Intraperitoneal Injection Of Hypertonic Saline Research Articles

Systemic hypertonic saline enhances glymphatic spinal cord delivery of lumbar intrathecal morphine

The blood-brain barrier significantly limits effective drug delivery to central nervous system (CNS) targets. The recently characterized glymphatic system offers a perivascular highway for intrathecally (i.t.) administered drugs to reach deep brain structures. Although periarterial cerebrospinal fluid (CSF) influx and concomitant brain drug delivery can be enhanced by pharmacological or hyperosmotic interventions, their effects on drug delivery to the spinal cord, an important target for many drugs, have not been addressed. Hence, we studied in rats whether enhancement of periarterial flow by systemic hypertonic solution might be utilized to enhance spinal delivery and efficacy of i.t. morphine. We also studied whether the hyperosmolar intervention affects brain or cerebrospinal fluid drug concentrations after systemic administration. Periarterial CSF influx was enhanced by intraperitoneal injection of hypertonic saline (HTS, 5.8%, 20 ml/kg, 40 mOsm/kg). The antinociceptive effects of morphine were characterized, using tail flick, hot plate and paw pressure tests. Drug concentrations in serum, tissue and microdialysis samples were determined by liquid chromatography-tandem mass spectrometry. Compared with isotonic solution, HTS increased concentrations of spinal i.t. administered morphine by 240% at the administration level (T13–L1) at 60 min and increased the antinociceptive effect of morphine in tail flick, hot plate, and paw pressure tests. HTS also independently increased hot plate and paw pressure latencies but had no effect in the tail flick test. HTS transiently increased the penetration of intravenous morphine into the lateral ventricle, but not into the hippocampus. In conclusion, acute systemic hyperosmolality is a promising intervention for enhanced spinal delivery of i.t. administered morphine. The relevance of this intervention should be expanded to other i.t. drugs and brought to clinical trials.

Extracellular signal‐regulated kinase phosphorylation in forebrain neurones contributes to osmoregulatory mechanisms

Vasopressin secretion from the magnocellular neurosecretory cells (MNCs) is crucial for body fluid homeostasis. Osmotic regulation of MNC activity involves the concerted modulation of intrinsic mechanosensitive ion channels, taurine release from local astrocytes as well as excitatory inputs derived from osmosensitive forebrain regions. Extracellular signal-regulated protein kinases (ERK) are mitogen-activated protein kinases that transduce extracellular stimuli into intracellular post-translational and transcriptional responses, leading to changes in intrinsic neuronal properties and synaptic function. Here, we investigated whether ERK activation (i.e. phosphorylation) plays a role in the functioning of forebrain osmoregulatory networks. We found that within 10min after intraperitoneal injections of hypertonic saline (3m, 6m) in rats, many phosphoERK-immunopositive neurones were observed in osmosensitive forebrain regions, including the MNC containing supraoptic nuclei. The intensity of ERK labelling was dose-dependent. Reciprocally, slow intragastric infusions of water that lower osmolality reduced basal ERK phosphorylation. In the supraoptic nucleus, ERK phosphorylation predominated in vasopressin neurones vs. oxytocin neurones and was absent from astrocytes. Western blot experiments confirmed that phosphoERK expression in the supraoptic nucleus was dose dependent. Intracerebroventricular administration of the ERK phosphorylation inhibitor U 0126 before a hyperosmotic challenge reduced the number of both phosphoERK-immunopositive neurones and Fos expressing neurones in osmosensitive forebrain regions. Blockade of ERK phosphorylation also reduced hypertonically induced depolarization and an increase in firing of the supraoptic MNCs recorded in vitro. It finally reduced hypertonically induced vasopressin release in the bloodstream. Altogether, these findings identify ERK phosphorylation as a new element contributing to the osmoregulatory mechanisms of vasopressin release.

5-HT neurons of the area postrema become c-Fos-activated after increases in plasma sodium levels and transmit interoceptive information to the nucleus accumbens.

