Neonatal endotoxin exposure changes neuroendocrine, cardiovascular function and mortality during polymicrobial sepsis in adult rats

Our aim was to investigate whether neonatal LPS challenge may improve hormonal, cardiovascular response and mortality, this being a beneficial adaptation when adult rats are submitted to polymicrobial sepsis by cecal ligation and puncture (CLP). Fourteen days after birth, pups received an intraperitoneal injection of lipopolysaccharide (LPS; 100μg/kg) or saline. After 8-12 weeks, they were submitted to CLP, decapitated 4, 6 or 24h after surgery and blood was collected for vasopressin (AVP), corticosterone and nitrate measurement, while AVP contents were measured in neurohypophysis, supra-optic (SON) and paraventricular (PVN) nuclei. Moreover, rats had their mean arterial pressure (MAP) and heart rate (HR) evaluated, and mortality and bacteremia were determined at 24h. Septic animals with neonatal LPS exposure had higher plasma AVP and corticosterone levels, and higher c-Fos expression in SON and PVN at 24h after surgery when compared to saline treated rats. The LPS pretreated group showed increased AVP content in SON and PVN at 6h, while we did not observe any change in neurohypophyseal AVP content. The nitrate levels were significantly reduced in plasma at 6 and 24h after surgery, and in both hypothalamic nuclei only at 6h. Septic animals with neonatal LPS exposure showed increase in MAP during the initial phase of sepsis, but HR was not different from the neonatal saline group. Furthermore, neonatally LPS exposed rats showed a significant decrease in mortality rate as well as in bacteremia. These data suggest that neonatal LPS challenge is able to promote beneficial effects on neuroendocrine and cardiovascular responses to polymicrobial sepsis in adulthood.     

Mineralocorticoid treatment attenuates activation of oxytocinergic and vasopressinergic neurons by icv ANG II

Central oxytocin (OT) neurons limit intracerebroventricular (icv) ANG II-induced NaCl intake. Because mineralocorticoids synergistically increase ANG II-induced NaCl intake, we hypothesized that mineralocorticoids may attenuate ANG II-induced activation of inhibitory OT neurons. To test this hypothesis, we determined the effect of deoxycorticosterone (DOCA; 2 mg/day) on icv ANG II-induced c-Fos immunoreactivity in OT and vasopressin (VP) neurons in the supraoptic (SON) and paraventricular (PVN) nuclei of the hypothalamus and also on pituitary OT and VP secretion in male rats. DOCA significantly decreased the percentage of c-Fos-positive (%c-Fos+) OT neurons in the SON and PVN, both in the magnocellular and parvocellular subdivisions, and the %c-Fos+ VP neurons in the SON after a 5-ng icv injection of ANG II. DOCA also significantly reduced the %c-Fos+ OT neurons in the SON after 10 ng ANG II and tended to attenuate 10 ng ANG II-induced OT secretion. However, the %c-Fos+ OT neurons in DOCA-treated rats was greater after 10 ng ANG II, and DOCA did not affect the %c-Fos+ OT neurons in the PVN nor VP secretion or c-Fos immunoreactivity in either the SON or PVN after 10 ng ANG II. DOCA also did not significantly alter the effect of intraperitoneal (ip) cholecystokinin (62 microg) on %c-Fos+ OT neurons or of ip NaCl (2 ml of 2 M NaCl) on the %c-Fos+ OT and VP neurons. These findings indicate that DOCA attenuates the responsiveness of OT and VP neurons to ANG II without completely suppressing the activity of these neurons and, therefore, support the hypothesis that attenuation of OT neuronal activity is one mechanism by which mineralocorticoids enhance NaCl intake.     

