Excitatory action of vasopressin in the brain of the rat: role of cAMP signaling.

Brain vasopressin plays a role in behavioral and cognitive functions and in pathological conditions. Relevant examples are pair bonding, social recognition, fear responses, stress disorders, anxiety and depression. At the neuronal level, vasopressin exerts its effects by binding to V1a receptors. In the brainstem, vasopressin can excite facial motoneurons by generating a sustained inward current which is sodium-dependent, tetrodotoxin-insensitive and voltage-gated. This effect is independent of intracellular calcium mobilization and is unaffected by phospholipase Cß (PLCß) or protein kinase C (PKC) inhibitors. There are two major unsolved problems. (i) What is the intracellular signaling pathway activated by vasopressin? (ii) What is the exact nature of the vasopressin-sensitive cation channels? We performed recordings in brainstem slices. Facial motoneurons were voltage-clamped in the whole-cell configuration. We show that a major fraction, if not the totality, of the peptide effect was mediated by cAMP signaling and that the vasopressin-sensitive cation channels were directly gated by cAMP. These channels appear to exclude lithium, are suppressed by 2-aminoethoxydiphenylborane (2-APB) and flufenamic acid (FFA) but not by ruthenium red or amiloride. They are distinct from transient receptor channels and from cyclic nucleotide-regulated channels involved in visual and olfactory transduction. They present striking similarities with cation channels present in a variety of molluscan neurons. To our knowledge, the presence in mammalian neurons of channels having these properties has not been previously reported. Our data should contribute to a better knowledge of the neural mechanism of the central actions of vasopressin, and may be potentially significant in view of clinical applications.

Oxytocin and vasopressin enhance synaptic transmission in the hypoglossal motor nucleus of young rats by acting on distinct receptor types.

Hypoglossal (XII) motoneurons innervate extrinsic and intrinsic muscles of the tongue and control behaviors such as suckling, swallowing, breathing or chewing. In young rats, XII motoneurons express V1a vasopressin and oxytocin receptors. Previous studies have shown that activation of these receptors induces direct powerful excitation in XII motoneurons. In addition, by activating V1a receptors vasopressin can also enhance inhibitory synaptic transmission in the XII nucleus. In the present work, we have further characterized the effect of these neuropeptides on synaptic transmission in the XII nucleus. We have used brainstem slices of young rats and whole-cell patch clamp recordings. Oxytocin enhanced the frequency of spontaneous inhibitory postsynaptic currents by a factor of two and a half. GABAergic and glycinergic events were both affected. The oxytocin effect was mediated by uterine-type oxytocin receptors. Vasopressin and oxytocin also increased the frequency of excitatory synaptic currents, the enhancement being sixfold for the former and twofold for the latter compound. These effects were mediated by V1a and oxytocin receptors, respectively. Miniature synaptic events were unaffected by either vasopressin or oxytocin. This indicates that the peptide-dependent facilitation of synaptic currents was mediated by receptors located on the somatodendritic membrane of interneurons or premotor neurons, and not by receptors sited on axon terminals contacting XII motoneurons. Accordingly, recordings obtained from non-motoneurons located near the border of the XII nucleus showed that part of these cells possess functional V1a and oxytocin receptors whose activation leads to excitation. Some of these neurons could be antidromically activated following electrical stimulation of the XII nucleus, suggesting that they may act as premotor neurons. We propose that in young rats, oxytocin and vasopressin may function as neuromodulators in brainstem motor circuits responsible of tongue movements.

Action of tachykinins in the hippocampus: facilitation of inhibitory drive to GABAergic interneurons.

By acting on neurokinin 1 (NK1) receptors, neuropeptides of the tachykinin family can powerfully excite rat hippocampal GABAergic interneurons located in the CA1 region and by this way indirectly inhibit CA1 pyramidal neurons. In addition to contact pyramidal neurons, however, GABAergic hippocampal interneurons can also innervate other interneurons. We thus asked whether activation of tachykinin-sensitive interneurons could indirectly inhibit other interneurons. The study was performed in hippocampal slices of young adult rats. Synaptic events were recorded using the whole-cell patch clamp technique. We found that substance P enhanced GABAergic inhibitory postsynaptic currents in a majority of the interneurons tested. Miniature, action potential-independent inhibitory postsynaptic currents were unaffected by substance P, as were evoked inhibitory synaptic currents. This suggests that the peptide acted at the somatodendritic membrane of interneurons, rather than at their axon terminals. The effect of substance P was mimicked by a selective NK1 receptor agonist, but not by neurokinin 2 (NK2) or neurokinin 3 (NK3) receptor agonists, and was suppressed by a NK1 selective receptor antagonist. In contrast to substance P, oxytocin, another peptide capable of activating hippocampal interneurons, had no effect on the inhibitory synaptic drive onto interneurons. We conclude that tachykinins, by acting on NK1 receptors, can influence the hippocampal activity by indirectly inhibiting both pyramidal neurons and GABAergic interneurons. Depending on the precise balance between these effects, tachykinins may either activate or depress hippocampal network activity.

