Central noradrenergic neurons signal via ATP to elicit vasopressin responses to haemorrhage.

It is now clear that ATP acts as a neurotransmitter in both the peripheral and central nervous systems. In the periphery, purinergic transmission has been best studied at certain sympathetic neuroeffector junctions where ATP, co-localized with noradrenaline, is used to elicit the primary post-junctional response. More recently, several groups have raised the possibility that central catecholaminergic neurons might use ATP in a similar fashion. Accordingly, we now present findings from immediate early gene expression and electrophysiological studies which indicate that ATP, acting through P2 purinoreceptors, is used as a transmitter by caudal brainstem noradrenergic neurons, the A1 group, in their interaction with vasopressinergic neurosecretory cells. Supraoptic nucleus vasopressin cell responses to moderate haemorrhage, known to be generated by the A1 projection, were suppressed by hypothalamic application of the P2 receptor antagonist suramin. However, suramin did not alter vasopressin cell responses to osmotic challenge or severely hypotensive haemorrhage, two stimuli known to excite vasopressin cells independently of the A1 projection. These data are consistent with an identity of action between the A1 input to vasopressin cells and the activation of ATP receptors on vasopressin cells. The use of ATP as a transmitter by other catecholamine neurons in the brain awaits further confirmation, but the present findings suggest that in certain instances the therapeutic manipulation of central catecholamine neuron output might best be achieved with pharmacological agents which target purinergic rather than adrenergic transmission.

Rat vasopressin cell responses to simulated hemorrhage: stimulus-dependent role for A1 noradrenergic neurons.

c-fos expression mapping and electrophysiological recording experiments were done to clarify the role of the A1 noradrenergic cell group in the vasopressin response to hypotensive hemorrhage. In pentobarbital-anesthetized rats, moderate and severe hypotensive hemorrhages were simulated by brief occlusion of the inferior vena cava sufficient to reduce mean arterial pressure to approximately 50 or 30 mmHg, respectively. Both stimuli significantly increased the number of A1 region catecholamine cells displaying Fos-like immunoreactivity, this effect being most prominent at the level of the area postrema. Both stimuli also increased the number of supraoptic nucleus vasopressin cells displaying Fos-like immunoreactivity. Accordingly, electrophysiological studies involving separate animals confirmed that both moderate and severe caval occlusion significantly increased the firing of functionally identified vasopressin cells recorded in the supraoptic nucleus. However, although interruption of A1 region neuronal function by injection of gamma-aminobutyric acid at the level of the area postrema eliminated the increase in vasopressin cell firing elicited by moderate caval occlusion, it did not block the response to severe caval occlusion. These findings suggest that, in the rat, the vasopressin response to an acute reduction in central blood volume, such as that produced by hemorrhage, depends on the A1 projection only if the stimulus is of moderate intensity. Severe stimuli appear to involve activation of both the A1 projection and an additional vasopressin-stimulatory pathway that bypasses the A1 region.

Initiation of rat vasopressin cell responses to simulated hypotensive hemorrhage.

Hypotensive hemorrhage is a major stimulus for vasopressin (VP) release, but in rats it is uncertain which receptors initiate this response. We have investigated this issue using transient occlusion of the inferior vena cava to simulate hypotensive hemorrhage. Single-unit recording experiments done in the supraoptic nucleus of pentobarbital-anesthetized rats demonstrated that severe caval occlusion, sufficient to drop mean arterial pressure (MAP) below 30 mmHg, excited 88% of putative VP neurosecretory cells and a similar proportion of putative oxytocin (OT) cells. Responsive VP cells increased their firing by 8.5 +/- 0.6 spikes/s within 11.2 +/- 0.8 s of the fall in MAP. This response was unrelated to the size of the fall in MAP and was unchanged by combined sinoaortic denervation (SAD) and vagal denervation, by T1 spinal section, or by administration of the angiotensin-converting enzyme inhibitor captopril, except that spinal section decreased the response latency. Moderate caval occlusion, sufficient to drop MAP to approximately 50 mmHg, did not excite any of the OT cells tested but did excite 65% of VP cells, causing a 3.8 +/- 0.3 spikes/s increase in firing after a delay of 9.0 +/- 1.3 s. This response was proportional to the size of the preceding fall in MAP, and after combined SAD and vagal denervation only 20% of VP cells still responded. Elimination of sinoaortic or vagal afferents alone had no effect on VP cell responses to moderate caval occlusion, except that SAD significantly increased the response latency. These data suggest that in rat the mechanisms that initiate the VP response to hypotensive hemorrhage depend on stimulus intensity.(ABSTRACT TRUNCATED AT 250 WORDS)