Reciprocal pathway between medullary visceral zone and hypothalamic supraoptic nucleus or paraventricular nucleus involved in hyperosmotic regulation

In this study we try to simultaneously investigate the response of neurons and astrocytes of rats following hyperosmotic stimulation and test the possibility that the reciprocal pathways between medullary visceral zone (MVZ) and hypothalamic paraventricular nucleus (PVN) or supraoptic nucleus (SON). Hyperosmotic pressure animal model was established by administering 3% sodium chloride as drinking water to rats. The distribution and expression of the HRP retrogradely labeled neurons, Fos, tyrosine hydroxylase (TH) or vasopressin (VP) positive neuron and glial fibrillary acidic protein (GFAP) positive astrocytes in the MVZ, SON and PVN were observed by quadruplicate-labeling methods of WGA-HRP retrograde tracing combined with anti-Fos, TH (or VP) and GFAP immunohistochemical technique. Fos positive neurons within the MVZ, PVN and SON increased markedly. There were also a large number of GFAP positive structures in the brain and their distribution pattern was fundamentally similar or analogous to Fos positive neurons in the above-mentioned areas. The augmented GFAP reactivities took on hypertrophic cell bodies, thicker and longer processes. Quadruplicate immunohistochemical staining showed that a neuron could be closely surrounded by many astrocytes and they formed neuron-astrocytic complex (N-ASC). Fos+/TH+/HRP+/GFAP+ and Fos+/VP+/HRP+/GFAP+ quadruplicate labeled N-ASC could be found in the MVZ, PVN and SON, respectively. The present results indicated that the neurons and astrocytes might be very active following hyperosmotic pressure and N-ASC as a functional unit might serve to modulate osmotic pressure. There were reciprocal osmoregulation pathways between the MVZ and SON or PVN in the brain.     

Peptides and transmitter enzymes in hypothalamic magnocellular neurons after administration of hyperosmotic stimuli: comparison between messenger RNA and peptide/protein levels

Summary In situ hybridization histochemistry and indirect immunofluorescence histochemistry were used to study changes in the expression of vasopressin (VP), oxytocin (OXY), tyrosine hydroxylase (TH), galanin (GAL), dynorphin (DYN) and cholecystokinin (CCK) in hypothalamic magnocellular neurons of the paraventricular (PVN) and supraoptic (SON) nuclei of rats. After prolonged administration of 2% sodium chloride as drinking water (salt-loading), the treatment increased the levels of VP, OXY, TH, GAL, DYN and CCK mRNA in the PVN and SON. The increase in CCK mRNA was, however, proportionally higher in the PVN than in the SON. Within cell bodies of the PVN and SON of salt-loaded rats, a depletion of VP- and OXY-like immunoreactivity (LI) and an increase in TH-LI were seen. In salt-loaded/colchicine-treated rats, a marked decrease in GAL- and DYN-LI, but no specific changes in CCK-LI were observed. Within nerve fibers of the posterior pituitary of salt-loaded rats, a marked depletion of VP-, GAL- and DYN-LI was found. Less pronounced depletion was observed in OXY- and CCK-LI, and no specific changes in TH-LI were seen. The results show that high plasma osmolality induces increased mRNA levels for VP, OXY, TH, GAL, DYN and CCK, presumably indicating increased synthesis, an increased export from cell somata of VP, OXY, GAL and DYN, and a decrease in levels of these peptides in the posterior pituitary, suggesting increased release. The catecholamine-synthesizing enzyme TH, however, which has a cytoplasmic localization and is not released from nerve endings, remains high in the cell bodies and nerve endings during this state of increased activity.     

Opioids affect acquisition of LiCl-induced conditioned taste aversion: involvement of OT and VP systems

Aversive properties of lithium chloride (LiCl) are mediated via pathways comprising neurons of the nucleus of the solitary tract (NTS) and oxytocin (OT) and vasopressin (VP) cells in the hypothalamic paraventricular (PVN) and supraoptic (SON) nuclei. Because opioids act on brain regions that mediate effects of LiCl, we evaluated whether administration of opioids shortly before LiCl in rats influences 1) development of conditioned taste aversion (CTA) and 2) activation of NTS neurons and OT/VP cells. Neuronal activation was assessed by applying c-Fos immunohistochemical staining. Three opioids were used: morphine (MOR), a mu-agonist, butorphanol tartrate (BT), a mixed mu/kappa-agonist, and nociceptin/orphanin FQ (N/OFQ), which binds to an ORL1 receptor. BT and N/OFQ completely blocked acquisition of CTA. MOR alleviated but did not eliminate the aversive effects. Each of the opioids decreased LiCl-induced activation of NTS neurons as well as OT and VP cells in the PVN and SON. We conclude that opioids antagonize aversive properties of LiCl, presumably by suppressing activation of pathways that encompass OT and VP cells and NTS neurons.     

