Pharmacology of reflex blinks in the rat: a novel model for headache research.

BACKGROUND: Migraineurs are highly sensitive to the nitric oxide donor glyceryl trinitrate which triggers attacks in many sufferers. In animal studies, glyceryl trinitrate increases neuronal activity in the trigeminovascular pathway and elevates neurotransmitter levels in the brainstem. Many migraineurs also display alterations in blink reflexes, known to involve brainstem circuits. We investigated the effect of GTN on evoked blinks in the anaesthetised rat to determine whether such reflexes may prove useful as the basis for a novel animal model to evaluate potential anti-migraine therapeutic agents. METHOD: In anaesthetised rats the electromyogram associated with the reflex blink evoked by corneal airpuff was recorded. Rats were infused with glyceryl trinitrate, sumatriptan plus glyceryl trinitrate or vehicle control. Changes in the magnitude of the reflex blink-associated electromyogram following these treatments were measured. RESULTS: Glyceryl trinitrate potentiated the evoked reflex blink-associated EMG response from 2 h after infusion. That effect was abolished by simultaneous infusion of sumatriptan with glyceryl trinitrate. CONCLUSIONS: These results show that simple skin surface measurements of evoked electromyographic activity in the rat can reliably detect the evoked blink reflex that can be potentiated by nitric oxide donors. This novel model may be an effective tool for evaluating putative anti-migraine therapeutic agents.

Insulin activates ATP-sensitive K+ channels in hypothalamic neurons of lean, but not obese rats.

Insulin and leptin receptors are present in hypothalamic regions that control energy homeostasis, and these hormones reduce food intake and body weight in lean, but not obese, Zucker rats. Here we demonstrate that insulin, like leptin, hyperpolarizes lean rat hypothalamic glucose-responsive (GR) neurons by opening KATP channels. These findings suggest hypothalamic K ATP channel function is crucial to physiological regulation of food intake and body weight.

Pharmacological and molecular characterization of ATP-sensitive K(+) conductances in CART and NPY/AgRP expressing neurons of the hypothalamic arcuate nucleus.

The role of hypothalamic ATP-sensitive potassium channels in the maintenance of energy homeostasis has been extensively explored. However, how these channels are incorporated into the neuronal networks of the arcuate nucleus remains unclear. Whole-cell patch-clamp recordings from rat arcuate nucleus neurons in hypothalamic slice preparations revealed widespread expression of functional ATP-sensitive potassium channels within the nucleus. ATP-sensitive potassium channels were expressed in orexigenic neuropeptide Y/agouti-related protein (NPY/AgRP) and ghrelin-sensitive neurons and in anorexigenic cocaine-and-amphetamine regulated transcript (CART) neurons. In 70% of the arcuate nucleus neurons recorded, exposure to glucose-free bathing medium induced inhibition of electrical excitability, the response being characterized by membrane hyperpolarization, a reduction in neuronal input resistance and a reversal potential consistent with opening of potassium channels. These effects were reversible upon re-introduction of glucose to the bathing medium or upon exposure to the ATP-sensitive potassium channel blockers tolbutamide or glibenclamide. The potassium channel opener diazoxide, but not pinacidil, also induced a tolbutamide and glibenclamide-sensitive inhibition of electrical excitability. Single-cell reverse transcription-polymerase chain reaction revealed expression of mRNA for sulfonylurea receptor 1 but not sulfonylurea receptor 2 subunits of ATP-sensitive potassium channels. Thus, rat arcuate nucleus neurons, including those involved in functionally antagonistic orexigenic and anorexigenic pathways express functional ATP-sensitive potassium channels which include sulfonylurea receptor 1 subunits. These data indicate a crucial role for these ion channels in central sensing of metabolic and energy status. However, further studies are needed to clarify the differential roles of these channels, the organization of signaling pathways that regulate them and how they operate in functionally opposing cell types.

The impact of ageing, fasting and high-fat diet on central and peripheral glucose tolerance and glucose-sensing neural networks in the arcuate nucleus.

Obesity and ageing are risk factors for diabetes. In the present study, we investigated the effects of ageing, obesity and fasting on central and peripheral glucose tolerance and on glucose-sensing neuronal function in the arcuate nucleus of rats, with a view to providing insight into the central mechanisms regulating glucose homeostasis and how they change or are subject to dysfunction with ageing and obesity. We show that, following a glucose load, central glucose tolerance at the level of the cerebrospinal fluid (CSF) and plasma is significantly reduced in rats maintained on a high-fat diet (HFD). With ageing, up to 2 years, central glucose tolerance was impaired in an age-dependent manner, whereas peripheral glucose tolerance remained unaffected. Ageing-induced peripheral glucose intolerance was improved by a 24-hour fast, whereas central glucose tolerance was not corrected. Pre-wean, immature animals have elevated basal plasma glucose levels and a delayed increase in central glucose levels following peripheral glucose injection compared to mature animals. Electrophysiological recording techniques revealed an energy-status-dependent role for glucose-excited, inhibited and adapting neurones, along with glucose-induced changes in synaptic transmission. We conclude that ageing affects central glucose tolerance, whereas HFD profoundly affects central and peripheral glucose tolerance and, in addition, glucose-sensing neurones adapt function in an energy-status-dependent manner.