Differing effects of electrical and chemical parabrachial nucleus stimulation on supraoptic vasopressin cells.

Electrical stimulation of the parabrachial nucleus stimulates vasopressin secretion. To determine whether this is due to activation of intrinsic projection neurons or fibers of passage we compared, in anaesthetized rats, the effects of electrical and chemical parabrachial nucleus stimulation on the activity of neurosecretory vasopressin cells of the supraoptic nucleus. Electrical stimulation excited 35% and inhibited 14% of vasopressin cells tested (257 tests). Parabrachial nucleus injections of the neuroexcitant glutamate (0.2, 10 or 200 mM) altered the activity of only 6% of vasopressin cells tested (118 tests). At the highest glutamate concentration, which produced clear changes in blood pressure, all responsive units were inhibited (4/22 cells). These data suggest that previous reports indicating a facilitatory role for parabrachial nucleus neurons in the regulation of vasopressin secretion are likely to be attributable to effects on fibers of passage.

Neuropeptide Y modulation of A1 noradrenergic neuron input to supraoptic vasopressin cells.

A1 noradrenaline (NA) neurons provide a direct excitatory input to supraoptic nucleus (SON) vasopressin (VP) cells. Many A1 cells contain neuropeptide Y (NPY) and past studies have established that NPY exerts excitatory postsynaptic effects on VP cell activity. We have now investigated whether NPY might also modulate A1 input to VP cells via presynaptic mechanisms. Experiments done in pentobarbitone-anesthetized rats demonstrated that SON application of NPY (10 microM) excited VP cells but also depressed their response to activation of the A1 input. These two effects were not correlated, suggesting independent mechanisms. The putative Y1 agonist [Leu31,Pro34]NPY (10 microM) also excited VP cells but did not alter their response to activation of the A1 input. In contrast, the putative Y2 receptor agonist Ac-[Leu28,Leu31]NPY24-36 mimicked the synaptic depression produced by NPY but did not significantly alter spontaneous activity. These data are consistent with the proposal that NPY acts on Y1-like receptors to excite VP cells but can also act on a presynaptic Y2-like receptor to depress A1-VP cell synaptic transmission.

Alpha 2-adrenoceptor modulation of A1 noradrenergic neuron input to supraoptic vasopressin cells.

The A1 noradrenaline (NA) cell group of the caudal medulla provides a direct excitatory input to supraoptic nucleus (SON) vasopressin (VP) cells. We have now investigated the possibility that NA released from A1 terminals acts presynaptically to modulate A1 input to VP cells. Initial experiments done in pentobarbitone anaesthetized rats established that SON application of NA excited VP cells but also depressed their response to activation of the A1 input. Moreover, the latter effect was mimicked by the alpha 2-adrenoceptor agonist clonidine and antagonised by intravenous administration of the alpha 2-adrenoceptor antagonist yohimbine. Further studies showed that yohimbine also prevented the relative decline in synaptic excitation of VP cells normally observed as A1 activation frequency increases. These data are consistent with the proposal that NA, released from the SON terminals of A1 NA cells, acts via alpha 2-adrenoceptors to depress A1 transmitter release and thus A1 influence on VP cells.

ATP mediates an excitatory noradrenergic neuron input to supraoptic vasopressin cells.