Pudendal motoneurons of the rat located in separated spinal nuclei possess nicotinic acetylcholine receptors having distinct pharmacological profiles.

Pudendal motoneurons are located in the ventral horn of the caudal lumbar spinal cord and innervate striated pelvic muscles implicated in sexual and eliminative functions. In rats they are distributed in the dorsomedial (DM) and dorsolateral (DL) nucleus. In male rats, dorsomedial motoneurons innervate the bulbocavernosus, the levator ani and the external anal sphincter, whereas dorsolateral motoneurons control the ischiocavernosus and external urethral sphincter. Using spinal cord slices of young male rats and whole-cell patch-clamp recordings, we investigated the sensitivity of pudendal motoneurons to nicotinic cholinergic agonists. Motoneurons were identified following 1,1'-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine, 4-chlorobenzenesulphonate retrograde labelling. In the presence of atropine, both dorsomedial and dorsolateral motoneurons responded to acetylcholine (ACh) by generating a rapidly activating inward current. By using selective nicotinic antagonists and a nicotinic positive allosteric modulator, we found that nicotinic ACh receptors present in dorsomedial and dorsolateral motoneurons display distinct pharmacological profiles. Whereas the former are of the heteromeric type, the latter are predominantly of the alpha7-containing type. These data were confirmed by light microscopic autoradiography. In young rats, a ligand for heteromeric nicotinic receptors labelled all laminae of the central grey matter, whereas in the ventral part of the central grey, a ligand selective for alpha7-containing nicotinic receptors labelled the DL but not the DM. Dorsolateral and dorsomedial motoneurons innervate two distinct groups of pelvic muscles. A difference in their nicotinic pharmacology may be clinically relevant, as it might allow a selective pharmacological intervention in view of influencing the activity of one or the other set of muscles.

Vasopressin modulates lateral septal network activity via two distinct electrophysiological mechanisms.

The lateral septal area is rich in vasopressin V(1A) receptors and is densely innervated by vasopressinergic axons, originating mainly from the bed nucleus of the stria terminalis and the amygdala. Genetic and behavioral studies provide evidence that activation of vasopressin receptors in this area plays a determinant role in promoting social recognition. What could be the neuronal mechanism underlying this effect? Using rat brain slices and whole-cell recordings, we found that lateral septal neurons are under the influence of a basal GABAergic inhibitory input. Vasopressin, acting via V(1A) but not V(1B) receptors, greatly enhanced this input in nearly all neurons. The peptide had no effect on miniature inhibitory postsynaptic currents, indicating that it acted on receptors located in the somatodendritic membrane, rather than on axon terminals, of GABAergic interneurons. Cell-attached recordings showed that vasopressin can cause a direct excitation of a subpopulation of lateral septal neurons by acting via V(1A) but not V(1B) receptors. The presence in the lateral septum of V(1A) but not of V(1B) receptors was confirmed by competition binding studies using light microscopic autoradiography. In conclusion, vasopressin appears to act in the lateral septum in a dual mode: (i) by causing a direct excitation of a subpopulation of neurons, and (ii) by causing an indirect inhibition of virtually all lateral septal neurons. This modulation by vasopressin of the lateral septal circuitry may be part of the neuronal mechanism by which the peptide, acting via V(1A) receptors, promotes social recognition.

The vasopressin-induced excitation of hypoglossal and facial motoneurons in young rats is mediated by V1a but not V1b receptors, and is independent of intracellular calcium signalling.