Integration of metabolic stimuli in the hypothalamic arcuate nucleus.

Integration of peripheral and central anabolic and catabolic inputs within the hypothalamic arcuate nucleus (ARC) is believed to be central to the maintenance of energy balance. In order to perform this complex task, neurons in the ARC express receptors for all major humoral and central transmitters involved in the maintenance of energy homeostasis. The integration of these inputs occurs at the cellular and circuit level and the resulting electrical output forms the origins for the activation of feeding and energy balance-related networks. Here, we discuss the role that active intrinsic membrane conductances, K(ATP) channels and intracellular second messenger systems play in the integration of metabolic stimuli at the cellular level in the ARC. We conclude that the research into the integration of hunger and satiety signals in the ARC has made substantial progress in the last decade, but we are far from unraveling the complex neuronal networks involved in the maintenance of energy homeostasis. The diverse range of inputs, neuronal integrative properties, targets, output signals and how these signals relate to the physiological output provides us with a colossal challenge for years to come. However, to battle the current obesity epidemic, target-specific drugs need to be developed for which the knowledge of neuronal pathways involved in the maintenance of energy homeostasis will be crucial.

Cardiovascular risk management by blocking the endocannabinoid system.

The specific blockade of endocannabinoids at the level of the cannabinoid receptor 1 (CB (1) receptor) is a new therapeutic option to reduce body weight and manage cardiovascular risk. Although clinical trials are underway to document the safety and efficacy of this approach, much is still unknown about this endogenous system. Endocannabinoids and their receptors are expressed in the central nervous system as well as in the periphery and regulate the central neural circuits for food uptake and peripheral metabolic circuits. Within the context of food uptake, the stimulation of the CB (1) receptor with Delta (9)-tetrahydrocannabinol (Delta (9)-THC) enhances food consumption, while its blockade with receptor antagonists is an emerging relevant therapeutic means to reduce body weight. Rimonabant is the first of a new class of drugs that interferes with the endocannabinoid system by blocking the CB (1) receptor. In recent clinical studies, a substantial reduction in body weight and waist circumference was associated with an improvement of the cardiovascular risk profile. In particular, increased HDL cholesterol, decreased serum triglycerides and improved insulin sensitivity were observed. Further research will serve to establish the role of these compounds in cardiovascular risk management.

Activation of hypothalamic ATP-sensitive K+ channels by the aminoguanidine carboxylate BVT.12777.

Derivatives of 3-guanidinopropionic acid, such as leptin, reduce body weight in obese, diabetic mice. We have assessed whether one of these analogues, BVT.12777 activates intracellular signalling pathways in the arcuate nucleus in a manner analogous to leptin and insulin. In addition, because these hormones have been shown to activate K(ATP) channels in a subset of arcuate neurones, we examined whether this channel is also a functional endpoint for BVT.12777 in the arcuate nucleus. BVT.12777 transiently increased phosphorylation of MAPK, STAT3, PKB and GSK3, in a manner identical to that observed for leptin and insulin. BVT.12777 also hyperpolarized glucose-responsive neurones by increasing the activity of K(ATP) channels. The increase in K(ATP) activity driven by BVT.12777 was PI3-kinase independent, unlike leptin and insulin activation of this channel, and could also be elicited in isolated patches. However, K(ATP) activity induced by BVT.12777 was dependent on actin filament dynamics, both in intact neurones and isolated patches. Thus, BVT.12777 modulates arcuate neurone K(ATP) activity by re-organization of the cytoskeleton, a mechanism that has also been ascribed to leptin and insulin. Consequently, BVT.12777 appears to act as a leptin and insulin mimetic with respect to at least some elements of arcuate neurone intracellular signalling and the activation of K(ATP) channels. Resistance to leptin and insulin, associated with obesity has, at least in part, been postulated to be due to aberrant intracellular signalling in arcuate neurones. The data presented here indicate that it may be possible to develop drugs, which by-pass up-stream signalling components associated with adiposity hormone resistance, such as PI3-kinase, but can still induce functional outputs from arcuate neurones by targeting downstream components of the leptin and insulin signalling cascades.

Noradrenergic receptor mRNA expression in adult rat superficial dorsal horn and dorsal root ganglion neurons.