Serotonergic (5-hydroxytryptamine, 5-HT) neurons of the area postrema (AP) represent one neuronal phenotype implicated in the regulation of salt appetite. Tryptophan hydroxylase (Tryp-OH, synthetic enzyme-producing 5-HT) immunoreactive neurons in the AP of rats become c-Fos-activated following conditions in which plasma sodium levels are elevated; these include intraperitoneal injections of hypertonic saline and sodium repletion. Non-Tryp-OH neurons also became c-Fos-activated. Sodium depletion, which induced an increase in plasma osmolality but caused no significant change in the plasma sodium concentration, had no effect on the c-Fos activity in the AP. Epithelial sodium channels are expressed in the Tryp-OH-immunoreactive AP neurons, possibly functioning in the detection of changes in plasma sodium levels. Since little is known about the neural circuitry of these neurons, we tested whether the AP contributes to a central pathway that innervates the reward center of the brain. Stereotaxic injections of pseudorabies virus were made in the nucleus accumbens (NAc), and after 4 days, this viral tracer produced retrograde transneuronal labeling in the Tryp-OH and non-Tryp-OH AP neurons. Both sets of neurons innervate the NAc via a multisynaptic pathway. Besides sensory information regarding plasma sodium levels, the AP→NAc pathway may also transmit other types of chemosensory information, such as those related to metabolic functions, food intake, and immune system to the subcortical structures of the reward system. Because these subcortical regions ultimately project to the medial prefrontal cortex, different types of chemical signals from visceral systems may influence affective functions.

An inhibitor of leukotriene synthesis affects vasopressin secretion following osmotic stimulus in rats

Previous studies revealed the presence of LTC4 synthase in paraventricular vasopressinergic neurons, suggesting a role for leukotrienes (LTs) in certain neuroendocrine system functions. Our aim was to study the effect of an inhibitor of LT synthesis in the release of arginine vasopressin (AVP) following an osmotic stimulus in rats. Male Wistar rats received an intra-cerebroventricular injection of 2μl of the LT synthesis inhibitor MK-886 (1, 2, or 4μg/kg), or vehicle (DMSO 5%), 1h before an intraperitoneal injection of hypertonic saline (NaCl 2M) or isotonic saline (NaCl 0.01M) in a volume corresponding to 1% of body weight. Thirty minutes after the osmotic stimulus, the animals were decapitated and blood was collected for determining hematocrit, plasma osmolality and plasma AVP levels. As expected, the injection of hypertonic saline significantly increased (P<0.05) the hematocrit, plasma osmolality and plasma AVP levels. While inhibiting LT synthesis by central administration of MK-886 did not cause any additional increase in hematocrit or osmolality, plasma AVP levels were augmented (P<0.05). We conclude that central leukotrienes may have a modulatory role in AVP secretion following an osmotic stimulus, this deserving future studies.

Osmoregulatory fluid intake but not hypovolemic thirst is intact in mice lacking angiotensin

Water intakes in response to hypertonic, hypovolemic, and dehydrational stimuli were investigated in mice lacking angiotensin II as a result of deletion of the angiotensinogen gene (Agt-/- mice), and in C57BL6 wild-type (WT) mice. Baseline daily water intake in Agt-/- mice was approximately threefold that of WT mice because of a renal developmental disorder of the urinary concentrating mechanisms in Agt-/- mice. Intraperitoneal injection of hypertonic saline (0.4 and 0.8 mol/l NaCl) caused a similar dose-dependent increase in water intake in both Agt-/- and WT mice during the hour following injection. As well, Agt-/- mice drank appropriate volumes of water following water deprivation for 7 h. However, Agt-/- mice did not increase water or 0.3 mol/l NaCl intake in the 8 h following administration of a hypovolemic stimulus (30% polyethylene glycol sc), whereas WT mice increased intakes of both solutions during this time. Osmoregulatory regions of the brain [hypothalamic paraventricular and supraoptic nuclei, median preoptic nucleus, organum vasculosum of the lamina terminalis (OVLT), and subfornical organ] showed an increased number of neurons exhibiting Fos-immunoreactivity in response to intraperitoneal hypertonic NaCl in both Agt-/- mice and WT mice. Polyethylene glycol treatment increased Fos-immunoreactivity in the subfornical organ, OVLT, and supraoptic nuclei in WT mice but only increased Fos-immunoreactivity in the supraoptic nucleus in Agt-/- mice. These data show that brain angiotensin is not essential for the adequate functioning of neural pathways mediating osmoregulatory thirst. However, angiotensin II of either peripheral or central origin is probably necessary for thirst and salt appetite that results from hypovolemia.