As a hormone, vasopressin binds to three distinct receptors: V1a and V1b receptors, which induce phospholipase-Cbeta (PLCbeta) activation and Ca2+ mobilization; and V2 receptors, which are coupled to adenylyl cyclase. V1a and V1b receptors are also present in neurons. In particular, hypoglossal (XII) and facial (VII) motoneurons are excited following vasopressin-V1a receptor binding. The aim of the present study was double: (i) to determine whether V1b receptors contribute to the excitatory effect of vasopressin in XII and VII motoneurons; and (ii) to establish whether the action of vasopressin on motoneurons is mediated by Ca2+ signalling. Patch-clamp recordings were performed in brainstem slices of young rats. Vasopressin depolarized the membrane or generated an inward current. By contrast, [1-deamino-4-cyclohexylalanine] arginine vasopressin (d[Cha4]AVP), a V1b agonist, had no effect. The action of vasopressin was suppressed by Phaa-D-Tyr(Et)-Phe-Gln-Asn-Lys-Pro-Arg-NH2, a V1a antagonist, but not by SSR149415, a V1b antagonist. Thus, the vasopressin-induced excitation of brainstem motoneurons was exclusively mediated by V1a receptors. Light microscopic autoradiography failed to detect V1b binding sites in the facial nucleus. In motoneurons loaded with GTP-gamma-S, a non-hydrolysable analogue of GTP, the effect of vasopressin was suppressed, indicating that neuronal V1a receptors are G-protein-coupled. Intracellular Ca2+ chelation suppressed a Ca2+-activated potassium current, but did not affect the vasopressin-evoked current. H7 and GF109203, inhibitors of protein kinase C, were without effect on the vasopressin-induced excitation. U73122 and D609, PLCbeta inhibitors, were also without effect. Thus, excitation of brainstem motoneurons by V1a receptor activation is probably mediated by a second messenger distinct from that associated with peripheral V1a receptors.

GABA(B) receptor-activation inhibits GABAergic synaptic transmission in parvocellular neurones of rat hypothalamic paraventricular nucleus.

The paraventricular nucleus of the hypothalamus contains three classes of neurones: (i) magnocellular and (ii) parvocellular neurosecretory neurones and (iii) nonendocrine projection neurones. The present study aimed to determine whether functional GABA(B) receptors are present on axon terminals that synapse with parvocellular neurosecretory and nonendocrine paraventricular neurones and to determine how activation of GABA(B) receptors control GABAergic input to these neurones. Whole-cell recordings were performed in coronal hypothalamic slices of the rat containing the paraventricular nucleus. GABA(A) receptor-mediated inhibitory postsynaptic currents (i.p.s.c.) were isolated pharmacologically in the presence of antagonists of glutamatergic ionotropic receptors. We found that baclofen, an agonist of GABA(B) receptors, decreased the frequency of spontaneous and miniature i.p.s.c. It also decreased the amplitude of evoked i.p.s.c. These effects were suppressed by CGP55845A, a competitive antagonist of GABA(B) receptors. CGP55845A also increased the frequency of miniature i.p.s.c. and the amplitude of evoked i.p.s.c., suggesting that, in physiological conditions, presynaptic GABA(B) receptors exert a tonic inhibition on GABA release. Baclofen had no effect on GABA-evoked postsynaptic currents, suggesting that the baclofen-dependent suppression of GABAergic i.p.s.c. was exclusively due to a presynaptic action of the agonist. Our data indicate that GABA(B) receptors are present on axon terminals of GABAergic presynaptic neurones contacting parvocellular neurosecretory and nonendocrine paraventricular neurones, and suggest that GABA(B) receptors exert a tonic inhibition of GABA release from GABAergic terminals. Activation of these receptors causes disinhibition of parvocellular neurosecretory and nonendocrine paraventricular neurones.

Vasopressin facilitates glycinergic and GABAergic synaptic transmission in developing hypoglossal motoneurons.