Although A1 noradrenaline (NA) neurons of the caudal medulla provide a direct, excitatory input to supraoptic vasopressin cells, they do not use NA as their primary transmitter. We have now tested the possibility that adenosine 5'-triphosphate (ATP) may fulfill this role. Extracellular recordings from rat supraoptic nucleus demonstrated that locally applied ATP excites neurosecretory vasopressin cells and that this effect is mimicked by the ATP receptor-agonist alpha,beta-methylene ATP and blocked by the ATP receptor-blocker suramin. Suramin did not block the excitatory effect of locally applied NA on vasopressin cells but did block excitations produced by vagus nerve stimulation, such excitations having previously been shown to involve a pathway in which the final relay is an input from the A1 cell group. These results indicate that certain central NA neurons use ATP as a transmitter and also provide the first demonstration of a specific physiological role for central purinergic neurons, i.e. regulation of secretion of the neurohormone vasopressin.

Locus coeruleus effects on baroreceptor responsiveness and activity of neurosecretory vasopressin cells.

The locus coeruleus (LC) has previously been implicated in the regulation of vasopressin secretion. To further investigate this issue experiments were done in which extracellular recordings were obtained from functionally identified neurosecretory vasopressin (VP) cells of the rat supraoptic nucleus. Electrolytic lesions of the ipsilateral LC reduced the proportion of VP cells inhibited by carotid baroreceptor activation from 93% to 35%; the inhibitory effect of aortic depressor nerve stimulation was unchanged. Electrical stimulation of the LC altered the discharge probability of 20% of VP cells tested, the predominant effect being excitation. In contrast to the effects of electrolytic lesions and electrical stimulation, neither chemical inhibition nor stimulation of the LC, by local injection of neuroactive amino acids, altered VP cell baroreceptor responsiveness or spontaneous discharge. These data indicate that while fibres of passage in the LC region can influence VP cell excitability, particularly responses to carotid baroreceptor activation, LC cells do not regulate VP cell function or, by implication, the secretion of this vasoactive and antidiuretic hormone.

A1 neurons and excitatory amino acid receptors in rat caudal medulla mediate vagal excitation of supraoptic vasopressin cells.

Extracellular recordings from the supraoptic nucleus of the rat established that vasopressinergic neurosecretory cells were excited by stimulation of cervical but not abdominal vagal afferents. This response was absent or significantly attenuated after microinjection of gamma-aminobutyric acid into a region of the caudal medulla known to contain the A1 noradrenaline cell group. Consistent with the possible involvement of the A1 group, vagal stimulation approximately doubled the frequency of proto-oncogene expression in A1 noradrenaline neurons, as indicated by the occurrence of nuclear Fos-like immunoreactivity in tyrosine hydroxylase-positive neurons of the caudal ventrolateral medulla. Finally, A1 region microinjection of either the N-methyl-D-aspartic acid (NMDA) receptor antagonist DL-2-amino-5-phosphonovaleric acid (APV), or the non-NMDA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), significantly reduced vasopressin cell responses to vagal stimulation. These findings suggest that: (i) the A1 group is an essential component in a pathway which relays facilitatory vagal input of cardiopulmonary origin to neurosecretory vasopressin cells, and (ii) the activation of A1 neurons in this pathway involves both NMDA and non-NMDA excitatory amino acid receptors, an observation consistent with an input to A1 cells which generates 'mixed' excitatory postsynaptic potentials.

Noxious somatic stimuli excite neurosecretory vasopressin cells via A1 cell group.

Activation of nociceptive somatic afferents excites hypothalamic neurosecretory cells and stimulates the release of vasopressin. To investigate the possibility that relevant afferent information is relayed through the A1 norepinephrine cell group of the caudal ventrolateral medulla, single-unit recording experiments were performed in pentobarbital sodium-anesthetized rats. The effects of somatic nerve stimulation, application of noxious somatic stimuli, and A1 region stimulation on the activity of putative vasopressin-secreting neurosecretory cells of the supraoptic nucleus were compared. The predominant effect of femoral and sciatic nerve stimulation on these cells was excitation, 54% (n tested = 113) displaying a marked increase in discharge probability, which had a mean onset latency of 72 +/- 3 ms and a mean duration of 114 +/- 9 ms. Almost all cells (96%) responding to somatic nerve stimulation were also excited by pinching of the ipsilateral or contralateral hindlimb paw, and the majority (84%) displayed a matching but shorter latency response to A1 region stimulation (mean onset 35 +/- 4 ms, duration 55 +/- 9 ms). A1 region injections of the inhibitory neurotransmitter gamma-aminobutyric acid reversibly blocked the effects of both somatic nerve stimulation (n = 14) and paw pinch (n = 9) on putative vasopressin cells. These results indicate that excitation of vasopressinergic neurosecretory cells by noxious somatic stimuli requires activation of neurons of the caudal ventrolateral medulla and hence are consistent with the proposal of a role for the A1 norepinephrine cell group.