Sodium depletion activates the aldosterone-sensitive neurons in the NTS independently of thirst

Thirst and sodium appetite are both critical for restoring blood volume. Because these two behavioral drives can arise under similar physiological conditions, some of the brain sensory sites that stimulate thirst may also drive sodium appetite. However, the physiological and temporal dynamics of these two appetites exhibit clear differences, suggesting that they involve separate brain circuits. Unlike thirst-associated sensory neurons in the hypothalamus, the 11-beta-hydroxysteroid dehydrogenase type 2 (HSD2) neurons in the rat nucleus tractus solitarius (NTS) are activated in close association with sodium appetite (16). Here, we tested whether the HSD2 neurons are also activated in response to either of the two physiological stimuli for thirst: hyperosmolarity and hypovolemia. Hyperosmolarity, produced by intraperitoneal injection of hypertonic saline, stimulated a large increase in water intake and a substantial increase in immunoreactivity for the neuronal activity marker c-Fos within the medial NTS, but not in the HSD2 neurons. Hypovolemia, produced by subcutaneous injection of hyperoncotic polyethylene glycol (PEG), stimulated an increase in water intake within 1-4 h without elevating c-Fos expression in the HSD2 neurons. The HSD2 neurons were, however, activated by prolonged hypovolemia, which also stimulated sodium appetite. Twelve hours after PEG was injected in rats that had been sodium deprived for 4 days, the HSD2 neurons showed a consistent increase in c-Fos immunoreactivity. In summary, the HSD2 neurons are activated specifically in association with sodium appetite and appear not to function in thirst.

Aging-related changes of microglia and astrocytes in hypothalamus after intraperitoneal injection of hypertonic saline in rats

To examine the aging-related changes of microglia and astrocytes in hypothalamus of rats after intraperitoneal injection of hypertonic saline in rats, old- and young-aged rats were injected with hypertonic saline solution into peritoneal cavity. Lectin histochemical techniques using Ricinus communis agglutinin-1 (RCA-1) and immunocytochemical method employing antibody against glial fibrillary acidic protein (GFAP) were used to demonstrate microglia and astrocytes in the hypothalamus of the rats, and the positively-stained cells were analyzed by computer-assisted image analysis system. Our results showed that the numbers of microglia and astrocytes were significantly increased in the hypothalamus of old-aged rats. After intraperitoneal injection of hypertonic saline, the number of microglia was significantly decreased in the hypothalamus of both young- and old-aged groups. After introperitoneal injection of hypertonic saline, the number of GFAP positive cells was significantly increased in the hypothalamus of young rats, but the number of GFAP positive cells did not show significant change in the hypothalamus of old rats. It is concluded that in the hypothalamus of old-aged rats, the increase of microglia may be related with the aging or degeneration of neurons, and the increase of astrocytes may provide more nourishment required by the aged neurons. The microglia and astrocytes in the hypothalamus of the two group rats may be affected by hypertonic saline, and the response of these cells to the stimuli is characterized by some aging-related changes.