The hypoglossal nucleus of young rats contains vasopressin binding sites and vasopressin can directly excite hypoglossal motoneurons. In addition, indirect evidence suggests that vasopressin can enhance the synaptic input to motoneurons. We have characterized this latter effect by using brainstem slices and whole-cell recordings. We found that, in the presence of blockers of fast glutamatergic transmission, vasopressin strongly facilitated inhibitory synaptic activity. On average, vasopressin caused a six-fold increase in the frequency and a 1.5-fold increase in the amplitude of GABAergic postsynaptic currents. The effect of vasopressin on glycinergic postsynaptic currents was similar in magnitude. Vasopressin did not affect the frequency of GABAergic or glycinergic miniature postsynaptic currents, indicating that the peptide-induced facilitation of inhibitory transmission was mediated by receptors located on the somatodendritic region rather than on axon terminals of presynaptic neurons. The pharmacological profile of these receptors was determined by using d[Cha4]AVP and dVDAVP, selective agonists of V1b and V2 vasopressin receptors, respectively, and Phaa-D-Tyr-(Et)-Phe-Gln-Pro-Arg-Arg-NH2, a selective antagonist of V1a vasopressin receptors. The two agonists had no effect on the frequency of inhibitory postsynaptic currents. By contrast, the antagonist suppressed the vasopressin-induced facilitation of these currents, indicating that the receptors involved were exclusively of the V1a type. Thus, vasopressin exerts a dual action on hypoglossal motoneurons: a direct excitatory action and an indirect action mediated by GABAergic and glycinergic synapses. By virtue of this dual effect, vasopressin could alter the input-output properties of these motoneurons. Alternatively, it could play a role in generating or modulating specific motor patterns.

Identified spinal motoneurons of young rats possess nicotinic acetylcholine receptors of the heteromeric family.

The aim of the present study was to determine whether, in young rats, spinal motoneurons possess functional nicotinic acetylcholine receptors. Motoneurons were identified either by retrograde labelling or by choline acetyltransferase immunohistochemistry. Whole-cell recordings were performed in spinal cord slices cut at the lumbar level. In voltage clamp, acetylcholine evoked a rapidly activating inward current. In current clamp, it depolarized the motoneuron membrane and induced action potential firing. The acetylcholine-evoked current was strongly reduced by d-tubocurarine or dihydro-beta-erythroidine, broad spectrum nicotinic antagonists, but was almost insensitive to methyllycaconitine, a nicotinic antagonist selective for receptors containing the alpha7 subunit. Moreover, exo-2-(2-pyridyl)-7-azabicyclo[2.2.1]heptane, an alpha7-specific agonist, was without effect. In young animals, light-microscopic autoradiography showed that in the central grey matter all laminae were intensely and equally labelled by [3H]epibatidine. A dense [125I]-alpha-bungarotoxin binding was also found in all laminae, with slightly lower levels in the superficial layers of the dorsal horns and in the ventral part of the grey matter. In adults, the density of [3H]epibatidine binding sites was much lower in the entire grey matter, except in layer 2 of the dorsal horn, and [125I]-alpha-bungarotoxin binding sites were present only in some selected areas. Our data indicate that spinal motoneurons possess functional nicotinic receptors of the heteromeric type and suggest that nicotinic cholinergic transmission may play a significant role in the developing spinal cord.

Comparative distribution of nicotinic receptor subtypes during development, adulthood and aging: an autoradiographic study in the rat brain.

The distribution in the rat brain of high affinity nicotinic heteromeric acetylcholine receptors and of low affinity nicotinic, alpha7-containing, homomeric receptors was studied using in vitro light microscopic autoradiography. As ligands, we used [3H]epibatidine, or [125I]epibatidine, and [125I]alpha-bungarotoxin, respectively. In adult animals, the two types of binding sites were widely distributed in many different brain structures, including the brainstem, cerebellum, mesencephalic structures, limbic system and cortex, but their anatomical distribution differed markedly. Only in rare instances could a co-localization be observed, for example in the superficial layer of the superior colliculus. In developing animals, both types of labeling were strongly expressed during embryonic and postnatal phases. Their distributions were qualitatively similar to those observed in adult animals, with a few noticeable exceptions in the cerebral cortex, hippocampus and brain stem. In aging animals, neither the distribution nor the density of nicotinic binding sites was significantly altered. Our conclusions are the following. (a) There is little overlap in the distribution of heteromeric and alpha7-containing homomeric nicotinic receptors in the rat brain. (b) The abundance of neuronal nicotinic receptors during embryonic and postnatal development suggests that they may play a role in the establishment of neuronal connectivity. (c) The expression of neuronal nicotinic receptors is unaltered in middle aged animals, suggesting that in the rat these receptors do not play any major role in aging process.

Action of tachykinins in the rat hippocampus: modulation of inhibitory synaptic transmission.