Involvement of the A1 cell group in baroreceptor inhibition of neurosecretory vasopressin cells.

Extracellular recording experiments were done to investigate the proposal that arterial baroreceptor inhibition of vasopressinergic (AVP) neurosecretory cells involves the A1 noradrenaline cell group of the caudal medulla. The responsiveness of functionally identified AVP cells of the rat supraoptic nucleus to baroreceptor activation was not discernibly altered by A1 region lesions even though AVP cell responses to certain other stimuli were abolished. Acute blockade of A1 region function by injection of gamma-aminobutyric acid sometimes impaired AVP cell responsiveness to baroreceptor stimulation, although more commonly there was no effect. These data suggest that the A1 cell group is not an essential component in pathways mediating baroreceptor inhibition of neurosecretory AVP cells, but may contribute to the sensitivity of AVP cells to this input.

Excitation of supraoptic vasopressin cells by stimulation of the A1 noradrenaline cell group: failure to demonstrate role for established adrenergic or amino acid receptors.

The effects of adrenergic and excitatory amino acid antagonists on supraoptic nucleus (SON) neurosecretory cell responses to stimulation of the A1 noradrenaline (NA) cell group were examined in anaesthetized male rats. As in previous studies, delivery of cathodal pulses (100 microA, 1 ms pulses, 1 Hz) to the A1 region of the caudal ventrolateral medulla excited spontaneously active, antidromically identified neurosecretory cells, the majority of which were identified as arginine vasopressin (AVP) secreting on the basis of basal discharge patterns and responses to abrupt increases in arterial blood pressure. Administration of alpha- and beta-adrenoreceptor antagonists, by systemic or intracerebroventricular delivery of a bolus, or by direct pressure injection into the SON, did not alter neurosecretory cell responses to A1 stimulation, even when doses applied exceeded that required for blockade of excitations elicited by local application of NA. Application of the broad spectrum excitatory amino acid antagonist kynurenic acid (5-40 mM) blocked the excitatory effects of locally applied glutamate (100 microM) and transiently inhibited spontaneous activity, but failed to alter the excitatory effects of A1 region stimulation on SON cells. Identical effects were obtained with a selective kainate/quisqualate receptor antagonist. These data indicate that neurosecretory cell responses to activation of the A1 cell group are unaltered by antagonists of alpha- and beta-adrenoreceptors, or excitatory amino acid receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Stimulation of the subcallosal fornix excites neurones in the cat preoptic region which project to the medial basal hypothalamus and in the medial forebrain bundle.

The projection of neurones in the cat preoptic region driven by stimulation of the subcallosal fornix was systematically explored. We found 19% projected to the medial basal hypothalamus (MBH) and 10% projected in the medial forebrain bundle (MFB). Neurones projecting to the MBH were driven more often by stimulation of the lateral aspect of the fornix than the medial aspect (P = 0.006) and these neurones were thought to lie in the medial division of the preoptic nucleus (MPNm) since they were found significantly more often in the medial 0.6 mm of the preoptic region than more laterally (P = 0.028). A reverse projection from the preoptic region in the fornix is also suggested based on the finding of 24 antidromically activated neurones in the preoptic region following stimulation of the fornix.

A1 cell group mediates solitary nucleus excitation of supraoptic vasopressin cells.