Ethics and Animal Welfare Related to in vivo Pharmacology and Toxicology in Laboratory Animals

Ethics and Animal Welfare Related to <i>in vivo</i> Pharmacology and Toxicology in Laboratory Animals

Antiserum Against Neuropeptide FF Augments Vasopressin Release in Conscious Rats

Yokoi, H., H. Arima, K. Kondo, T. Murase, Y. Iwasaki, H.-Y. T. Yang and Y. Oiso. Antiserum Against Neuropeptide FF Augments Vasopressin Release in Conscious Rats. Peptides 19(2) 393–395, 1998.—We previously reported that centrally administered neuropeptide FF (NPFF) inhibited arginine vasopressin (AVP) release. In this study, immunoneutralization of central NPFF was performed to evaluate the role of endogenous NPFF in the regulation of AVP release. Intracerebroventricular (ICV) injection of antiserum against NPFF (Anti-NPFF) significantly augmented the plasma AVP increase induced by hyperosmolality [intraperitoneal injection of hypertonic saline (600 mOsm/kg, 2% BW)] at 60 min after ICV injection compared with normal rabbit serum (NRS) (NRS: 4.20 ± 0.30 pg/ml, Anti-NPFF: 5.83 ± 0.46 pg/ml, p < 0.01). Anti-NPFF did not cause significant change in plasma osmolality, plasma volume or arterial blood pressure. This evidence indicates that endogenous NPFF might be physiologically involved in osmoregulation of the plasma AVP level through its inhibitory action.

Fos and Jun expression in rat supraoptic nucleus neurons after acute vs. repeated osmotic stimulation

Using a double-label immunofluorescence method, we analyzed the time course of the appearance of Fos and Jun in the nuclei of supraoptic nucleus (SON) neurons following intraperitoneal injection of hypertonic saline. Fos and Jun immunostaining appeared within 30 min, peaked at 90–120 min, and disappeared 4–5 h later. At all time points (30, 60, 120, 180, 240 min postinjection), colocalized Fos and Jun immunostaining was observed (>90% colocalized staining in labeled neurons). At 4 h post-saline injection, some rats received a second injection of normal or hypertonic saline. A second injection of normal saline resulted in no Fos/Jun immunostaining 90 min later, while hypertonic saline induced combined Fos/Jun staining in only 17% of SON neurons. Of the remaining SON cells, 23% had staining to Fos alone and 4% of the cells stained for Jun only. In spite of the delivery of an effective second osmotic stimulus, determined by assessment of plasma osmolality and sodium content, SON neurons exhibited less colocalized Fos/Jun immunostaining, dramatically less Jun expression, and substantial, but attenuated, immunostaining for Fos. These results are discussed in the context of known negative feedback mechanisms that control the re-expression of these transcription factors.

Effect of AV3V lesions on Fos expression and cell size increases in magnocellular neurons of the rat hypothalamus during chronic dehydration

The effects of osmotic stimulation on Fos expression and cell size increase in the supraoptic nucleus were evaluated in intact, sham-operated, and AV3V-lesioned rats. Fos-positive neurons were found mainly in the AV3V regions and the hypothalamic magnocellular neurons in the forebrain in dehydrated intact rats. Intraperitoneal injection of hypertonic saline and chronic dehydration induced a significant increase in number of Fos-positive neurons in the supraoptic nucleus of intact and sham-operated rats. AV3V lesions completely abolished the expression of Fos in SON neurons of rats that were intraperitoneally injected with hypertonic saline and were chronically dehydrated. Chronic dehydration increased significantly cell size of the OXT and AVP magnocelluar neurons in intact and sham-operated rats. However, there was no increase in cell size of those in the AV3V-lesioned rats. These results demonstrate that neural input derived from AV3V regions plays a significant role in causing Fos expression and structural changes such as cell size increase in the hypothalamic magnocellular neurons with osmotic stimulation.

Role of organic osmolytes in myelinolysis. A topographic study in rats after rapid correction of hyponatremia.

