Pharmacology of neuronal elements in the cat spinal dorsal horn

Immunohistochemical, fluorescence and a number of biochemical techniques have demonstrated that high concentrations of cholecystokinin (CCK), somatostatin (SS), substance P (SP), vasoactive intestinal polypeptide (VIP), L-glutamate, 5-hydroxytryptamine (5-HT) and norepinephrine (NE) are present in the superficial parts of the spinal dorsal horn, the same area where small myelinated (A(delta)) and unmyelinated (C) primary afferent fibers are known to terminate. The intent of this project was to study the actions of these substances on the excitability of functionally identified dorsal horn neurons and single cutaneous primary afferent fibers in the cat spinal cord in vivo;Applied iontophoretically and/or by pressure microinjection, CCK-8 and VIP caused a moderate to strong excitation of a majority of tested neurons located in laminae I-VII of intact cat spinal cord. The excitation was not limited to a single population of neurons but was observed in all categories of units recognized in spinal preparations of cats in this area on the basis of their excitability by different kinds of cutaneous afferent input. These results are consistent with the possibility that CCK-8 and VIP act in the dorsal horn of the spinal cord as neurotransmitters or neuromodulators. While in intact cats SP excited about 31% of units activated by innocuous stimulation of the skin and about 80% of units activated by noxious stimulation, this peptide caused excitation of all spinal cord neurons tested in p-chlorophenylalanine (p-CPA)-pretreated cats. Thus, irrespective of their excitatory input, the sensitivity of spinal neurons to SP appears to be increased in the p-CPA-pretreated cats;In order to determine more directly whether SS, L-glutamate and NE exert presynaptic action on the primary afferent fibers, we have measured the electrical excitability changes of intraspinal single cutaneous primary afferent C- and A-fibers during their iontophoretic and/or micropressure application into the spinal cord. SS and L-glutamate increased excitability in about 50% of all fibers tested. NE decreased excitability in a majority of fibers tested. In addition, we have found that both (alpha)- and (beta)-receptor agonists cause a reduction of the excitability of C- and A-fibers, clonidine being the most potent in this respect and isoproterenol the least potent. These results indicate that SS, L-glutamate and NE, in addition to previously shown postsynaptic actions, also modify excitability of intraspinal cutaneous primary afferent fibers

Ghrelin Stimulates Porcine Somatotropes

Ghrelin is an endogenous ligand for growth hormone secretagogue receptor (GHS-R) and is predominantly produced by the stomach and lower amounts in the hypothalamus and various peripheral tissues. Ghrelin is a potent stimulator of growth hormone (GH) secretion from the pituitary in vivo and in vitro. GH secretion from the pituitary also is controlled by two hypothalamic peptides: stimulatory GH-releasing hormone (GHRH) and inhibitory somatostatin-14 (SRIH). GH participates in its own rhythmic secretion through feedback action on GHRH and SRIH neurons. The mechanism of action of GHS is not established. The present study examined the signal transduction pathways of ghrelin in isolated porcine somatotropes. The ability of ghrelin to induce an increase in the intracellular Ca2+ concentration – [Ca2+]i – somatotropes was examined in dispersed porcine pituitary cells using a calcium imaging system. Somatotropes were functionally identified by application of human growth hormone releasing hormone (hGHRH). Ghrelin increased the [Ca2+]i in a dose-dependent manner in 98% of the cells that responded to. In the presence of (D-Lys3)-GHRP-6, a specific receptor antagonist of GHS-R, the increase in [Ca2+]i evoked by ghrelin was decreased. Pretreatment of cultures with somatostatin or neuropeptide Y reduced the ghrelin-induced increase of [Ca2+]i. The stimulatory effect of ghrelin on somatotropes was greatly attenuated in lowcalcium saline and blocked by nifedipine, an L-type calcium channel blocker, suggesting involvement of calcium channels. In a zero Na+ solution, the stimulatory effect of ghrelin on somatotropes was decreased, suggesting that besides calcium channels, sodium channels are also involved in ghrelin-induced calcium transients. Either SQ-22536, an adenylyl cyclase inhibitor, or U73122, a phospholipase C inhibitor, decreased the stimulatory effects of ghrelin on [Ca2+]i transiently, indicating the involvement of adenylyl cyclase-cyclic adenosine monophosphate and phospholipase C inositol 1,4,5-trisphosphate pathways. The non-peptidyl GHS, L-692,585 (L-585), induced changes in [Ca2+]i similar to those observed with ghrelin. Application of L-585 after ghrelin did not have additive effects on [Ca2+]i. Preapplication of L-585 blocked the stimulatory effect of ghrelin on somatotropes. Our results suggest that the actions of ghrelin and synthetic GHS closely parallel each other, in a manner that is consistent with an increase of hormone secretion. An understanding of the molecular mechanisms by which ghrelin and GHS modulate GH secretion is of particular interest in the regulation of GH for muscle accretion and somatic growth