Noradrenaline (NAdr) has well documented analgesic actions at the level of the spinal cord. Released from bulbospinal projections onto superficial dorsal horn (SDH) neurons, NAdr modulates the excitability of these neurons through the activation of alpha1, alpha2 or beta adrenoceptors. This study utilised in situ hybridisation to determine the specific expression of adrenoceptors within adult rat lumbar SDH and dorsal root ganglion (DRG) neurons, and reports the presence of alpha1A, alpha1B, alpha2B, beta1 and beta2 adrenoceptor mRNA within SDH neurons, and the presence of alpha1A, alpha1B and alpha2C adrenoceptor mRNA within DRG neurons. The present study provides an insight into the modulation of sensory processing at the level of the spinal cord following adrenoceptor activation.

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.

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.

Carbenoxolone depresses spontaneous epileptiform activity in the CA1 region of rat hippocampal slices.

An important contributor to the generation of epileptiform activity is the synchronization of burst firing in a group of neurons. The aim of this study was to investigate whether gap junctions are involved in this synchrony using an in vitro model of epileptiform activity. Hippocampal slices (400 microm) were prepared from female Sprague-Dawley rats (120-170 g). The perfusion of slices with a medium containing no added magnesium and 4-aminopyridine (50 microM) resulted in the generation of spontaneous bursts of population spikes of a fast frequency along with less frequent negative-going bursts. The frequency of the bursts produced was consistent over a 3h period. Carbenoxolone (100 microM), a gap junction blocker and mineralocorticoid agonist, perfused for 75 min, reduced the frequency of both types of spontaneous burst activity. Perfusion of spironolactone (1 microM), a mineralocorticosteroid antagonist, for 15 min prior to and during carbenoxolone perfusion did not alter the ability of carbenoxolone to depress the frequency of spontaneous activity. The incubation of hippocampal slices in carbenoxolone prior to recording increased the time taken for the spontaneous activity to start on change to the zero magnesium/4-aminopyridine medium and decreased the total number of spontaneous bursts over the first 60 min period. The ability of carbenoxolone to delay induction of epileptiform activity and reduce established epileptiform activity suggests that gap junctions contribute to the synchronization of neuronal firing in this model.

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)

Melatonin generates an outward potassium current in rat suprachiasmatic nucleus neurones in vitro independent of their circadian rhythm.

The present study investigated the membrane mechanisms underlying the inhibitory influence of melatonin on suprachiasmatic nucleus (SCN) neurones in a hypothalamic slice preparation. Perforated-patch recordings were performed to prevent the rapid rundown of spontaneous firing rate as observed during whole cell recordings and to preserve circadian rhythmicity in SCN neurones. In current-clamp mode melatonin (1 microM or 1 nM) application, in the presence of agents that block action potential generation and fast synaptic transmission, resulted in a membrane hyperpolarisation accompanied with a decrease in input resistance in the majority of SCN neurones (71-86%). The amplitude of the hyperpolarisation was not found to be significantly different between circadian time 5-12 and 14-21. In voltage-clamp mode melatonin (1 microM or 1 nM) induced an outward current accompanied with an increase in membrane conductance. The current was found to be mainly potassium driven with voltage kinetics resembling those of an open rectifying potassium conductance. Investigations into the signal transduction mechanism revealed melatonin-induced inhibition of SCN neurones to be sensitive to pertussis toxin but independent of intracellular cAMP levels and phospholipase C activity. The present study shows that melatonin, at night-time physiological concentrations, reduces the neuronal excitability of the majority of SCN neurones independent of the time of application in the circadian cycle. Thus in vivo melatonin may be important for circadian time-keeping by amplifying the circadian rhythm in SCN neurones, by lowering their sensitivity to phase-shifting stimuli occurring at night.

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)

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.

Serotonin receptor mRNA expression in rat dorsal root ganglion neurons.

In the present study, we have used in situ hybridization to examine the distribution of serotonin (5-HT) receptors in rat dorsal root ganglion (DRG) neurons. Within DRG neurons, mRNAs for 5-HT1B, 5-HT1D, 5-HT2A, 5-HT2B, 5-HT3B and 5-HT4 receptors were readily detected in small (<25 microm), medium (25-45 microm) and large (>45 microm) diameter neurons. In contrast mRNAs for 5-HT1A, 5-HT1E, 5-HT2C, 5-HT5A, 5-HT5B, 5-HT6 and 5-HT7 receptors were undetectable in these neurons. The present study provides an insight into the molecular profile of 5-HT receptor subtypes in neurons responsible for modulating sensory information.

Aß oligomer toxicity inhibitor protects memory in models of synaptic toxicity.