Organic osmolytes have been implicated in the pathogenesis of myelinolysis because some of them are accumulated slowly during correction of chronic hyponatremia. I investigated whether there was a topographic correlation between demyelinative lesions and the regional changes of organic osmolytes after rapid correction of chronic hyponatremia. In normal female Sprague-Dawley rats, concentrations of glutamate, glutamine, taurine, and betaine were highest in the cerebral cortex and decreased toward the brain stem. Conversely, glycine level was highest in the brainstem, and decreased toward the cortex. Myoinositol, glycerophosphorylcholine, glycerophosphorylethanolamine, and creatine were distributed more evenly. In chronic hyponatremic rats (plasma Na 110 +/- 4 meq/liter), organic osmolytes decreased globally with the total loss ranging from 13 (medulla) to 24 (cerebellum) mmol/kg H2O. After rapid correction with intraperitoneal injection of hypertonic saline, the recovery of the loss of organic osmolytes was 48% in the cerebral cortex, cerebellum, and medulla oblongata, 44% in pons, but only 17% in midbrain and 36% in striatum. Histopathology of the brain was examined in nine rats 2-7 d after correction of hyponatremia. Large demyelinative lesions were seen persistently in the midbrain and striatum, and smaller lesions in cerebrum, cerebellum, and pons were found less frequently. This is the first report of regional distribution of brain organic osmolytes. After rapid correction of chronic hyponatremia, a topographic correlation between demyelination lesions and delayed accumulation of organic osmolytes exists.

Functional identification of central afferent projections conveying information of acute "stress" to the hypothalamic paraventricular nucleus

Intraperitoneal administration of hypertonic saline is a potent stimulus to the "stress" responsive hypophysiotrophic parvicellular neurons of the hypothalamic paraventricular nucleus (PVN), as well as to magnocellular neurons of the hypothalamo-neurohypophysial system. Precise identification of the pathways gating information about the "stress" of intraperitoneal hypertonic saline to the PVN has not been ascertained earlier. In this study we demonstrate that intraperitoneal injection of hypertonic saline induces increased expression of c-fos immunoreactivity within neurons of the medial parvicellular division of the PVN, within the circumventricular organs surrounding the anteroventral tip of the third ventricle, and within the magnocellular neurons of the PVN and supraoptic nucleus. Also, neurons involved in conveying visceral information to the PVN, including the parabrachial nucleus, the nucleus of the solitary tract and the ventrolateral medulla responded with increased c-fos expression to the "stress" paradigm. Combined c-fos immunocytochemistry and retrograde tracing experiments with cholera toxin B (ChB) was used to identify neurons projecting to the PVN activated by the applied "stressor." Neither the mere intracerebral presence of ChB nor intraperitoneal administration of isotonic saline influenced the number of c-fos immunoreactive nuclei in the brain. Dual immunocytochemistry revealed that intraperitoneal administration of hypertonic saline induced expression of c-fos immunoreactive nuclei in approximately half of the retrogradely labeled neurons projecting to the PVN from the anteroventral tip of the third ventricle (AV3v), including the subfornical organ (SFO) and the organum vasculosum laminae terminalis (OVLT). In the brainstem, the "stressor" induced expression of c-fos-IR nuclei in almost all of the retrogradely labeled cells of the ventrolateral part of the medulla oblongata (A1 and C1), while only about 25% of the ChB-labeled cells of the caudal part of the nucleus of the solitary tract (A2) were concomitantly immunoreactive to c-fos. Within the parabrachial nucleus, only 20% of the ChB-labeled cells were also immunoreactive for c-fos. The present results provide evidence that information about the "stress" of intraperitoneal hypertonic saline is conveyed to both magnocellular neurons projecting to the neurohypophysis and hypophysiotrophic parvicellular neurons the PVN via afferent projections from a variety of neurons in the osmosensitive anteroventral tip of the third ventricle and visceromotor neurons of the parabrachial nucleus, the ventrolateral medulla and the nucleus of the solitary tract.

Simultaneous microdialysis in blood and brain: oxytocin and vasopressin release in response to central and peripheral osmotic stimulation and suckling in the rat.