Stimulation of the nucleus tractus solitarius (NTS) excites putative vasopressin-secreting cells of the supraoptic nucleus (SON) via a catecholaminergic projection to hypothalamus. Despite recent evidence of a direct catecholaminergic projection from NTS to SON, we have performed single-unit recording experiments in pentobarbital sodium-anesthetized rats to investigate the possibility that NTS stimulation effects on SON vasopressin cells are indirect, being relayed via the A1 noradrenergic cell group of the caudal ventrolateral medulla. The effects of single-pulse NTS and A1 region stimulation on the activity of antidromically identified SON neurosecretory cells that had been functionally characterized as vasopressin secreting were compared. NTS stimulation excited 81% of all putative vasopressin-secreting cells tested (n = 83), with a mean onset latency of 51 +/- 1 ms. A1 region stimulation excited 76% of all cells tested and 90% of units responsive to NTS stimulation, with a mean latency of 39 +/- 1 ms. Consistent with previous work NTS stimulation excited only a minority of oxytocin cells tested (3/13), and of these two-thirds also responded to A1 stimulation. Bilateral electrolytic lesions of the A1 region abolished the effects of NTS stimulation on putative vasopressin cells. Ipsilateral A1 region injections of the inhibitory neurotransmitter gamma-amino-butyric acid reversibly blocked NTS stimulation effects on putative vasopressin cells in animals where the contralateral A1 region had already been lesioned. These results support the proposal that excitation of SON vasopressin-secreting cells after NTS stimulation is due to activation of a relay projection through the A1 noradrenergic cell group of the caudal ventrolateral medulla.

Neuropeptide Y potentiates excitation of supraoptic neurosecretory cells by noradrenaline.

The effects of neuropeptide Y (NPY) and noradrenaline (NA) on the activity of rat supraoptic nucleus (SON) neurosecretory cells were examined using perfused hypothalamic slices. Bath application of either NPY (10(-9)-10(-6) M) or NA (10(-6)-10(-3) M) excited SON cells, although only NA elicited consistent, dose-dependent effects. Application of NPY at a dose having virtually no direct effects (10(-8) M) produced a 5-fold increase in SON cell responsiveness to NA at the sub-maximal response dose of 10(-5) M, but did not alter the minimum concentration of NA required to excite SON cells or increase the maximal response elicited by higher NA concentrations. The effects of NA, alone or in combination with NPY, were abolished by alpha-adrenoreceptor blockade. These data suggest that NPY has only weak direct effects on neurosecretory cells, but may have important neuromodulatory actions, significantly enhancing the excitatory effects of NA.

Subfornical organ lesions in rats abolish hyperdipsic effects of isoproterenol and serotonin.

Isoproterenol (300 micrograms/ml/kg) and serotonin (2 mg/ml/kg) given SC to rats (n = 27) caused significant drinking (Fisher PLSD, Scheffe F test, Dunnett t) in the 1 and 2 hours after injection. Such drinking was completely prevented in rats later shown to have complete lesions of their subfornical organs (n = 7). In contrast a response not significantly different from the prelesion response was found in rats later given cortical lesions (n = 11) or other lesions which did not damage the subfornical organ (n = 7). We conclude that drinking evoked by SC injection of serotonin and isoproterenol is brought about by peripheral production of angiotensin II. Blood borne angiotensin II in turn stimulates neurons in subfornical organ which initiate the neural organization of a drinking response.

Responses from osmosensitive neurons of the rat subfornical organ in vitro.

