Water transport in cultured cells from the rat inner medullary collecting duct.

In this study, cells derived from rat inner medullary collecting duct formed a polarized monolayer when grown in wells on a membrane. When the membrane was sealed in an Ussing apparatus, the cells passed water from their apical to basolateral surfaces in the presence of an osmotic gradient. This was detected by the movement of the meniscus (1 mm = 1 microL) in a capillary tube connected to the basolateral chamber. The movements were measured by a travelling microscope, and the effects of vasopressin, oxytocin, and kappa opioids were explored at 37 +/- 1 degrees C. In the presence of vasopressin and oxytocin, water movement increased to between the threshold of 0.2 pM and the maxima of 100 pM (vasopressin) and 20 pM (oxytocin). Higher concentrations had a lesser effect (vasopressin) or no greater effect (oxytocin). It was possible to get a similar effect with each of two doses of 10 pM vasopressin at intervals as short as 3 min, but tachyphyllaxis lasting 70 min followed a dose of 20 pM oxytocin. The effects of vasopressin and oxytocin could be blocked completely by the cAMP antagonist adenosine-3',5'-cyclic monophosphorothioate Rp isomer. The benzenacetamide kappa agonist U-50488 had no effect on the response to 1 pM vasopressin but the kappa antagonist norbinaltorphimine significantly increased the effects of 1 pM vasopressin; this action was exerted earlier on in the initiation of water transport, as norbinaltorphimine did not affect the response to Sp-cAMPS, an activator of cAMP-dependent kinases.

An NMDA receptor antagonist reduces ethanol preference in untrained but not trained rats.

Rats were exposed to a potent antagonist of NMDA receptors, 2-amino-5 phosphonovalerate (AP5) by implanting osmotic pumps delivering AP5 (test group) or the vehicle for AP5 (control group) into the cerebrospinal fluid through previously implanted cannulas. A week later they were given the choice of 6% ethanol or water for 1 h. The control group had a significantly greater preference for ethanol than the test group after the first 5 days. Another group of rats was trained almost to a 100% criterion in preferring ethanol to water in the hour the choice was available. Then they were implanted with osmotic pumps and cannulas and administered AP5. These trained rats showed no significant difference between control and test groups in preference for ethanol after the first 5 days. The results support the hypothesis that activation of NMDA receptors is involved in learning to drink ethanol.

The increased ethanol preference in rats induced by choice, darkness, or drugs is reduced by ritanserin.

We tested the hypothesis that ritanserin, a serotonin S2 antagonist, reduces voluntary and induced forms of ethanol drinking. We gave 10 mg/kg ritanserin IP or SC to groups of rats given either a) a free choice between 3% ethanol and water, or b) kept in the dark for 5 weeks and given a choice between a range of ethanol concentrations (3-25%) and water, or c) implanted with osmotic pumps filled with tetrahydro-beta carboline and given a choice between a range of ethanol concentrations and water. In each case, ritanserin significantly reduced ethanol consumption and ethanol preference for 8-10 days after the last injection.

ANP and naloxone reduce postdeprivation drinking after subfornical organ lesions.

We tested a report that atrial natriuretic peptide (ANP) injected into, or near, the subfornical organ (SFO) will reduce the water consumption of previously water deprived rats and that suggested ANP acts on neurons in the SFO to bring about this action. We tested this suggestion and the hypothesis that the SFO is involved in the facilitation of drinking produced by opioids. ANP (5 nmol in 4 microliters, IVT) or naloxone (2 mg/ml/kg, SC, or 200 micrograms in 2 microliters, IVT) when given to rats deprived of water for 16 h (SC treatment) or 23 h (IVT treatment) significantly depressed postdeprivation drinking measured at 15 and 60 min. Rats with complete, partial, or control lesions of the SFO, after the same treatment, also showed a significant depression of postdeprivation drinking and, after 23-h deprivation, a significant hyperdipsia. There was no interaction between drug effects and lesion effects (two-factor analysis of variance, Tukey's post-hoc tests). The hyperdipsia declined exponentially and was lost 45-50 days after lesioning. Our results do not support the hypothesis that the SFO is involved in the actions of ANP or of opioids on postdeprivation drinking.