Activation of brain renin-angiotensin-aldosterone system by central sodium in Wistar rats

Functional studies indicate that the sympathoexcitatory and pressor responses to an increase in cerebrospinal fluid (CSF) [Na+] by central infusion of Na+-rich artificial cerebrospinal fluid (aCSF) in Wistar rats are mediated in the brain by mineralocorticoid receptor (MR) activation, ouabain-like compounds (OLC), and AT1-receptor stimulation. In the present study, we examined whether increasing CSF [Na+] by intracerebroventricular infusion of Na+-rich aCSF activates MR and thereby increases OLC and components of the renin-angiotensin system in the brain. Male Wistar rats received via osmotic minipump an intracerebroventricular infusion of aCSF or Na+-rich aCSF, in some groups combined with intracerebroventricular infusion of spironolactone (100 ng/h), antibody Fab fragments (to bind OLC), or as control gamma-globulins. After 2 wk of infusion, resting blood pressure and heart rate were recorded, OLC and aldosterone content in the hypothalamus were assessed by a specific ELISA or radioimmunoassay, and angiotensin-converting enzyme (ACE) and AT1-receptor binding densities in various brain nuclei were measured by autoradiography using 125I-labeled 351 A and 125I-labeled ANG II. When compared with intracerebroventricular aCSF, intracerebroventricular Na+-rich aCSF increased CSF [Na+] by approximately 5 mmol/l, mean arterial pressure by approximately 20 mmHg, heart rate by approximately 65 beats/min, and hypothalamic content of OLC by 50% and of aldosterone by 33%. Intracerebroventricular spironolactone did not affect CSF [Na+] but blocked the Na+-rich aCSF-induced increases in blood pressure and heart rate and OLC content. Intracerebroventricular Na+-rich aCSF increased ACE and AT1-receptor-binding densities in several brain nuclei, and Fab fragments blocked these increases. These data indicate that in Wistar rats, a chronic increase in CSF [Na+] may increase hypothalamic aldosterone and activate CNS pathways involving MR, and OLC, leading to increases in AT1-receptor and ACE densities in brain areas involved in cardiovascular regulation and hypertension.     

Increased brain uptake and brain to blood efflux transport of 14C-GABA in spontaneously hypertensive rats

The brain uptake and brain to blood efflux transport of (14)C-GABA were studied in spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto (WKY) rats using 20 min bilateral in situ brain perfusion in rats anesthetized using urethane. The volume of distribution (Vd) of (14)C-GABA into cerebrospinal fluid (CSF) and brain regions (cortex, diencephalon, cerebellum, and brain stem) was significantly greater in SHR than in the corresponding regions in WKY rats (p<0.05). The estimated Vd value of (14)C-GABA in CSF of SHR was 3.4 fold greater than that in WKY. Also compared to WKY, the Vd of (14)C-GABA into cerebellum and cortex of SHR was 15.3 fold and 19.4 fold greater, respectively. Although the study of blood-brain barrier (BBB) integrity using (3)H-mannitol revealed increased paracellular permeability at the brain capillaries of SHR when compared to WKY rats, this was found to be only partially responsible for the increased (14)C-GABA uptake. The study of brain to blood efflux transport of (14)C-GABA (after loading of brain with (14)C-GABA by vascular perfusion) revealed that the half-time of elimination was significantly shorter in SHR (5.35+/-0.66 min) than in WKY rats (14.83+/-1.94 min), (p<0.001). HPLC analysis revealed that GABA concentrations in brain extracts and CSF of SHR were similar to those in WKY rats (p>0.05). The faster efflux in SHR might be, at least partially, responsible to compensate for increased uptake of this neurotransmitter and to preserve the protective function of BBB towards GABA. The protective function of the BCSFB towards GABA appears to be also preserved, since systemic infusion of GABA within a wide range of administered doses (0.004-5.00 mg/kg) produced an increase in GABA CSF concentration from around 0.5 microM to only 11 microM, and the obtained pattern of CSF GABA concentrations under these conditions did not differ between SHR and WKY rats, as revealed by HPLC.     

