. A high degree of correlation was found to exist between docking energies and experimental K d values of peptides that blocked hSKCa2 and/or hSKCa3 channels, suggesting it could be appropriate to predict K d values of other bioactive peptides. The best scoring complexes were also used to identify key residues of both interacting partners, indicating that such an approach should help the design of more active and/or selective peptide blockers of targeted ion channels.

GABA receptor-mediated inhibition of neuronal activity in rat SCN in vitro: pharmacology and influence of circadian phase.

The effect of gamma-aminobutyric acid (GABA) on neuronal firing rate in rat suprachiasmatic nucleus (SCN) slices was examined using continuous recording methods. GABA inhibited neuronal discharge during both the subjective day and the subjective night in a concentration-dependent manner characterized by two apparent affinity states. The GABAA receptor agonist muscimol caused potent inhibition regardless of circadian time; repeated applications of the agonist did not reverse the direction of effect. The GABAA receptor antagonists bicuculline and picrotoxin increased excitability when applied during either subjective day or subjective night. A significant increase in GABAA receptor- mediated inhibition, as well as endogenous GABAergic tone, was observed on the second day after slice preparation. The GABAB receptor agonist baclofen inhibited cell firing during subjective day and night, but the GABAB antagonist phaclofen had no significant effect. These data provide additional strong support for a predominantly inhibitory role of GABA in the rat SCN, regardless of the time of application in relation to the circadian rhythm, and demonstrate an important level of plasticity of this system in vitro.

5-HT 1A agonist potential of pindolol: electrophysiologic studies in the dorsal raphe nucleus and hippocampus

Background: The ability of pindolol to block 5-HT 1A autoreceptors on serotonin-containing neurons in the raphe nuclei is thought to underlie the clinical reports of enhanced efficacy and rate of improvement in depressed patients treated with pindolol/selective serotonin reuptake inhibitor (SSRI) combinations. Selectivity for somatodendritic 5-HT 1A autoreceptors is a crucial requirement, as blockade of postsynaptic 5-HT 1A sites may jeopardize the therapeutic response. Previous investigators have probed the effects of pindolol on serotonergic dorsal raphe cell firing in animal species; here we confirm their findings and extend them to include observations on postsynaptic 5-HT 1A receptors in the hippocampus. Methods: Extracellular single-unit recordings were made in rats using standard electrophysiologic techniques. Firing rates of serotonin-containing neurons in the dorsal raphe nucleus and CA3 hippocampal pyramidal neurons were monitored and the effects of pindolol given alone or in combination with an SSRI (fluoxetine) or a 5-HT 1A antagonist (WAY-100,635) were determined. Results: Pindolol inhibited the firing rates of serotonergic dorsal raphe neurons in a dose-dependent manner. Recovery to baseline firing rates was gradual, but this inhibition could be acutely reversed by WAY-100,635. A range of pindolol doses failed to block the inhibitory effects of fluoxetine on dorsal raphe cell firing. In the hippocampus, pindolol also inhibited cell firing as a function of dose, although these effects were insensitive to WAY-100,635 treatment. Conclusions: The ability of pindolol to inhibit serotonergic dorsal raphe cell firing is indicative of its agonist potential and is consistent with previous studies. The lack of observable antagonism of the SSRI-induced slowing of raphe unit activity casts doubt on the suitability of this mechanism of action to account for the positive findings in clinical studies utilizing pindolol/SSRI combinations. The 5-HT 1A-independent inhibition of hippocampal CA3 cell firing by pindolol suggests that this compound invokes multiple pharmacologic actions, all of which need to be assimilated into any proposed mechanism of action.

Effects of neurotensin receptor antagonists on the firing rate of rat ventral pallidum neurons.