Synergistic effects of physical and chemical guidance cues on neurite alignment and outgrowth on biodegradable polymer substrates

This article demonstrates that directional outgrowth of neurites is promoted by applying a combination of physical and chemical cues to biodegradable polymer substrates. Films of poly-D,L-lactic acid and poly(lactide-co-glycolide) were micropatterned to form grooves on substrate surfaces, using novel indirect transfer techniques developed specifically for biodegradable polymers that cannot be micropatterned directly. Laminin was selectively adsorbed in the grooves. Whole and dissociated dorsal root ganglia were seeded on the substrates and neurite outgrowth and alignment along the microgrooves were measured. The microgrooves provide physical guidance, whereas laminin provides chemical cues to the neurons. The groove depth and spacing were found to significantly influence neurite alignment. The presence of laminin was found to promote neurite adhesion and outgrowth along the grooves. Using a combination of optimized physical and chemical cues, excellent spatial control of directional neurite outgrowth, with up to 95% alignment of neurites, was obtained. The synergistic effect of physical and chemical guidance cues was found to be more effective than individual cues in promoting directional outgrowth of neurites

Subpopulations of Chicken Somatotropes with Differing Intracellular Calcium Concentrations Responses to Secretagogues

Multiple secretagogues stimulate the release of growth hormone (GH). The present studies examined the ability of chicken somatotropes to respond to GH secretagogues with increased intracellular calcium concentrations ([Ca 2+ ]i ). It was hypothesized that there are subsets of the somatotrope population with different responsiveness to the various secretagogues. Avian somatotropes were identified and distinguished from other anterior pituitary cells, by their unique ability to respond to GH-releasing hormone with increased [Ca 2+ ]i with immunocytochemistry used as a post-hoc confirmatory test. Large increases in [Ca 2+ ]i (222 ± 16 nm) were evoked by thyrotropin-releasing hormone in only 73% of the somatotropes. Similarly, [Ca 2+ ]i was increased by perifusion with pituitary adenylate cyclase-activating peptide in 85% and by leptin but only in 51% of somatotropes. Ghrelin acutely increased [Ca 2+ ]i in only 21% of somatotropes. Perfusion with gonadotropinreleasing hormone elevated [Ca 2+ ]i , but in only 40% of somatotropes. The kinetics of calcium transients and the magnitude of the response differed from those observed in the presumptive gonadotropes. It is concluded that there are subsets of the somatotrope population in the anterior pituitary gland with differences in their ability to respond to various secretagogue

Glutamate May Be an Efferent Transmitter That Elicits Inhibition in Mouse Taste Buds