Substance P and other neuropeptides of the tachykinin family can powerfully excite CA1 hippocampal interneurons present in the CA1 region. In the present work we show that, by exciting hippocampal interneurons, tachykinins can indirectly inhibit pyramidal neurons. We found that tachykinins caused a decrease in the inhibitory synaptic current interval and an increase in the inhibitory synaptic current amplitude in almost all pyramidal neurons tested. This effect was tetrodotoxin sensitive. Tachykinins did not alter the frequency or amplitude of miniature inhibitory synaptic currents and were without effect on evoked inhibitory synaptic currents. Thus, these neuropeptides acted at the somatodendritic membrane of GABAergic interneurons, rather than at their axon terminals. The effect of substance P on spontaneous inhibitory synaptic currents could be mimicked by a selective agonist of NK1 receptors, but not by selective agonists of NK2 and NK3 receptors. It was suppressed by an NK1 receptor antagonist. In CA1 interneurons located in stratum radiatum, substance P generated a sustained tetrodotoxin-insensitive inward current or induced membrane depolarization and action potential firing. This direct excitatory action was mediated by NK1 receptors. Current-voltage relationships indicate that the net tachykinin-evoked current reversed in polarity at or near the K+ equilibrium potential, suggesting that a suppression of a resting K+ conductance was involved. By increasing the excitability of CA1 GABAergic interneurons, tachykinins can powerfully facilitate the inhibitory synaptic input to pyramidal neurons. This indirect inhibition could play a role in regulating short-term and/or long-term synaptic plasticity, promoting neuronal circuit synchronization or, in some physiopathological situations, influencing epileptogenesis.

Nicotinic acetylcholine receptors: from structure to brain function.

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated ion channels and can be divided into two groups: muscle receptors, which are found at the skeletal neuromuscular junction where they mediate neuromuscular transmission, and neuronal receptors, which are found throughout the peripheral and central nervous system where they are involved in fast synaptic transmission. nAChRs are pentameric structures that are made up of combinations of individual subunits. Twelve neuronal nAChR subunits have been described, alpha2-alpha10 and beta2-beta4; these are differentially expressed throughout the nervous system and combine to form nAChRs with a wide range of physiological and pharmacological profiles. The nAChR has been proposed as a model of an allosteric protein in which effects arising from the binding of a ligand to a site on the protein can lead to changes in another part of the molecule. A great deal is known about the structure of the pentameric receptor. The extracellular domain contains binding sites for numerous ligands, which alter receptor behavior through allosteric mechanisms. Functional studies have revealed that nAChRs contribute to the control of resting membrane potential, modulation of synaptic transmission and mediation of fast excitatory transmission. To date, ten genes have been identified in the human genome coding for the nAChRs. nAChRs have been demonstrated to be involved in cognitive processes such as learning and memory and control of movement in normal subjects. Recent data from knockout animals has extended the understanding of nAChR function. Dysfunction of nAChR has been linked to a number of human diseases such as schizophrenia, Alzheimer's and Parkinson's diseases. nAChRs also play a significant role in nicotine addiction, which is a major public health concern. A genetically transmissible epilepsy, ADNFLE, has been associated with specific mutations in the gene coding for the alpha4 or beta2 subunits, which leads to altered receptor properties.

Presence of functional vasopressin receptors in spinal ventral horn neurons of young rats: a morphological and electrophysiological study.

The objective of the present work was double. (i) Light microscopic autoradiography was used to determine the distribution of vasopressin and oxytocin binding sites in the spinal cord of rats. (ii) Whole-cell recordings were performed in lumbar spinal cord slices in order to assess whether these receptors are functional, whether they are located pre- or postsynaptically and whether they are present in motoneurons. In newborns, vasopressin binding sites of the V1a type were present in all laminae of the central gray at all segmental levels, whereas oxytocin binding sites were found only in the superficial layers of the dorsal horn. In adults, binding sites for both neuropeptides were also present, but were less dense. The dissociation constants for vasopressin were similar in newborns and adults. Whole-cell recordings showed that in identified motoneurons vasopressin exerted a direct effect, by inducing a membrane depolarization or by generating a sustained inward current, and an indirect effect, by enhancing glycinergic and GABAergic inhibitory transmission. Vasopressin-induced facilitation of inhibitory transmission could also be demonstrated in unidentified ventral horn neurons. All these effects were mediated by V1a but not V1b receptors. In some neurons, glycinergic transmission was also facilitated by a selective oxytocin receptor agonist. Our data, together with data obtained previously in brainstem motor nuclei, suggest that vasopressin of hypothalamic origin could play a role in motricity. The neuropeptide could act as a neuromodulator, because it would not directly activate motoneurons, but rather render them more responsive to incoming excitatory inputs. Vasopressin may thus act as a regulator of muscular force.