Using anaesthetized adult rats, we compared the effects of i.v. injections of neurotensin receptor antagonists (SR48692 and SR142948), haloperidol and clozapine on neuronal firing rate in the two ventral pallidal areas. SR48692 (0.5, 1 and 2 mg/kg, i.v.) induced a dose-dependent inhibition of firing rate in two thirds of neurons in the ventromedial part without any effect in the ventrolateral part. These effects are in keeping with the preferential neurotensin immunoreactivity distribution reported in the ventral pallidum. The classical antipsychotic drug haloperidol (0.5 mg/kg, i.v.) induced an inhibition of neuronal firing rate in both ventral pallidal areas whereas the atypical antipsychotic drug clozapine (20 mg/kg, i.v.), like the neurotensin receptor antagonist SR48692, inhibited cell firing only in the ventromedial part.

Small-conductance calcium-activated potassium channels.

SK channels play a fundamental role in all excitable cells. SK channels are potassium selective and are activated by an increase in the level of intracellular calcium, such as occurs during an action potential. Their activation causes membrane hyperpolarization, which inhibits cell firing and limits the firing frequency of repetitive action potentials. The intracellular calcium increase evoked by action potential firing decays slowly, allowing SK channel activation to generate a long-lasting hyperpolarization termed the slow afterhyperpolarization (sAHP). This spike-frequency adaptation protects the cell from the deleterious effects of continuous tetanic activity and is essential for normal neurotransmission. Slow AHPs can be classified into two groups, based on sensitivity to the bee venom toxin apamin. In general, apamin-sensitive sAHPs activate rapidly following a single action potential and decay with a time constant of approximately 150 ms. In contrast, apamin-insensitive sAHPs rise slowly and decay with a time constant of approximately 1.5 s. The basis for this kinetic difference is not yet understood. Apamin-sensitive and apamin-insensitive SK channels have recently been cloned. This chapter will compare with different classes of sAHPs, discuss the cloned SK channels and how they are gated by calcium ions, describe the molecular basis for their different pharmacologies, and review the possible role of SK channels in several pathological conditions.

Serotonin inhibits trigeminal nucleus activity evoked by craniovascular stimulation through a 5HT1B/1D receptor: a central action in migraine?

The development of serotonin (5HT1B/1D) agonists as treatments for the acute attack of migraine has resulted in considerable interest in their mechanism of action and, to some extent, renewed interest in the role of serotonin (5-hydroxytryptamine; 5HT) in the disorder. The initial synthesis of this class of compounds was predicated on the clinical observation that intravenous 5HT terminated acute attacks of migraine. In this study the superior sagittal sinus was isolated in the alpha-chloralose (60 mg/kg i.p. and 20 mg/kg i.v. injection supplementary 2 hourly) anesthetized cat. The sinus was stimulated electrically (120V, 250 microsec duration, 0.3 Hz), and neurons of the trigeminocervical complex in the dorsal C2 spinal cord were monitored using electrophysiological methods. After baseline recordings in each animal, 5HT (15 microg/kg/min) was infused for 5 minutes in the presence of either vehicle (group A) or the 5HT1B/1D antagonist GR127935 (100 microg/kg i.v. injection; group B). The baseline probability of cell firing after sagittal sinus stimulation was 0.61 +/- 0.1 at a latency to the fastest peak of 11.1 +/- 0.4 msec. In group A, 5HT infusion alone had a small effect of increasing mean blood pressure (12 +/- 3 mm Hg), which in itself did not alter cell firing. In group A, 5HT alone had an inhibitory effect on evoked trigeminal activity, which developed 15 to 20 minutes after commencement of the infusion. The inhibition of cell firing lasted for 20 minutes, after which the activity returned to baseline. In group B, the combination of 5HT and GR127935 had no effect on trigeminal cell firing, although the small hypertensive effect was still present. These data indicate that 5HT inhibits evoked trigeminal nucleus firing via the 5HT1B/1D receptor at which GR127935 is an antagonist. It is likely that some part of the effect of 5HT in migraine relates to inhibition of trigeminal nucleus activity, just as it is likely that some part of the effect of the triptans is also mediated at this central site and may be complementary to their nonneuronal actions. Moreover, the data highlight the case for describing this class of headache as neurovascular headaches rather than vascular headaches, to recognize the implicit contribution of the trigeminovascular system to their pathophysiology.