Recent studies suggest that l-glutamate may be an efferent transmitter released from axons innervating taste buds. In this report, we determined the types of ionotropic synaptic glutamate receptors present on taste cells and that underlie this postulated efferent transmission. We also studied what effect glutamate exerts on taste bud function. We isolated mouse taste buds and taste cells, conducted functional imaging using Fura 2, and used cellular biosensors to monitor taste-evoked transmitter release. The findings show that a large fraction of Presynaptic (Type III) taste bud cells (∼50%) respond to 100 µM glutamate, NMDA, or kainic acid (KA) with an increase in intracellular Ca2+. In contrast, Receptor (Type II) taste cells rarely (4%) responded to 100 µM glutamate. At this concentration and with these compounds, these agonists activate glutamatergic synaptic receptors, not glutamate taste (umami) receptors. Moreover, applying glutamate, NMDA, or KA caused taste buds to secrete 5-HT, a Presynaptic taste cell transmitter, but not ATP, a Receptor cell transmitter. Indeed, glutamate-evoked 5-HT release inhibited taste-evoked ATP secretion. The findings are consistent with a role for glutamate in taste buds as an inhibitory efferent transmitter that acts via ionotropic synaptic glutamate receptors

Botulinum Neurotoxin for Pain Management: Insights from Animal Models

The action of botulinum neurotoxins (BoNTs) at the neuromuscular junction has been extensively investigated and knowledge gained in this field laid the foundation for the use of BoNTs in human pathologies characterized by excessive muscle contractions. Although much more is known about the action of BoNTs on the peripheral system, growing evidence has demonstrated several effects also at the central level. Pain conditions, with special regard to neuropathic and intractable pain, are some of the pathological states that have been recently treated with BoNTs with beneficial effects. The knowledge of the action and potentiality of BoNTs utilization against pain, with emphasis for its possible use in modulation and alleviation of chronic pain, still represents an outstanding challenge for experimental research. This review highlights recent findings on the effects of BoNTs in animal pain models

P2 receptors are involved in the mediation of motivation-related behavior

The importance of purinergic signaling in the intact mesolimbic–mesocortical circuit of the brain of freely moving rats is reviewed. In the rat, an endogenous ADP/ATPergic tone reinforces the release of dopamine from the axon terminals in the nucleus accumbens as well as from the somatodendritic region of these neurons in the ventral tegmental area, as well as the release of glutamate, probably via P2Y1 receptor stimulation. Similar mechanisms may regulate the release of glutamate in both areas of the brain. Dopamine and glutamate determine in concert the activity of the accumbal GABAergic, medium-size spiny neurons thought to act as an interface between the limbic cortex and the extrapyramidal motor system. These neurons project to the pallidal and mesencephalic areas, thereby mediating the behavioral reaction of the animal in response to a motivation-related stimulus. There is evidence that extracellular ADP/ATP promotes goal-directed behavior, e.g., intention and feeding, via dopamine, probably via P2Y1 receptor stimulation. Accumbal P2 receptor-mediated glutamatergic mechanisms seem to counteract the dopaminergic effects on behavior. Furthermore, adaptive changes of motivation-related behavior, e.g., by chronic succession of starvation and feeding or by repeated amphetamine administration, are accompanied by changes in the expression of the P2Y1 receptor, thought to modulate the sensitivity of the animal to respond to certain stimuli

P2 receptor-mediated modulation of neurotransmitter release—an update

Presynaptic nerve terminals are equipped with a number of presynaptic auto- and heteroreceptors, including ionotropic P2X and metabotropic P2Y receptors. P2 receptors serve as modulation sites of transmitter release by ATP and other nucleotides released by neuronal activity and pathological signals. A wide variety of P2X and P2Y receptors expressed at pre- and postsynaptic sites as well as in glial cells are involved directly or indirectly in the modulation of neurotransmitter release. Nucleotides are released from synaptic and nonsynaptic sites throughout the nervous system and might reach concentrations high enough to activate these receptors. By providing a fine-tuning mechanism these receptors also offer attractive sites for pharmacotherapy in nervous system diseases. Here we review the rapidly emerging data on the modulation of transmitter release by facilitatory and inhibitory P2 receptors and the receptor subtypes involved in these interactions