Evidence for inhibitory amino acid receptors on guinea pig medial vestibular nucleus neurons in vitro.

There is little evidence to indicate the identity of the inhibitory receptors which mediate inhibitory interaction between the two medial vestibular nuclei ('brainstem commissural inhibition'). In the present study we tested the hypothesis that medial vestibular nucleus (MVN) neurons have gamma-aminobutyric acid (GABA) or glycine receptors by recording from single MVN neurons in isolated guinea pig MVN slices maintained in vitro while superfusing with GABA (10(-8) M) and the non-competitive GABAA antagonist picrotoxin (10(-6) M or 2 x 10(-6) M), or glycine (10(-6) M) and the competitive glycine antagonist strychnine (10(-6) M). Forty-four % (16/36) of the neurons tested with GABA showed a decrease in firing; in 7 out of 8 cases in which a decrease in firing occurred, the addition of the antagonist picrotoxin completely blocked the effect of the GABA alone. Fifty % (7/14) of the neurons tested with glycine showed a decrease in firing; in 4 out of 6 cases where a decrease occurred, the addition of the antagonist strychnine completely blocked the effect of the glycine alone. In one case only did a cell respond both to GABA and glycine (8 neurons tested with both). These results are consistent with the hypothesis that some MVN neurons have GABA or glycine receptors (but in most cases not both), which may mediate brainstem commissural inhibition.

Luteinizing hormone release in the anesthetized cat following stimulation in the diagonal band of Broca, dorsal septum and fornix.

The release of luteinizing hormone (LH) in response to electrical stimulation of septal nuclei (the diagonal band of Broca, DBB, and dorsal septal nucleus) and the subcallosal fornix has been studied in gonadectomized female cats. The cats were anesthetized with Althesin. Electrodes were placed on the medial and lateral aspects of the subcallosal fornix and paired bipolar stimulating electrodes were aimed at the DBB or dorsal septal nucleus. The effect of electrical stimulation of these regions on the secretion of LH was studied by radioimmunoassay of LH in serial blood samples taken before, during and after stimulation. Stimulation in the DBB or in the dorsal septal nucleus resulted in a peak of LH release during stimulation followed by further spontaneous peaks. All peaks showed an exponential decline. The frequency of spontaneous peaks following stimulation could reach the level found in unanesthetized cats. Stimulation of the subcallosal fornix produced a significant depression in the amplitude of LH release. The effects of DBB, dorsal septal and fornix stimulation are all exerted, we suggest, by projections to LHRH containing neurons in the preoptic region.

Responses in the diagonal band of Broca, adjacent septal nuclei and the islands of Calleja of cats to stimulation of the subcallosal fornix, medial basal hypothalamus and medial forebrain bundle.

The projection of neurons in the septal nuclei and the insula magna of the islands of Callaja (IC) was explored together with their response to stimulation of the fornix. The septal nuclei all contained neurons projecting in the medial forebrain bundle (MFB). Only the diagonal band of Broca (DBB) and the lateral septal nucleus (LS) contained many neurons projecting toward the medial basal hypothalamus (MBH). The spatial distribution of neurons excited by stimulation of the fornix in the DBB was almost identical with the distribution of neurons projecting toward the MBH and there was considerable overlap (10/28 cells). In the medial septal nucleus the spatial distribution of neurons excited by stimulation of the fornix and neurons projecting in the MFB was similar and there was considerable overlap (6/21 cells). The connectivity of the IC resembled that of the MS but there was little overlap between the neurons excited by fornix stimulation and those projecting in the MFB (1/27 cells). In the LS there were almost equal numbers of neurons projecting in the MFB and toward the MBH but there was very little input from the fornix. Neurons were significantly more often excited by stimulation of the lateral fornix, carrying axons from the subiculum, than they were by medial stimulation exciting axons from Ammon's horn. Axons projecting toward the MBH or in the MFB had conduction velocities less than 1 m/s.

Evidence that NMDA receptors contribute to synaptic function in the guinea pig medial vestibular nucleus.