Phase Shifting of Circadian Rhythms and Depression of Neuronal Activity in the Rat Suprachiasmatic Nucleus by Neuropeptide Y: Mediation by Different Receptor Subtypes

Neuropeptide Y (NPY) has been implicated in the phase shifting of circadian rhythms in the hypothalamic suprachiasmatic nucleus (SCN). Using long-term, multiple-neuron recordings, we examined the direct effects and phase-shifting properties of NPY application in rat SCN slices in vitro (n = 453). Application of NPY and peptide YY to SCN slices at circadian time (CT) 7.5-8.5 produced concentration-dependent, reversible inhibition of cell firing and a subsequent significant phase advance. Several lines of evidence indicated that these two effects of NPY were mediated by different receptors. NPY-induced inhibition and phase shifting had different concentration-response relationships and very different phase-response relationships. NPY-induced phase advances, but not inhibition, were blocked by the GABAA antagonist bicuculline, suggesting that NPY-mediated modulation of GABA may be an underlying mechanism whereby NPY phase shifts the circadian clock. Application of the Y2 receptor agonists NPY 13-36 and (Cys2,8-aminooctanoic acid5,24,D-Cys27)-NPY advanced the peak of the circadian rhythm but did not inhibit cell firing. The Y1 and Y5 agonist [Leu31,Pro34]-NPY evoked a substantial inhibition of discharge but did not generate a phase shift. NPY-induced inhibition was not blocked by the specific Y1 antagonist BIBP-3226; the antagonist also had no effect on the timing of the peak of the circadian rhythm. Application of the Y5 agonist [D-Trp32]-NPY produced only direct neuronal inhibition. These are the first data to indicate that at least two functional populations of NPY receptors exist in the SCN, distinguishable on the basis of pharmacology, each mediating a different physiological response to NPY application.

Functional anatomical and behavioral consequences of dopamine receptor stimulation.

Taken together with electrophysiological data, these results suggest that in states of DA deficiency, systemically administered L-dopa or DA agonist drugs inhibit cell firing in the major output nuclei of the basal ganglia (GPI and SNPR). We propose that DA produces this net effect by a direct influence on the striato-GPI and striato-SNPR neurons, as well as indirectly via the striato-GPE-STN-GPI/SNPR circuit. The RCGU data suggest that DA activates the direct pathway by stimulating D1 receptor-bearing striatal GABAergic neurons projecting to GPI and SNPR. Supportive evidence includes the ability of D1 agonists to facilitate GABA release in the striatum and to increase the firing rates of striatonigral neurons. The RCGU data also support a net stimulatory action of DA on the GPE output on STN, consistent with the excitatory effects of DA on GPE neuronal firing rates. This effect may be mediated by both D1 and D2 receptors. Stimulation of this pathway should physiologically inhibit the STN resulting in dysfacilitation of the GPI and SNPR. According to this scheme, DA exerts complementary actions via both direct and indirect anatomical pathways to decrease tonic firing rates of intrinsic neurons in the major output nuclei of the basal ganglia (i.e., GPI and SNPR).

The role of 5-HT 1B/1D receptors in the modulation of 5-hydroxytryptamine levels in the frontal cortex of the conscious guinea pig