Nicotinic cholinergic activation of magnocellular neurons of the hypothalamic paraventricular nucleus.

The aim of the present work was to determine whether paraventricular neurons possess functional acetylcholine nicotinic receptors. Using infrared videomicroscopy and differential interference contrast optics, we performed whole-cell recordings in hypothalamic slices containing the paraventricular nucleus. Acetylcholine, locally applied by pressure microejection in the presence of the muscarinic antagonist atropine, evoked a rapidly rising inward current in paraventricular magnocellular endocrine neurons. This current persisted in the presence of blockers of synaptic transmission. It could be reversibly suppressed by nanomolar concentrations of methyllycaconitine, a selective antagonist of alpha 7-containing nicotinic receptors, but was insensitive to micromolar concentrations of dihydro-beta-erythroidine, an antagonist acting preferentially on non-alpha 7 nicotinic receptors. In addition, the effect of acetylcholine could be mimicked by exo-2-(2-pyridyl)-7-azabicyclo[2.2.1]heptane, a recently synthesized nicotinic agonist specific for alpha 7 receptors. Acetylcholine also desensitized paraventricular nicotinic receptors. Desensitization was pronounced and recovery from desensitization was rapid, consistent with the notion that paraventricular nicotinic receptors contain the alpha 7 subunit. Nicotinic currents could not be evoked in paraventricular parvocellular neurons, suggesting that these neurons are devoid of functional nicotinic receptors. The electrophysiological data were corroborated by light microscopic autoradiography, showing that [(125)I]alpha-bungarotoxin binding sites are present in all the magnocellular divisions of the paraventricular nucleus but are undetectable in other areas of this nucleus. Immunohistochemistry, performed using antibodies directed against vasopressin and oxytocin, indicated that responsiveness to nicotinic agonists was a property of vasopressin as well as of oxytocin magnocellular endocrine neurons, in both the paraventricular and the supraoptic nucleus. We conclude that nicotinic agonists can influence the magnocellular neurosecretory system by directly increasing the excitability of magnocellular neurons. By contrast, they are probably without direct effects on paraventricular parvocellular neurons.

Effect of vasopressin on the input-output properties of rat facial motoneurons.

Vasopressin can directly excite facial motoneurons in young rats and mice. It acts by generating a persistent inward current, which is Na(+)-dependent, tetrodotoxin-insensitive and voltage-gated. This peptide-evoked current is unaffected by Ca(++) or K(+) channel blockade and is modulated by extracellular divalent cations. In the present work, we determined how vasopressin alters the input-output properties of facial motoneurons. Whole-cell recordings were obtained from these neurons in the current clamp mode, in brainstem slices of young rats. Repetitive firing was evoked by injecting depolarizing current pulses. Steady-state frequency-current (f-I) relationships were constructed and the effect of vasopressin on these relationships was studied. We found that vasopressin caused a parallel shift to the left of the cell steady-state f-I relationship. This effect persisted in the presence of blockers of K(+) or Ca(++) channels. The peptide effect was distinct from that brought about by Ca(++) channel suppression or by apamin, a blocker of the mAHP. These latter manipulations resulted in an increase in the slope of the steady-state f-I relationship. We conclude that the vasopressin-induced modification of the input-output properties of facial motoneurons is probably exclusively caused by the sodium-dependent, voltage-modulated inward current elicited by the peptide, rather than being due to indirect effects of the peptide on Ca(++) channels, K(+) channels or Ca(++)-dependent K(+) channels. Computer simulation, based on a simple model of facial motoneurons, indicates that the introduction of a conductance having the properties of the vasopressin-dependent conductance can entirely account for the observed peptide-induced shift of the f-I relationship.

Vasopressin- and oxytocin-induced activity in the central nervous system: electrophysiological studies using in-vitro systems.