Single medial vestibular nucleus neurons were recorded from guinea pig brainstem slices in vitro while superfusing with the selective N-methyl-D-aspartate (NMDA) antagonists, MK801 and CPP. The majority of neurons tested showed a decrease in firing rate in response to these NMDA antagonists, suggesting that NMDA receptors may contribute to the resting activity of MVN neurons.

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.

Stimulation of the LH release by naloxone in anaesthetized cats after ovariectomy.

The effect of intravenous injections or infusions of the opioid receptor antagonist naloxone on the secretion of luteinizing hormone (LH) was studied in 18 spayed cats anaesthetized with Althesin. Effective injections significantly increased the LH concentration of plasma samples (taken every 10-15 min and measured by radio-immunoassay) to a peak 20-30 min after injection. The concentration thereafter declined exponentially (ke = 0.42), and, in 4/8 trials rose again significantly and declined again without further injection. The threshold dose was between 0.4 and 0.5 mg/kg. There did not appear to be a dose dependence of the effect above threshold. Infusion of naloxone at levels up to 5 mg/kg/h was effective in producing a pulsatile release of LH and repeated injections of threshold doses (0.5 mg/kg) could produce a maintained plateau and pulsatile release at frequencies comparable to pulse frequencies in vivo.

Guinea pig medial vestibular nucleus neurons in vitro respond to ACTH4-10 at picomolar concentrations.

The responses of single guinea pig medial vestibular nucleus (MVN) neurons in vitro to adrenocorticotropic hormone, fragment 4-10 (ACTH4-10) were recorded extracellularly. In coronal slices and in the isolated MVN, neurons were found which responded to ACTH4-10 at picomolar concentrations (10(-12)M), indicating that ACTH4-10 acts directly on MVN neurons and suggesting the possibility that ACTH4-10 may act as a neurotransmitter in the MVN. In most cases where neurons responded to ACTH4-10 (37/74 neurons), the effect was a decrease in firing. Whether or not the depressive action of ACTH4-10 on the firing rate of MVN neurons in vitro is related to the acceleration of behavioral recovery from unilateral labyrinthectomy (vestibular compensation), which has been reported previously (Flohr and Luneburg 1982; Igarashi et al. 1985), is unclear.

Neuronal activity in the guinea pig medial vestibular nucleus in vitro following chronic unilateral labyrinthectomy.

Unilateral labyrinthectomy (UL) causes ocular motor and postural disorders which disappear over time in a process of recovery known as vestibular compensation. Vestibular compensation is due to CNS plasticity which generates a partial recovery of resting activity in the vestibular nucleus ipsilateral to the UL, however the mechanism of this neural recovery is unknown. It has been suggested that other areas of the CNS may substitute non-vestibular sensory inputs for the missing labyrinthine input, thereby causing vestibular compensation. The present results show that resting activity can be recorded from medial vestibular nucleus (MVN) neurons in vitro, in brainstem slices from guinea pigs which have compensated for an ipsilateral UL. This result suggests that MVN neurons are capable of generating resting activity without inputs from many other CNS areas. Perfusion with high Mg2+ solution did not abolish resting activity in most cases, suggesting that part of the resting activity may be generated spontaneously by the neurons, possibly through changes in the electrical excitability of the cell membrane.

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.

Central respiratory inhibition by angiotensin II in anesthetized cats.

Observations were made on respiration, alveolar concentration of CO2, arterial blood pressure and heart rate in barbiturate-anesthetized cats subjected routinely to section of the vagus and carotid sinus nerves and maintained with pure O2 for inhalation. Bolus i.v. injection of angiotensin II (AII), 0.6 to 6.0 micrograms/kg, evoked a prompt shortlasting suppression of breathing, manifested mainly as a reduction in tidal volume susceptible to the development of tachyphylaxis if injections were spaced more closely than 30 min apart. Ablation of the area postrema failed to eliminate the respiratory effect of All. The response also persisted after midbrain transection, spinal cord transection at C8, dorsal rhizotomy of spinal segments C1 to C8 and interruption of cranial nerves V, VII, VIII, IX, X, XI and XII. No influence of All was evident on indirectly evoked tibialis muscle contractions and on pulmonary compliance. A reduction in phrenic nerve impulse traffic coincided with the respiratory response to All. Cranial i.a. injection of All initiated the respiratory effect in its first pass through the brain, before any change occurred in the blood pressure. The degree of All-induced respiratory inhibition was relatively constant at different magnitudes of breathing produced by CO2 inhalation or by electrical stimulation of the respiratory center in the medulla oblongata. It is concluded that All given by bolus intravascular injection causes respiratory inhibition through a direct central action at the final processing step in the CO2-tidal volume controller.

