Biomedical subjects
David Spanswick
Publications and source records attributed to David Spanswick.
Histamine excites neonatal rat sympathetic preganglionic neurons in vitro via activation of H1 receptors.
The role of histamine in regulating excitability of sympathetic preganglionic neurons (SPNs) and the expression of histamine receptor mRNA in SPNs was investigated using whole-cell patch-clamp electrophysiological recording techniques combined with single-cell reverse transcriptase polymerase chain reaction (RT-PCR) in transverse neonatal rat spinal cord slices. Bath application of histamine (100 microM) or the H1 receptor agonist histamine trifluoromethyl toluidide dimaleate (HTMT; 10 microM) induced membrane depolarization associated with a decrease in membrane conductance in the majority (70%) of SPNs tested, via activation of postsynaptic H1 receptors negatively coupled to one or more unidentified K+ conductances. Histamine and HTMT application also induced or increased the amplitude and/or frequency of membrane potential oscillations in electrotonically coupled SPNs. The H2 receptor agonist dimaprit (10 microM) or the H3 receptor agonist imetit (100 nM) were without significant effect on the membrane properties of SPNs. Histamine responses were sensitive to the H1 receptor antagonist triprolidine (10 microM) and the nonselective potassium channel blocker barium (1 mM) but were unaffected by the H2 receptor antagonist tiotidine (10 microM) and the H3 receptor antagonist, clobenpropit (5 microM). Single cell RT-PCR revealed mRNA expression for H1 receptors in 75% of SPNs tested, with no expression of mRNA for H2, H3, or H4 receptors. These data represent the first demonstration of H1 receptor expression in SPNs and suggest that histamine acts to regulate excitability of these neurons via a direct postsynaptic effect on H1 receptors.
Angiotensin II induces calcium-dependent rhythmic activity in a subpopulation of rat hypothalamic median preoptic nucleus neurons.
Whole cell patch-clamp recordings revealed a subpopulation (16%, n = 18/112) of rat median preoptic nucleus (MnPO) neurons responded to bath-applied angiotensin II (Ang II; 100 nM to 5 microM; 30-90 s) with a prolonged TTX-resistant membrane depolarization and rhythmic bursting activity. At rest, cells characteristically displayed relatively low input resistance and negative resting potentials. Ang-II-induced responses featured increased input resistance, a reversal potential of -95 +/- 2 mV, an increase in action potential duration from 2.9 +/- 0.5 to 4.3 +/- 0.8 ms, and the appearance of a rebound excitation at the offset of membrane responses to hyperpolarizing current injection. The latter was sensitive to Ni2+ (0.5-1 mM; n = 5), insensitive to extracellular Cs+ (1 mM, n = 7), and intracellular QX-314 (4 mM, n = 5), consistent with activation of a T-type Ca2+ conductance. Coincident with the Ang-II-induced depolarization was the appearance of rhythmic depolarizing shifts at a frequency of 0.14 +/- 0.09 Hz with superimposed bursts of 4-22 action potentials interspersed with silent periods persisting for >1 h after washout. These TTX-resistant depolarizing shifts increased in amplitude and decreased in frequency with membrane hyperpolarization with activity ceasing beyond approximately -80 mV, and were abolished in low-Ca(2+)/high-Mg2+ bathing medium (n = 6), Co2+ (1 mM; n = 6), or Ni2+ (0.5-1 mM; n = 8). Thus in a subpopulation of MnPO neurons, Ang II induces "pacemaker-like" activity by reducing a K(+)-dependent leak conductance that contributes to resting membrane potential and promoting of Ca(2+)-dependent regenerative auto-excitation mediated, in part, by a T-type Ca2+ conductance.
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.
Nitric oxide inhibits NMDA currents in a subpopulation of substantia gelatinosa neurons of the adult rat spinal cord.
In the present study, the actions of nitric oxide (NO) on NMDA mediated excitatory neurotransmission in substantia gelatinosa (SG) neurons of the adult rat spinal cord were investigated. Bath application of the NO donor sodium-nitroso-N-acetylpenicillamine (SNAP) reversibly inhibited NMDA receptor mediated excitatory postsynaptic currents evoked by electrical stimulation of the dorsal root. These effects of SNAP were prevented by pre-incubation with the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy PTIO) or the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-alpha]quinoxalin-1-one, and were mimicked by the membrane permeable cGMP analogue dibutyryl cGMP. Finally, these effects were shown to be mediated at a postsynaptic level using acutely applied exogenous NMDA. In conclusion, we demonstrate for the first time the inhibition of NMDA mediated excitatory neurotransmission by NO in spinal cord SG neurons, an action mediated at least in part via the activation of guanylate cyclase.
Activation and integration of bilateral GABA-mediated synaptic inputs in neonatal rat sympathetic preganglionic neurones in vitro.