Simultaneous microdialysis in blood and brain has been used to monitor the release of both oxytocin and vasopressin into the systemic circulation (jugular vein/right atrium) and within the hypothalamic supraoptic nucleus of rats. Both home-made probes for blood and brain microdialysis revealed detectable nonapeptide concentrations under basal conditions and differential responses to a variety of stimuli. In urethane-anesthetized male rats, bilateral stimulation of the supraoptic nucleus by microdialyzing hypertonic medium (1 M NaCl) not only significantly increased the intranuclear release of both oxytocin and vasopressin (p < 0.05), but also their release from the neurohypophysis into blood (p < 0.05). In poststimulation microdialysates sampled from blood, the nonapeptides reached basal levels again, whereas intranuclear levels were further elevated. Intraperitoneal injection of hypertonic saline, on the other hand, resulted not only in the well-known increased peripheral release of oxytocin and vasopressin (p < 0.01 each), but also in a delayed increase in intranuclear oxytocin (p < 0.05). In contrast, intranuclear vasopressin release failed to change within the 90-min period following osmotic stimulation. In conscious lactating rats, suckling increased oxytocin contents in microdialysates sampled simultaneously in blood and the supraoptic nucleus (p < 0.05 each) further validating the microdialysis techniques used. The in vivo recovery in blood of approximately 65% determined using both radiolabeled and endogenous oxytocin provides a rough estimate to assess nonapeptide concentrations in plasma from 30-min or even 10-min blood microdialysis data.(ABSTRACT TRUNCATED AT 250 WORDS)

Intraperitoneal administration of recombinant human interleukin-1β inhibits osmotic thirst in the rat

Decrease in water intake after intraperitoneal injection of interleukin-1β (IL-1β) was studied in the rat. Administration of IL-1β at a dose of 20 μg/kg attenuated osmotic thirst induced by intraperitoneal injection of hypertonic saline, but did not affect hypovolemic thirst induced by subcutaneous injection of either polyethylene glycol or angiotensin II. Interleukin-1β also decreased spontaneous intake of water but not that of 1.8% saline. The results suggest that the decrease in water intake by IL-1β is caused, at least in part, by suppression of osmotic thirst but not by general suppression of behavior. The effects of IL-1β were not secondary responses accompanied by feeding behavior, since food supply was removed during the experiments. Pretreatment with indomethacin blocked the decrease in water intake by IL-1β, suggesting the involvement of production of prostaglandins.

Chronic stress elevates enkephalin expression in the rat paraventricular and supraoptic nuclei

Numerous studies have implicated opioids in the regulation of hypothalamic functions. Dynorphin, which is co-expressed with vasopressin in the magnocellular neurons of the paraventricular and supraoptic nuclei, is co-regulated with vasopressin in response to hyperosmolality and appears to inhibit vasopressin and oxytocin release from the posterior pituitary. Enkephalin is present in paraventricular parvocellular neurons and its expression is elevated in response to various stresses. However, enkephalin's presence and roles in paraventricular and supraoptic magnocellular neurons are uncertain. By giving rats daily intraperitoneal injections of hypertonic saline for up to 12 days, we induced a marked increase in enkephalin expression in magnocellular neurons of the paraventricular and supraoptic nuclei, beyond what develops from drinking hypertonic saline. Our results suggest that enkephalin expression in both vasopressin and oxytocin neurons may increase in response to chronic stresses and provide another source of enkephalin in addition to the parvocellular neurons.

Vasopressin receptors in the brain, liver and kidney of rats following osmotic stimulation

The binding site concentration ( B max) and equilibrium dissociation constant ( K d) for [ 3H]-arginine vasopressin (AVP) binding sites were measured in limbic brain areas (septum, dorsal hippocampus, amygdala) and liver and kidney of control and osmotically stimulated male Wistar rats. Membrane binding was performed in these five areas 30, 60 and 180 min following intraperitoneal injection of hypertonic saline. This paradigm resulted in no significant change in binding characteristics in the septum, dorsal hippocampus, amygdala and liver from control treated rats. In contrast, the kidney B max was significantly reduced 60min following osmotic stimulation, with no effect on affinity. These results also suggest that AVP receptors in the CNS are relatively resistant to regulatory effects of an acute AVP exposure.