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.

Noradrenaline excites neurons in the guinea pig cerebellar vermis in vitro.

Noradrenaline (NA) was applied to the solution bathing the cavy cerebellar vermis in vitro and the responses of 98 neurons were recorded extracellularly. Two thirds (23/35) of the responses were excitations and the remaining third were inhibitions. The lowest concentration of NA with which responses could be obtained was 10(-11) M NA. Responses were generally transient and occurred with a mean latency of 61 +/- 8 sec. The excitation was generally direct as most responses (9/11) survived synaptic blockade. The excitations were thought to be mediated by alpha 1 receptors because they could be mimicked by phenylephrine and antagonised by prazozin.

Excitatory effects of atrial natriuretic peptide on rat subfornical organ neurons in vitro.

Atrial natriuretic peptide (ANP) was applied in the solution bathing the rat subfornical organ in vitro and the response of 170 neurons recorded extracellularly. A significant change in firing rate was found in 20% of neurons tested at a concentration of 1 X 10(-10) M. At higher concentrations 45% of neurons responded. The response was usually a brief excitation. Tests of a range of concentrations on each of 6 neurons showed the maximal excitation was attained at 5 X 10(-9) M. The excitatory responses were generally (41/51) not affected by synaptic blockade indicating a direct action of ANP. The excitatory effect of ANP upon subfornical neurons can be contrasted with the generally inhibitory effect of changes in osmotic pressure. Neurons excited by ANP also responded to 5% osmotic pressure changes in the proportions (28/43) expected from the known response rate to osmotic pressure changes (66%). The commonest pattern of response (16/28) was excitation by hyper-osmotic solutions and inhibition by hypo-osmotic solutions.

Distribution and projection of single units in the cat preoptic region responding to stimulation of the medial amygdala.

Neuronal responses were recorded extracellularly in the preoptic region of 11 male castrate cats following stimulation in the 'medial' group of amygdalar nuclei (medial, basomedial, basolateral). The latency of responses varied with the site of stimulation and could be explained if connections to the preoptic region were made through both the stria terminalis and the ventral amygdalofugal pathway. Projection of 13% of orthodromically excited preoptic units to the medial basal hypothalamus was established by collision tests following stimulation in these regions. The amygdalar excitation was probably exerted through the amygdalofugal pathway. In 5/6 cats the amygdalar stimulation was successfully used to raise plasma LH levels. In one cat no preoptic units projecting to the medial basal hypothalamus were found and in the cat which failed to increase plasma LH in response to amygdala stimulation, 5 such units were found. We conclude the pathway from the amygdala through the preoptic region to the hypothalamus may not be the way in which amygdalar stimulation affects LH release in the cat. Projection of 10% of orthodromically excited preoptic units in the medial forebrain bundle was established by collision tests in 3 cats. These identified units were driven from amygdalar sites thought to project to the preoptic region by ventral amygdalofugal pathways.

Neuron responses to substance P and enkephalin in rat dorso-lateral septum in vitro.

Using an in vitro brain slice technique the responses of spontaneously active neurons in the rat dorso-lateral septum to 10 nM substance P (SP) and enkephalin were determined. Fewer neurons responded to SP (41%) than to enkephalin (55%). The SP responses were 13 excitations, 14 inhibitions, the enkephalin responses were 13 excitations, 14 inhibitions and 11 responded to both, 6 of these were inhibited by both. Immunocytochemical techniques have shown there is a discrete localisation of SP and enkephalin axons and terminals in the rat septum. SP responsive neurons were associated with the SP terminal-rich region (p = 0.01) but no association was found for enkephalin responses in the enkephalin terminal-rich region (p = 0.7).