The role of GABA receptors in synaptic transmission to neonatal rat sympathetic preganglionic neurones (SPNs) was investigated utilizing whole-cell patch clamp recording techniques in longitudinal and transverse spinal cord slice preparations. In the presence of glutamate receptor antagonists (NBQX, 5 microm and D-APV, 10 microm), electrical stimulation of the ipsilateral or contralateral lateral funiculi (iLF and cLF, respectively) revealed monosynaptic inhibitory postsynaptic potentials (IPSPs) in 75% and 65% of SPNs, respectively. IPSPs were sensitive to bicuculline (10 microM) in all neurones tested and reversed polarity around -55 mV, the latter indicating mediation via chloride conductances. In three neurones IPSPs evoked by stimulation of the iLF (n = 1) or cLF (n = 2) were partly sensitive to strychnine (2 microM). The expression of postsynaptic GABA(A) and GABA(B) receptors were confirmed by the sensitivity of SPNs to agonists, GABA (2 mm), muscimol (10-100 microM) or baclofen (10-100 microM), in the presence of TTX, each of which produced membrane hyperpolarization in all SPNs tested. Muscimol-induced responses were sensitive to bicuculline (1-10 microM) and SR95531 (10 microM) and baclofen-induced responses were sensitive to 2-hydroxy-saclofen (100-200 microM) and CGP55845 (200 nM). The GABA(C) receptor agonist CACA (200 microM) was without significant effect on SPNs. These results suggest that SPNs possess postsynaptic GABA(A) and GABA(B) receptors and that subsets of SPNs receive bilateral GABAergic inputs which activate GABA(A) receptors, coupled to a chloride conductance. At resting or holding potentials close to threshold either single or bursts (10-100 Hz) of IPSPs gave rise to a rebound excitation and action potential firing at the termination of the burst. This effect was mimicked by injection of small (10-20 pA) rectangular-wave current pulses, which revealed a time-dependent, Cs(+)-sensitive inward rectification and rebound excitation at the termination of the response to current injection. Synaptic activation of a rebound excitation mediated by a time-dependent inward rectification expressed intrinsically by SPNs may provide a novel mechanism enabling SPNs to be entrained to rhythms driven from the brainstem or higher centres.
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 micro M. 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.
Leading the charge--pioneering treatments in the fight against neuropathic pain.
Neuropathic pain, arising from nerve injury, results in a chronic and debilitating form of pain that in the past has been poorly diagnosed and treated. During the past few years, intensive research has resulted in major progress towards understanding the basic mechanisms that contribute to this condition, and the renewed possibility of safe and effective medicines. Indeed, the next few years should see the first fruits of this labour reaching the market place and, with them, the opportunity to assess whether the scientific advances achieved have resulted in significant clinical improvement. In this article, some of the key compounds that will lead this charge and the clinical results obtained so far are discussed.
Emerging antiobesity drugs.
The healthcare burden that the obesity epidemic now poses in highly significant, in part due to increased risk of secondary chronic diseases such as hypertension. A lack of physical activity and high fat diets are major factors contributing to this condition. However, increasingly apparent is the genetic predisposition of individuals and ethnic groups to obesity. Present treatment strategies are currently inadequate and unlikely to have a major effect on the future prevalence of obesity. To slow the obesity epidemic, the source needs to be tackled now through fundamental research into the mechanisms by which obesity is manifest, and education on the risks and how to prevent it. This article will describe current and emerging treatments for obesity and review the recent advances in research that may provide the antiobesity treatments of the future. Research into obesity has escalated at considerable pace, catalysed by the discovery of the obese gene product leptin. Leptin is secreted by adipose tissue and acts via specific receptors in the brain to engage central neural pathways involved in regulating energy homeostasis. Since this discovery, numerous significant advances have been made in our understanding of how the brain integrates and responds to central and peripheral signals involved in maintaining energy homeostasis, and how disruption of these signalling mechanisms can manifest as obesity. As a consequence of these findings, numerous potential sites for therapeutic intervention into this condition have and are materializing. The aim of this review is to highlight current treatment strategies for obesity, recent advances in our understanding of the central neural control of energy balance, and what the authors consider to be the most promising targets for the development of novel antiobesity drugs in the future. Thus, the review focuses on leptin, neuropeptide Y, melanocortin and ghrelin signalling at the level of the CNS, and strategies targeting the sympathetic innervation of fat cells at the periphery.
Active and passive membrane properties of rat sympathetic preganglionic neurones innervating the adrenal medulla.
The intravascular release of adrenal catecholamines is a fundamental homeostatic process mediated via thoracolumbar spinal sympathetic preganglionic neurones (AD-SPN). To understand mechanisms regulating their excitability, whole-cell patch-clamp recordings were obtained from 54 retrogradely labelled neonatal rat AD-SPN. Passive membrane properties included a mean resting membrane potential, input resistance and time constant of -62 +/- 6 mV, 410 +/- 241 MOmega and 104 +/- 53 ms, respectively. AD-SPN were homogeneous with respect to their active membrane properties. These active conductances included transient outward rectification, observed as a delayed return to rest at the offset of the membrane response to hyperpolarising current pulses, with two components: a fast 4-AP-sensitive component (A-type conductance), contributing to the after-hyperpolarisation (AHP) and spike repolarisation; a slower prolonged Ba(2+)-sensitive component (D-like conductance). All AD-SPN expressed a Ba(2+)-sensitive instantaneous inwardly rectifying conductance activated at membrane potentials more negative than around -80 mV. A potassium-mediated, voltage-dependent sustained outward rectification activated at membrane potentials between -35 and -15 mV featured an atypical pharmacology with a component blocked by quinine, reduced by low extracellular pH and arachidonic acid, but lacking sensitivity to Ba(2+), TEA and intracellular Cs(+). This quinine-sensitive outward rectification contributes to spike repolarisation. Following block of potassium conductances by Cs(+) loading, AD-SPN revealed the capability for autorhythmicity and burst firing, mediated by a T-type Ca(2+) conductance. These data suggest the output capability is dynamic and diverse, and that the range of intrinsic membrane conductances expressed endow AD-SPN with the ability to generate differential and complex patterns of activity. The diversity of intrinsic membrane properties expressed by AD-SPN may be key determinants of neurotransmitter release from SPN innervating the adrenal medulla. However, factors other than active membrane conductances of AD-SPN must ultimately regulate the differential ratio of noradrenaline (NA) versus adrenaline (A) release secreted in response to various physiological and environmental demands.