Extracellular recordings were made in vitro from 212 single units in the rat subfornical organ (SFO) and 54 single units in the rat medial preoptic area (MPO). Units were exposed to solutions made hyper-osmotic or hypo-osmotic by 1.4-11%. A reversible 30% or greater change in frequency followed the osmotic challenge in tests of 66% of units in the SFO and 46% of units in the MPO. Responses consisted of increases in frequency (excitations) or decreases in frequency (inhibitions) and were either sustained for the whole test period or of a transitory nature. Units responded to either hyperosmotic (SFO, 19%; MPO, 43%) or to hypo-osmotic changes (SFO, 30%; MPO, 28.5%) or to both (SFO, 51%; MPO, 28.5%). The response pattern of the SFO and MPO was significantly different (chi 2 54.0, 3df, P = 0.0001). In both the SFO and MPO the stimulus to which the units responded was a change in tonicity. This was indicated by the findings that similar responses were evoked by hyperosmotic changes made with either mannitol or NaCl and there was no response to solutions containing urea, either as an additive, or as a substitute for NaCl. In the SFO, in the presence of synaptic blockade produced by raising the Mg concentration in the bathing solution to 15 mM, the frequency of 19/27 units fell significantly. Responses of 40% of units to osmotic pressure changes were blocked indicating these responses were synaptically evoked. The responses which survived synaptic blockade when compared with pre-blockade responses were more often transient (P less than 0.02) and more often inhibitions. Post blockade there were also significantly more responses in the SFO to hypo-osmotic than to hyper-osmotic changes (P = 0.01). Our results suggest that while an ability to change their firing rate in response to small changes of osmotic pressure may be a general property of neurons, the neurons of the SFO are specialised for the detection of changes in the extracellular osmotic pressure.

Direct catecholaminergic projection from nucleus tractus solitarii to supraoptic nucleus.

To determine whether the supraoptic nucleus (SON) receives a direct projection from catecholamine cells of the nucleus tractus solitarii (NTS), retrograde transport of rhodamine-tagged latex microspheres was combined with a procedure for the fluorescence histochemical visualization of catecholamines. SON tracer injections, made via transpharyngeal approach, retrogradely labelled cells at all levels of NTS, although the majority were located caudal to obex with an ipsilateral predominance. Approximately half of these cells were also identified as catecholaminergic; the relatively caudal level in the dorsomedial medulla of most of these cells suggests that they probably correspond to the A2 catecholamine cell group.

Solitary nucleus excitation of supraoptic vasopressin cells via adrenergic afferents.

Recent studies confirm that the supraoptic nucleus (SON) receives a direct projection from the nucleus of the solitary tract (NTS). We have examined the effect of NTS stimulation on antidromically identified SON neurosecretory cells that were classified as arginine vasopressin (AVP) or oxytocin (OXY) secreting in accord with basal activity patterns and responsiveness to arterial baroreceptor activation. Medial NTS stimulation at the level of the obex or area postrema excited 78% (59 of 76) of putative AVP cells (onset latency 52 +/- 1 ms) but only 20% (3 of 15) of putative OXY cells. Commissural NTS stimulation did not excite AVP cells (n = 13). After complete SON catecholamine afferent disruption, achieved by local injection of 6-hydroxydopamine, AVP cells tested were unresponsive to medial NTS stimulation (12 of 13), although arterial baroreceptor activation inhibited four of four cells. These data indicate that medial NTS stimulation preferentially excites SON AVP cells and that this effect involves an adrenergic input to SON. A direct projection from the A2 catecholamine cell group of the NTS may be involved, although the long latency to excitation and the poor correspondence between effective NTS stimulation sites and the location of the A2 group within NTS raise the possibility that a relay projection, possibly through the A1 catecholamine cell group of the ventrolateral medulla, may be involved.

The influence of the estrous cycle, persistent estrus, ovariectomy and estrogen replacement, on the numbers and frequency of spontaneously active neurons in slices of the rat preoptic region in vitro.

Spontaneous single-unit activity was studied in the preoptic region of rat brain slices. Similar unit frequencies were recorded during the estrous cycle and for all ovariectomized (OVX) rats (median frequencies between 0.8 and 2.0 Hz). Higher frequencies were recorded in persistent estrus (PE) (median 3.5 Hz) and in males (median 3.4 Hz), P vs estrus less than 0.001. The mean percentage of tracks with units was low at estrus (15%), at diestrus (18%), in OVX rats (16%) and male rats (22%), and was significantly increased in metestrus (36%) and proestrus (33%) (P vs estrus less than 0.01) and in OVX rats after estrogen treatment (P less than 0.05 to P less than 0.01). It is suggested that the increased number of units found in OVX rats after estrogen treatment and in PE rats are both effects of prolonged elevated levels of estrogen in the brain.