The role of 5-HT 1B/1D receptors in modulating extracellular 5-hydroxytryptamine (5-HT) levels in the guinea pig was investigated with the non-selective 5-HT 1B/1D receptor inverse agonist, methiothepin, and the selective 5-HT 1B/1D receptor partial agonists, GR 127935 ( n-[4-methoxy-3-(4-methyl-1-piperizinyl)phenyl]-2′-methyl-4′-(5-methyl-1,2,4-oxadiazole-3-yl)[1,1′-biphenyl]-4-carboxamide) and GR 125743 ( n-[4-methoxy-3-(4-methyl-1-piperizinyl)phenyl]-3-methyl-4-(4-pyridinyl)benzamide). Extracellular 5-HT levels were measured using the technique of brain microdialysis, in the frontal cortex of the freely moving guinea-pig. Extracellular 5-HT was tetrodotoxin sensitive and calcium dependent, and increased when perfused with a high concentration of K +. In addition, extracellular 5-HT levels were lowered by the 5-HT 1B/1D receptor agonist, sumatriptan, and the 5-HT 1A receptor agonist, 8-hydroxy-2-(di- n-propylamino)tetralin, while perfusion of the selective serotonin re-uptake inhibitor, paroxetine, increased 5-HT in a concentration-dependent manner. Perfusion of methiothepin, GR 127935 and GR 125743 into the frontal cortex caused significant but transient increases of extracellular 5-HT. However, systemic administration of methiothepin, GR 127935 and GR 125743, at 0.3 mg/kg i.p., produced significant decreases in extracellular 5-HT, to minima of 27±3%, 31±12% and 27±13% of basal, respectively. The increase of extracellular 5-HT, following 5-HT 1B/1D receptor inverse and partial agonist perfusion into the frontal cortex, was probably a consequence of attenuation of an endogenous 5-HT tone at terminal 5-HT autoreceptors. The unexpected decrease in 5-HT levels following systemic administration may be a result of additional attenuation of endogenous 5-HT tone at cell body autoreceptors in the raphe. Such an increase in local 5-HT levels could then stimulate 5-HT 1A receptors to inhibit cell firing and hence decrease 5-HT levels in the terminal regions. This was confirmed when co-administration of the 5-HT 1A receptor antagonist, WAY 100635, significantly attenuated the GR 127935 decrease in 5-HT.

Antagonism of the effects of (+)-PD 128907 on midbrain dopamine neurones in rat brain slices by a selective D2 receptor antagonist L-741,626.

1. The ability of PD 128907 to activate dopamine receptors in the ventral tegmental area, substantia nigra pars compacta, and striatum was investigated by use of in vitro electrophysiological recording and fast cyclic voltammetry. The affinity of a novel D2 selective antagonist L-741,626 for receptors activated by this agonist was measured to determine if its effects were mediated by D2 or D3 receptors. 2. The active (+) enantiomer of PD 128907 bound with high affinity and selectivity to rat D3 dopamine receptors. The Ki values for (+)-PD 128907 were 620 nM at D2, 1 nM at D3 and 720 nM at D4 receptors. 3. (+)-PD 128907 inhibited cell firing in both the ventral tegmental area and substantia nigra pars compacta with EC50 values of 33 nM (pEC50 = 7.48 +/- 0.10, n = 10) and 38 nM (pEC50 = 7.42 +/- 0.15, n = 5), respectively. No effects of (+)-PD 128907 (100 nM) were observed on glutamate or GABA-mediated synaptic potentials elicited by focal bipolar stimulation. 4. L-741,626 antagonized these effects of (+)-PD 128907 in a concentration-dependent and surmountable manner with an affinity, determined from Schild analysis, of 20 nM (pKB = 7.71 +/- 0.14) in the ventral tegmental area and 11 nM (pKB = 7.95 +/- 0.18) in the substantia nigra pars compacta. 5. (+)-PD 128907 also inhibited dopamine release in the caudate-putamen with an EC50 of 66 nM (n = 5). The affinity of L-741,626 for these nerve terminal autoreceptors (pKB = 7.71 +/- 0.06; = 20 nM) was identical to that observed on midbrain dopamine neurones. 6. These data demonstrate that the D3 receptor ligand (+)-PD 128907 is a potent agonist on rat midbrain dopamine neurones. However, its lack of regional selectivity, and the high affinity of the selective D2 receptor antagonist L-741,626 for receptors activated by (+)-PD 128907, was more consistent with an action on D2 autoreceptors rather than upon a D3 dopamine receptor subtype.