David Spanswick

Insulin activates ATP-sensitive K + channels in hypothalamicneurons 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 KATP channel function is crucial to physiological regulation of food intake and body weight.

Orexigen-sensitive NPY/AgRP pacemaker neurons in the hypothalamic arcuate nucleus

The hypothalamic arcuate nucleus (ARC) integrates and responds to satiety and hunger signals and forms the origins of the central neural response to perturbations in energy balance. Here we show that rat ARC neurons containing neuropeptide Y (NPY) and agouti-related protein (AgRP), which are conditional pacemakers, are activated by orexigens and inhibited by the anorexigen leptin. We propose a neuron-specific signaling mechanism through which central and peripheral signals engage the central neural anabolic drive.

Leptin mediates the increase in blood pressure associated with obesity

Summary Obesity is associated with increased blood pressure (BP), which in turn increases the risk of cardiovascular diseases. We found that the increase in leptin levels seen in diet-induced obesity (DIO) drives an increase in BP in rodents, an effect that was not seen in animals deficient in leptin or leptin receptors (LepR). Furthermore, humans with loss-of-function mutations in leptin and the LepR have low BP despite severe obesity. Leptin’s effects on BP are mediated by neuronal circuits in the dorsomedial hypothalamus (DMH), as blocking leptin with a specific antibody, antagonist, or inhibition of the activity of LepR-expressing neurons in the DMH caused a rapid reduction of BP in DIO mice, independent of changes in weight. Re-expression of LepRs in the DMH of DIO LepR-deficient mice caused an increase in BP. These studies demonstrate that leptin couples changes in weight to changes in BP in mammalian species.

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.

Leptin activates ATP-sensitive potassium channels in the rat insulin-secreting cell line, CRI-G1

1 Whole‐cell current‐clamp recordings demonstrate that leptin (0.3–10 nm) hyperpolarizes CRI‐G1 insulin‐secreting cells. This effect is slow on onset and is not reversed on washout of the leptin. 2 Voltage‐clamp recordings indicate that leptin activates a potassium conductance in the presence of intracellular ATP (5 mm), but has no effect in its absence. Following activation of ATP‐sensitive K+ (KATP) current by diazoxide (0.2 mm), addition of leptin did not alter cell membrane potential or potassium current further. 3 The leptin‐induced hyperpolarization and increased potassium conductance are completely inhibited by the application of the sulphonylureas tolbutamide (100 μm) and glibenclamide (0.5 μm). 4 Cell‐attached and inside‐out single‐channel recordings indicate that leptin activates tolbutamide‐sensitive KATP channels in CRI‐G1 insulin‐secreting cells.

Orexins induce increased Excitability and Synchronisation of Rat Sympathetic Preganglionic Neurones

The neuropeptides orexin A and B are synthesised by perifornical and lateral hypothalamic (LH) neurones and exert a profound influence on autonomic sympathetic processes. LH neurones project to spinal areas containing sympathetic preganglionic neurones (SPNs) and therefore may directly modulate sympathetic output. In the present study we examined the possibility that orexinergic inputs from the LH influence SPN activity. Orexin‐positive neurones in the LH were labelled with pseudorabies virus injected into the liver of parasympathetically denervated animals and orexin fibres were found adjacent to the soma and dendrites of SPNs. Orexin A or B (10–1000 nm) directly and reversibly depolarised SPNs in spinal cord slices. The response to orexin A was significantly reduced in the presence of the orexin receptor 1 (OX1R) antagonist SB334867A at concentrations of 1–10 μm. Single cell reverse transcriptase‐polymerase chain reaction revealed expression of mRNA for both OX1R and OX2R in the majority of orexin‐sensitive SPNs. The orexin‐induced depolarisation involved activation of pertussis toxin‐sensitive G‐proteins and closure of a K+ conductance via a protein kinase A (PKA)‐dependent pathway that did not require an increase in intracellular Ca2+. Orexins also induced biphasic subthreshold membrane potential oscillations and synchronised activity between pairs of electrically coupled SPNs. Coupling coefficients and estimated junctional conductances between SPNs were not altered indicating synchronisation is due to activation of previously silent coupled neurones rather than modulation of gap junctions. These findings are consistent with a direct excitation and synchronisation of SPNs by orexinergic neurones that in vivo could increase the frequency and coherence of sympathetic nerve discharges and mediate LH effects on sympathetic components of energy homeostasis and cardiovascular control.

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.

Electrotonic coupling between rat sympathetic preganglionic neurones in vitro.

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

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.

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.