Rick L. Pieschl

Molecular Dissection of Two Distinct Actions of Melatonin on the Suprachiasmatic Circadian Clock

The pineal hormone melatonin elicits two effects on the suprachiasmatic nuclei (SCN): acute neuronal inhibition and phase-shifting. Melatonin evokes its biological effects through G protein-coupled receptors. Since the Mel1a melatonin receptor may transduce the major neurobiological actions of melatonin in mammals, we examined whether it mediates both melatonin effects on SCN function by using mice with targeted disruption of the Mel1a receptor. The Mel1a receptor accounts for all detectable, high affinity melatonin binding in mouse brain. Functionally, this receptor is necessary for the acute inhibitory action of melatonin on the SCN. Melatonin-induced phase shifts, however, are only modestly altered in the receptor-deficient mice; pertussis toxin still blocks melatonin-induced phase shifts in Mel1a receptor-deficient mice. The other melatonin receptor subtype, the Mel1b receptor, is expressed in mouse SCN, implicating it in the phase-shifting response. The results provide a molecular basis for two distinct, mechanistically separable effects of melatonin on SCN physiology.

Targeting acute ischemic stroke with a calcium-sensitive opener of maxi-K potassium channels

During ischemic stroke, neurons at risk are exposed to pathologically high levels of intracellular calcium (Ca++), initiating a fatal biochemical cascade. To protect these neurons, we have developed openers of large-conductance, Ca++-activated (maxi-K or BK) potassium channels, thereby augmenting an endogenous mechanism for regulating Ca++ entry and membrane potential. The novel fluoro-oxindoles BMS-204352 and racemic compound 1 are potent, effective and uniquely Ca++-sensitive openers of maxi-K channels. In rat models of permanent large-vessel stroke, BMS-204352 provided significant levels of cortical neuroprotection when administered two hours after the onset of occlusion, but had no effects on blood pressure or cerebral blood flow. This novel approach may restrict Ca++ entry in neurons at risk while having minimal side effects.

Targeted disruption of the mouse Mel(1b) melatonin receptor.

ABSTRACT Two high-affinity, G protein-coupled melatonin receptor subtypes have been identified in mammals. Targeted disruption of the Mel1a melatonin receptor prevents some, but not all, responses to the hormone, suggesting functional redundancy among receptor subtypes (Liu et al., Neuron 19:91-102, 1997). In the present work, the mouse Mel1b melatonin receptor cDNA was isolated and characterized, and the gene has been disrupted. The cDNA encodes a receptor with high affinity for melatonin and a pharmacological profile consistent with its assignment as encoding a melatonin receptor. Mice with targeted disruption of the Mel1b receptor have no obvious circadian phenotype. Melatonin suppressed multiunit electrical activity in the suprachiasmatic nucleus (SCN) in Mel1b receptor-deficient mice as effectively as in wild-type controls. The neuropeptide, pituitary adenylyl cyclase activating peptide, increases the level of phosphorylated cyclic AMP response element binding protein (CREB) in SCN slices, and melatonin reduces this effect. The Mel1a receptor subtype mediates this inhibitory response at moderate ligand concentrations (1 nM). A residual response apparent in Mel1a receptor-deficient C3H mice at higher melatonin concentrations (100 nM) is absent in Mel1a-Mel1b double-mutant mice, indicating that the Mel1b receptor mediates this effect of melatonin. These data indicate that there is a limited functional redundancy between the receptor subtypes in the SCN. Mice with targeted disruption of melatonin receptor subtypes will allow molecular dissection of other melatonin receptor-mediated responses.

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.

A Reexamination of the Role of GABA in the Mammalian Suprachiasmatic Nucleus

Three independent electrophysiological approaches in hypothalamic slices were used to test the hypothesis that [.gamma]-amino butyric acid (GABA)Areceptor activation excites suprachiasmatic nucleus (SCN) neurons during the subjective day, consistent with a recent report. First, multiple-unit recordings during either the subjective day or night showed that GABA or muscimol inhibited firing activity of the SCN population in a dose-dependent manner. Second, cell-attached recordings during the subjective day demonstrated an inhibitory effect of bath-or microapplied GABA on action currents of single SCN neurons. Third, gramicidin perforated-patch recordings showed that bicuculline increased the spontaneous firing rate during the subjective day. Therefore, electrophysiological data obtained by three different experimental methods provide evidence that GABA is inhibitory rather than excitatory during the subjective day.

Ex vivo assessment of binding site occupancy of monoamine reuptake inhibitors: Methodology and biological significance

The goal of this study was to develop and validate ex vivo binding assays for serotonin (SERT), norepinephrine (NET) and dopamine (DAT) transporters, and to use these assays to evaluate the binding site occupancy of triple and double monoamine reuptake inhibitors in rat brains. This study demonstrated that while autoradiographic methods provided anatomic precision and regional resolution, the homogenate binding method for site occupancy assessment yielded comparable sensitivity with markedly improved throughput. For ex vivo binding assays, the reduction of temperature and time during the in vitro process (primarily incubation with a radioligand) markedly decreased the dissociation of test agents from binding sites in brain tissues. This reduction, in turn, minimized the potential for underestimation of site occupancy in vivo especially for test compounds with affinity >10nM. The ratios of measured occupancy ED(50) values (doses at which 50% occupancy occurs) among SERT, NET and DAT sites for duloxetine, venlafaxine, nomifensine, indatraline, DOV 21,947 and DOV 216,303 were consistent with the ratios of the in vitro affinities between these target binding sites. The biological relevance of the monoamine transporter occupancy for these compounds is discussed.