BACKGROUND AND PURPOSE: Amyloid-ß (Aß) aggregation into synaptotoxic, prefibrillar oligomers is a major pathogenic event underlying the neuropathology of Alzheimer's disease (AD). The pharmacological and neuroprotective properties of a novel Aß aggregation inhibitor, SEN1269, were investigated on aggregation and cell viability and in test systems relevant to synaptic function and memory, using both synthetic Aß(1-42) and cell-derived Aß oligomers. EXPERIMENTAL APPROACH: Surface plasmon resonance studies measured binding of SEN1269 to Aß(1-42) . Thioflavin-T fluorescence and MTT assays were used to measure its ability to block Aß(1-42) -induced aggregation and reduction in cell viability. In vitro and in vivo long-term potentiation (LTP) experiments measured the effect of SEN1269 on deficits induced by synthetic Aß(1-42) and cell-derived Aß oligomers. Following i.c.v. administration of the latter, a complex (alternating-lever cyclic ratio) schedule of operant responding measured effects on memory in freely moving rats. KEY RESULTS: SEN1269 demonstrated direct binding to monomeric Aß(1-42) , produced a concentration-related blockade of Aß(1-42) aggregation and protected neuronal cell lines exposed to Aß(1-42) . In vitro, SEN1269 alleviated deficits in hippocampal LTP induced by Aß(1-42) and cell-derived Aß oligomers. In vivo, SEN1269 reduced the deficits in LTP and memory induced by i.c.v. administration of cell-derived Aß oligomers. CONCLUSIONS AND IMPLICATIONS: SEN1269 protected cells exposed to Aß(1-42) , displayed central activity with respect to reducing Aß-induced neurotoxicity and was neuroprotective in electrophysiological and behavioural models of memory relevant to Aß-induced neurodegeneration. It represents a promising lead for designing inhibitors of Aß-mediated synaptic toxicity as potential neuroprotective agents for treating AD.

Electrophysiological, pharmacological and molecular profile of the transient outward rectifying conductance in rat sympathetic preganglionic neurons in vitro.

Transient outward rectifying conductances or A-like conductances in sympathetic preganglionic neurons (SPN) are prolonged, lasting for hundreds of milliseconds to seconds and are thought to play a key role in the regulation of SPN firing frequency. Here, a multidisciplinary electrophysiological, pharmacological and molecular single-cell rt-PCR approach was used to investigate the kinetics, pharmacological profile and putative K+ channel subunits underlying the transient outward rectifying conductance expressed in SPN. SPN expressed a 4-aminopyridine (4-AP) sensitive transient outward rectification with significantly longer decay kinetics than reported for many other central neurons. The conductance and corresponding current in voltage-clamp conditions was also sensitive to the Kv4.2 and Kv4.3 blocker phrixotoxin-2 (1-10 µM) and the blocker of rapidly inactivating Kv channels, pandinotoxin-Kα (50 nM). The conductance and corresponding current was only weakly sensitive to the Kv1 channel blocker tityustoxin-Kα and insensitive to dendrotoxin I (200 nM) and the Kv3.4 channel blocker BDS-II (1 µM). Single-cell RT-PCR revealed mRNA expression for the α-subunits Kv4.1 and Kv4.3 in the majority and Kv1.5 in less than half of SPN. mRNA for accessory ß-subunits was detected for Kvß2 in all SPN with differential expression of mRNA for KChIP1, Kvß1 and Kvß3 and the peptidase homologue DPP6. These data together suggest that the transient outwardly rectifying conductance in SPN is mediated by members of the Kv4 subfamily (Kv4.1 and Kv4.3) in association with the ß-subunit Kvß2. Differential expression of the accessory ß subunits, which may act to modulate channel density and kinetics in SPN, may underlie the prolonged and variable time-course of this conductance in these neurons.

Short photoperiod-induced decrease of histamine H3 receptors facilitates activation of hypothalamic neurons in the Siberian hamster.

Nonhibernating seasonal mammals have adapted to temporal changes in food availability through behavioral and physiological mechanisms to store food and energy during times of predictable plenty and conserve energy during predicted shortage. Little is known, however, of the hypothalamic neuronal events that lead to a change in behavior or physiology. Here we show for the first time that a shift from long summer-like to short winter-like photoperiod, which induces physiological adaptation to winter in the Siberian hamster, including a body weight decrease of up to 30%, increases neuronal activity in the dorsomedial region of the arcuate nucleus (dmpARC) assessed by electrophysiological patch-clamping recording. Increased neuronal activity in short days is dependent on a photoperiod-driven down-regulation of H3 receptor expression and can be mimicked in long-day dmpARC neurons by the application of the H3 receptor antagonist, clobenproprit. Short-day activation of dmpARC neurons results in increased c-Fos expression. Tract tracing with the trans-synaptic retrograde tracer, pseudorabies virus, delivered into adipose tissue reveals a multisynaptic neuronal sympathetic outflow from dmpARC to white adipose tissue. These data strongly suggest that increased activity of dmpARC neurons, as a consequence of down-regulation of the histamine H3 receptor, contributes to the physiological adaptation of body weight regulation in seasonal photoperiod.