During the last two decades, it has become apparent that vasopressin and oxytocin, in addition to playing a role as peptide hormones, also act as neurotransmitters/neuromodulators. A number of arguments support this notion: (i) vasopressin and oxytocin are synthesized not only in hypothalamo-neurohypophysial cells, but also in other hypothalamic and extrahypothalamic cell bodies, whose axon projects to the limbic system, the brainstem and the spinal cord. (ii) Vasopressin and oxytocin can be shed from central axons as are classical neurotransmitters. (iii) Specific binding sites, i.e. membrane receptors having high affinity for vasopressin and oxytocin are present in the central nervous system. (iv) Vasopressin and oxytocin can alter the firing rate of selected neuronal populations. (v) In-situ injection of vasopressin and oxytocin receptor agonists and antagonists can interfere with behavior or physiological regulations. Morphological studies and electrophysiological recordings have evidenced a close anatomical correlation between the presence of vasopressin and oxytocin receptors in the brain and the neuronal responsiveness to vasopressin or oxytocin. These compounds have been found to affect membrane excitability in neurons located in the limbic system, hypothalamus, circumventricular organs, brainstem, and spinal cord. Sharp electrode intracellular recordings and whole-cell recordings, done in brainstem motoneurons or in spinal cord neurons, have revealed that vasopressin and oxytocin can directly affect neuronal excitability by opening non-specific cationic channels or by closing K(+) channels. These neuropeptides can also influence synaptic transmission, by acting either postsynaptically or upon presynaptic target neurons or axon terminals. Whereas, in cultured neurons, vasopressin and oxytocin appear to mobilize intracellular Ca(++), in brainstem slices, the action of oxytocin is mediated by a second messenger that is distinct from the second messenger activated in peripheral target cells. In this review, we will summarize studies carried out at the cellular level, i.e. we will concentrate on in-vitro approaches. Vasopressin and oxytocin will be treated together. Though acting via distinct receptors in distinct brain areas, these two neuropeptides appear to exert similar effects upon neuronal excitability.

Role of neuronal nicotinic receptors in the transmission and processing of information in neurons of the central nervous system.

The properties of nicotinic acetylcholine receptors (nAChRs) were studied following exogenous expression in a host system or using whole-cell recordings in brain slices, autoradiography and immunohistochemistry. When expressed in HEK-293 cells, alpha 4 beta 2 nAChRs displayed both a high and a low affinity component. The ratio of these two states was modified by chronic nicotine exposure, resulting in an enhanced sensitivity and a marked reduction in desensitization. Mutations in the gene coding for the alpha 4 subunit are responsible for a particular form of nocturnal epilepsy. When expressed in Xenopus oocytes, alpha 4 beta 2 nAChRs containing these mutations displayed distinct alterations in agonist affinity, desensitization and calcium permeability. Magnocellular endocrine neurons in the supraoptic (SO) nucleus of the hypothalamus were found to express functional alpha 7-containing nAChRs, which could play a role in regulating neurohypophysial peptide secretion. Facial (VII), hypoglossal (XII) and vagal (X) motoneurons of young rats responded to ACh by a fast inward current. The nAChRs present in VII and XII nuclei were of the non-alpha 7-containing type, whereas those present in the X nucleus contained the alpha 7 subunit. In Bcl-2 transgenic mice, facial nerve axotomy caused nAChRs downregulation by interfering negatively with the expression of the alpha 4 subunit. Binding sites corresponding to alpha 7-containing nAChRs were also detected in spinal motor nuclei and axotomy provoked a reduction of the binding. Together, these data indicate that long-term exposure to nicotine can promote neuroadaptive changes in nAChRs and that genetic alterations of neuronal nAChRs can result in transmissible neurological diseases. They also suggest that these receptors probably play a role in the central regulation of autonomic functions, as well as in motor control.

Nicotinic acetylcholine receptors in neonatal motoneurons are regulated by axotomy: an electrophysiological and immunohistochemical study in human bcl-2 transgenic mice.