Influence of hypergravity on hypothalamic vasopressin and oxytocin neurons in rats

This study was aimed to evaluate the reaction of the vasopressin (VP) and oxytocin (OT) neurons of the supraoptic nucleus (SON) in rats to single or repeated hypergravity (HG). Special attention was paid to the tyrosine hydroxylase (TH) expression in VP neurons as a marker of the neuron activation. Rats were revolved in a centrifuge with overloading 2G for 5 days or 34 days as well as for 34 days plus 5 days with an interval of 39 days between two rotations. Control rats were kept in a centrifuge room. Radioimmununoassay, quantitative and semi-quantitative immunocytochemistry and in situ hybridization were used to evaluate: a) VP concentration in the pituitary posterior lobe (PL) and in plasma; b) the number of VP-, OT- and TH-immunoreactive neurons in the SON; c) the optic density of VP-, OT- and TH-immunoreactive materials in cell bodies (SON) and distal axons (PL), d) the optic density of VP and OT mRNAs signals (S35) in the whole SON on microfilms. According to our data, VP neurons were strongly activated during HG (5 days or 34 days) that was manifested in the functional hypertrophy of the neurons, greatly increased concentrations of VP mRNA in the SON and VP in plasma, the onset of the TH expression. The neurons showed initially (5 days) the functional insufficiency (VP release > VP synthesis) followed by their adaptation (subsequent 29 days) to the increased need in VP (VP release < VP synthesis). No reaction of VP neurons was observed to repeated HG. In contrast to VP neurons, OT neurons did not react to short-term HG or showed functional depression after the long-term treatment.     

Osmotic regulation of estrogen receptor-beta expression in magnocellular vasopressin neurons requires lamina terminalis

Estrogen receptor-beta (ER-beta) expression in rat magnocellular vasopressin (VP) neurons of the supraoptic and paraventricular nuclei (SON and PVN, respectively) becomes undetectable after 72 h of 2% NaCl consumption. To test the hypothesis that osmosensitive mechanisms that originate in the region of the organum vasculosum lamina terminalis (OVLT) control ER-beta expression in the SON and PVN, animals were water deprived after electrolytic lesions were performed on the area anterior to the ventral third ventricle (AV3V). Such lesions prevent osmotic stimulation of VP release. Four weeks after surgery, male rats [lesioned (n = 16) or sham (n = 14)] were water deprived for 48 h or allowed water ad libitum. Water deprivation eliminated ER-beta-immunoreactivity (-ir) in SON and magnocellular PVN of sham-lesioned animals. Fos-ir was evident in these neurons, and plasma osmolality (Posm) and hematocrit (Ht) were significantly elevated compared with the sham-hydrated rats (Posm, 304 +/- 1 vs. 318 +/- 2 mosmol/kgH2O; P < 0.001; Ht, 49.6 +/- 0.6 vs. 55.0 +/- 0.9%; P < 0.001). ER-beta expression was comparable in sham-hydrated, AV3V-hydrated, and 6 of 8 AV3V-dehydrated rats despite significant increases in Posm in both groups (AV3V hydrated, 312 +/- 2; AV3V dehydrated, 380 +/- 10 mosmol/kgH2O; P < 0.001). OVLT was not ablated in the AV3V-dehydrated rats in which ER-beta was depleted. Fos-ir was low or undetectable in SON in the AV3V-hydrated animals despite elevated Posm values. In AV3V-dehydrated rats, Fos-ir was significantly less than in sham-dehydrated animals but was significantly increased compared with the sham-hydrated group. This could reflect activation by nonosmotic parameters that do not inhibit ER-beta expression. These data support the hypothesis that inhibition of ER-beta expression in the SON by osmotic stimulation is mediated by osmoreceptive neurons in the lamina terminalis.     