Stress‐Induced Oxytocin Release in the Rat After Lesion of the Paraventricular Nuclei; Possible Deficiency of Corticotrophin‐Releasing Factor

Abstract Oxytocin is released under stressful conditions and corticotrophin-releasing factor (CRF) is known to be involved in mediating general 'stress responses'. We therefore examined whether CRF neurons in the paraventricular nucleus participate in the stress-induced oxytocin release in the rat. CRF (0.02 to 2 nmol) injected into the third ventricle produced a dose-dependent rise in the plasma oxytocin concentration. The oxytocin release induced by CRF occurred without a change in blood pressure, and was not affected by dexamethasone pretreatment, which prevents adrenocorticotrophin release following CRF injection. Lesioning of the paraventricular nucleus reduced oxytocin release by immobilization stress, but did not alter the release of oxytocin in response to osmotic stimulation induced by intraperitoneal injection of hypertonic saline. Anti-CRF serum injection into the third ventricle reduced delayed oxytocin response to immobilization stress. These results are consistent with the hypothesis that CRF neurons in the paraventricular nucleus are involved in the oxytocin release during immobilization stress in the rat.

Sex-related differences in the effects of morphine and stress on visceral pain

The effect of morphine (in the absence of stress and after a type of naloxone-sensitive swim-stress) on the painful contractions induced by intraperitoneal injection of hypertonic saline was studied in four groups of rats: normally cycling females in oestrus, males, ovariectomized females and females subjected to constant light, to induce a hyperoestrogenemic state. In the absence of swim-stress, the sensitivity to morphine was maximum for males (i.e. analgesia was produced with the smallest doses). After swim-stress, both males and ovariectomized rats showed less sensitivity to the analgesic effect of morphine than normal females in oestrus, while females subjected to constant light were less sensitive. In females in natural oestrus, but not in the other groups, small doses (0.05 mg/kg) of morphine, paradoxically reduced the analgesic effect of swim-stress considerably. These results show the importance of sex-related factors in the sensitivity of visceral pain to morphine and stress. A selective role for endogenous androgens and oestrogens is likely.

In vivo stimulation of phosphoinositide metabolism in the brainstem of rats following osmotic stress

We have investigated the effects of osmotic stress on the activity of inositol phospholipid turnover in the central nervous system of rats. Intracerebroventricular injection of [ 3H]myo-inositol was used for metabolic labelling of brain phosphoinositides in vivo. The levels of radiolabelled inositol lipids increased in a time-dependent manner in several areas of the brain, reaching a maximum 24 h after the injection. Treatment with LiCl 20 h after the administration of tritiated myo-inositol did not modify the levels of inositol lipids, but resulted in a dose-dependent accumulation of inositol phosphates. In rats treated with 10mEq/kg LiCl, intraperitoneal injections of hypertonic saline (1.50 M NaCl) resulted in the stimulation of phosphoinositide turnover in the brainstem, but not in any of the other regions of the brain studied. This response was not prevented by unilateral cervical vagotomy, but was significantly lower in vasopressin-deficient Brattleboro rats. Brain phosphoinositide metabolism was not stimulated by acute blood volume depletion. We conclude that osmotic stress, but not acute hypovolemia, results in vasopressin-dependent activation of brainstem neurons by stimulating inositol phospholipid metabolism. In addition, metabolic labelling in vivo followed by treatment with LiCl provides a useful approach for assessing the physiological significance of the activation of polyphosphoinositide metabolism in the central nervous system in vivo. Our study provides evidence for a functional role of this second messenger system in the brainstem.