NR2B antagonist CP-101,606 abolishes pitch-mediated deviance detection in awake rats

Schizophrenia patients exhibit a decreased ability to detect change in their auditory environment as measured by auditory event-related potentials (ERP) such as mismatch negativity. This deficit has been linked to abnormal NMDA neurotransmission since, among other observations, non-selective channel blockers of NMDA reliably diminish automatic deviance detection in human subjects as well as in animal models. Recent molecular and functional evidence links NR2B receptor subtype to aberrant NMDA transmission in schizophrenia. However, it is unknown if NR2B receptors participate in pre-attentive deviance detection. We recorded ERP from the vertex of freely behaving rats in response to frequency mismatch protocols. We saw a robust increase in N1 response to deviants compared to standard as well as control stimuli indicating true deviance detection. Moreover, the increased negativity was highly sensitive to deviant probability. Next, we tested the effect of a non-selective NMDA channel blocker (ketamine, 30 mg/kg) and a highly selective NR2B antagonist, CP-101,606 (10 or 30 mg/kg) on deviance detection. Ketamine attenuated deviance mainly by increasing the amplitude of the standard ERP. Amplitude and/or latency of several ERP components were also markedly affected. In contrast, CP-101,606 robustly and dose-dependently inhibited the deviant’s N1 amplitude, and as a consequence, completely abolished deviance detection. No other ERPs or components were affected. Thus, we report first evidence that NR2B receptors robustly participate in processes of automatic deviance detection in a rodent model. Lastly, our model demonstrates a path forward to test specific pharmacological hypotheses using translational endpoints relevant to aberrant sensory processing in schizophrenia.

Effect of acute NR2B antagonist treatment on long-term potentiation in the rat hippocampus.

The long lasting antidepressant response seen following acute, i.v. ketamine administration in patients with treatment-resistant depression (TRD) is thought to result from enhanced synaptic plasticity in cortical and hippocampal circuits. Using extracellular field recordings in rat hippocampal slices, we show that a single dose of the non-selective NMDA receptor antagonist ketamine or CP-101,606, a selective antagonist of the NR2B subunit of the NMDA receptor, enhances hippocampal synaptic plasticity induced with high frequency stimulation (HFS) 24h after dosing - a time at which plasma concentrations of the drug are no longer detectable in the animal. These results indicate that acute inhibition of NMDA receptors containing the NR2B subunit can lead to long-lasting changes in hippocampal plasticity.

Development of New Benzenesulfonamides As Potent and Selective Nav1.7 Inhibitors for the Treatment of Pain

By taking advantage of certain features in piperidine 4, we developed a novel series of cyclohexylamine- and piperidine-based benzenesulfonamides as potent and selective Nav1.7 inhibitors. However, compound 24, one of the early analogs, failed to reduce phase 2 flinching in the mouse formalin test even at a dose of 100 mpk PO due to insufficient dorsal root ganglion (DRG) exposure attributed to poor membrane permeability. Two analogs with improved membrane permeability showed much increased DRG concentrations at doses of 30 mpk PO, but, confoundingly, only one of these was effective in the formalin test. More data are needed to understand the disconnect between efficacy and exposure relationships.

Differential effects of coadministration of fluoxetine and WAY‐100635 on serotonergic neurotransmission in vivo: Sensitivity to sequence of injections

Serotonin 5‐HT1A receptor antagonists potentiate the effects of serotonin reuptake inhibitors on extracellular serotonin levels in a variety of brain regions. These effects are quite variable, however, with reports indicating potentiations of anywhere from 100–1900%. One factor that might impact the magnitude of such potentiations is the timing of administration of the two agents; reports in which the reuptake inhibitor is given prior to the serotonin receptor antagonist consistently report larger potentiations than reports in which the antagonist is given first. To test this relationship directly, microdialysis and electrophysiology studies were performed to assess the magnitude of increase in extracellular serotonin and changes in cellular activity produced by the serotonin reuptake inhibitor fluoxetine and the 5‐HT1A receptor antagonist WAY‐100635 under various dosing regimens. In microdialysis studies, when WAY‐100635 (0.5 mg/kg s.c.) was administered 80 min after fluoxetine (10 mg/kg i.p.) the increase in serotonin was more than twice that observed when the compounds were coadministered. In electrophysiology studies in vivo, WAY‐100635 reversed the depression of cell firing produced by fluoxetine when administered 30 min after fluoxetine, but when the two compounds were coadministered, a depression in firing rate was observed comparable to that produced by fluoxetine alone. In contrast, slice recording studies showed that WAY‐100635 blocked the effects of fluoxetine regardless of the order of administration. These results indicate that fluoxetine and WAY‐100635 can interact in a fashion not predicted by the currently accepted model. It is likely that neuronal circuitry outside of the raphe nuclei underlies this relationship. Synapse 38:17–26, 2000.