Motoneuron axotomy was exploited as a model system for studying functional and morphological changes caused in motoneuron cell bodies by peripheral axon injury. Rodent facial motoneurons express functional nicotinic acetylcholine receptors. We have determined the effect of neonatal unilateral facial nerve transection on these receptors by using electrophysiological and immunohistochemical techniques. To avoid rapid apoptotic cell death of axotomized motoneurons, the study was done in mice overexpressing the human bcl-2 transgene. Intact motoneurons responded to acetylcholine by generating a rapidly rising inward current, which was insensitive to methyllycaconitine, a selective antagonist of alpha7-containing nicotinic receptors, but was suppressed by dihydro-beta-erythroidine, a broad-spectrum antagonist. This indicates that mouse facial motoneurons possess nicotinic receptors which are probably devoid of the alpha7 subunit. In striking contrast, axotomized motoneurons displayed little or no sensitivity to acetylcholine. Axotomy did not affect the sensitivity of facial motoneurons to the selective glutamate receptor agonist alpha-amino-3-hydroxy-5-methyl-4-isoxaxolepropionic acid. Immunohistochemical studies revealed that the alpha4 nicotinic receptor subunit was present in intact motoneurons but was undetectable in axotomized motoneurons. By contrast, the beta2 subunit was comparable in intact and axotomized motoneurons. alpha3 immunoreactivity was undetectable, both in intact and in axotomized motoneurons.Thus, mouse facial nicotinic receptors are possibly of the alpha4beta2 type and axotomy interferes negatively with the expression of the alpha4 subunit. By down-regulating nicotinic receptors, peripheral nerve injury may facilitate motoneuron degeneration. Alternatively, nicotinic receptor downregulation and motoneuron degeneration may be independent consequences of peripheral axotomy.

Oxytocin receptor agonists enhance inhibitory synaptic transmission in the rat hippocampus by activating interneurons in stratum pyramidale.

Oxytocin probably plays a role as a neurotransmitter/neuromodulator in the hippocampus of the rat. Oxytocin binding sites are present in the subiculum and CA1 region and oxytocin can excite a class of CA1 nonpyramidal neurons. In the present work we characterized the effect of oxytocin on hippocampal synaptic transmission. Whole-cell recordings were obtained from pyramidal neurons, in conditions of nearly symmetrical chloride concentrations. The selective oxytocin receptor agonist, [Thr4,Gly7]-oxytocin (TGOT), caused an increase in the frequency and amplitude of spontaneous inhibitory postsynaptic currents (IPSCs) in virtually all neurons. These peptide-enhanced IPSCs were blocked by bicuculline, but not by strychnine, and reversed near 0 mV, indicating that they were mediated by gamma-aminobutyric acid (GABA)A receptors. On average, TGOT caused a nearly threefold increase in the frequency and almost a doubling in the amplitude of spontaneous IPSCs. TGOT did not influence the frequency and the amplitude of miniature IPSCs or spontaneous excitatory postsynaptic currents (EPSCs), and had no effect on evoked IPSCs. The peptide did not affect the basic membrane properties of pyramidal neurons or their GABA sensitivity. Thus, TGOT facilitated inhibitory transmission by exerting an excitatory action on the soma and/or dendrites of GABAergic interneurons. Extracellular recordings were performed in interneurons located in various hippocampal strata. Their sensitivity to TGOT was compared to that of substance P (SP). Interneurons in stratum pyramidale were excited both by TGOT and by SP. By contrast, stratum radiatum interneurons responded to SP but not to TGOT. In stratum oriens, half of the interneurons responded to SP, but only a minority to TGOT. Thus, oxytocin-responsive interneurons appear to be preferentially located in close vicinity of pyramidal neurons.

Involvement of calmodulin-dependent protein kinase II in carbachol-induced rhythmic activity in the hippocampus of the rat.

The role of calcium and protein kinases in rhythmic activity induced by muscarinic receptor activation in the CA1 area in rat hippocampal slices was investigated. Extracellular recording showed that carbachol (20 microM) induced synchronized field potential activity with a dominant frequency of 7.39+/-0.68 Hz. Pretreatment with the membrane permeable Ca(2+) chelator BAPTA-AM (50 microM) or with thapsigargin (1 microM), a compound which depletes intracellular calcium stores, reduced the dominant power of carbachol-induced theta-like activity by 83% and 78%, respectively. Inhibition of calmodulin-dependent protein kinase II (CaMKII) by the cell permeable inhibitor KN-93 (10 microM) reduced the power of carbachol-induced theta-like activity by 80%. In contrast the protein kinase C (PKC) inhibitor calphostin C did not significantly (P>0.05) affect the effect of carbachol. Whole-cell recording indicated that KN-93 also blocked carbachol-induced suppression of slow I(AHP) and strongly inhibited the carbachol-induced plateau potential. Our data suggest that activation of CaMKII by carbachol is crucial for local theta-like activity in the CA1 area of the rat hippocampus in vitro. Furthermore, involvement of CaMKII in carbachol-induced suppression of the slow I(AHP) and the induction of plateau potentials could play a role in the induction of theta-like rhythmic activity by carbachol.