Altered astrocyte glutamate transporter regulation of hypothalamic neurosecretory neurons in heart failure rats

Neurohumoral activation, which includes augmented plasma levels of the neurohormone vasopressin (VP), is a common finding in heart failure (HF) that contributes to morbidity and mortality in this disease. While an increased activation of magnocellular neurosecretory cells (MNCs) and enhanced glutamate function in HF is well documented, the precise underlying mechanisms remain to be elucidated. Here, we combined electrophysiology and protein measurements to determine whether altered glial glutamate transporter function and/or expression occurs in the hypothalamic supraoptic nucleus (SON) during HF. Patch-clamp recordings obtained from MNCs in brain slices show that pharmacological blockade of astrocyte glutamate transporter 1 (GLT1) function [500 μM dihydrokainate (DHK)], resulted in a persistent N-methyl-D-aspartate receptor (NMDAR)-mediated inward current (tonic I(NMDA)) in sham rats, an effect that was significantly smaller in MNCs from HF rats. In addition, we found a diminished GLT1 protein content in plasma membrane (but not cytosolic) fractions of SON punches in HF rats. Conversely, astrocyte GLAST expression was significantly higher in the SON of HF rats, while nonselective blockade of glutamate transport activity (100 μM TBOA) evoked an enhanced tonic I(NMDA) activation in HF rats. Steady-state activation of NMDARs by extracellular glutamate levels was diminished during HF. Taken together, these results support a shift in the relative expression and function of two major glial glutamate transporters (from GLT1 to GLAST predominance) during HF. This shift may act as a compensatory mechanism to preserve an adequate basal glutamate uptake level in the face of an enhanced glutamatergic afferent activity in HF rats.     

Effect of Block of Catecholamine Synthesis on the Functional State of Vasopressinergic Neurons of Hypothalamus in Dehydrated Rats

Effect of catecholamines (CA) on the functional state of vasopressin (VP)-ergic neurons of hypothalamus at their stimulation produced by dehydration (salt diet and water deprivation) was studied in in vivo experiments on adult male Wistar rats. Quantitative assessment of VP-immunopositive substance and digoxigenin-labeled VP mRNA (hybridization in situ) in neurosecretory cells of supraoptic (SON) and paraventricular (PVN) nuclei was performed using measurements of optical density of the stained substance in perykaria and a computer digital television analyzer with PhotoM software. Hybridization in situ technique allowed evaluating intensity of VP synthesis, while comparison of the amount of VP mRNA and VP-immunoreactive substance in neurons of SON and PVN made it possible to evaluate release of VP from perykaria. In PVN, repeated saline administration (0.25 ml per 100 g weight) and severe dehydration led to activation both of synthesis and of release of VP from cell perikarya. Use of -methyl-p-tyrosine, an inhibitor of catecholamine (CA) synthesis on the background of dehydration was not accompanied by changes of the functional state of VP-ergic neurons of PVN as compared with dehydrated animals. No changes in functional state of VP-ergic neurosecretory cells in SON were found after saline administration, whereas dehydration activated synthesis and release of VP from perykaria, like in VP-ergic neurons of PVN. Inhibition of CA synthesis on the background of dehydration led to activation of VP release by SON neurons without affecting the level of VP synthesis. The data obtained indicate that CA is able to suppress the VP release from neurons of SON, which is produced caused by activation of the VP-ergic system under conditions of dehydration.     

Acidic amino acid clearance from CSF in the neonatal versus adult rat using ventriculo-cisternal perfusion

The acidic amino acids aspartate and glutamate are excitatory neurotransmitters in the CNS. The clearance of this group of amino acids from CSF of adult and neonatal (7-day-old) rats was investigated. Ventriculo-cisternal perfusions with 14C-amino acids and 3H-dextran were carried out for up to 90 min. Uptake of the amino acids by the whole brain was measured, and the loss to blood was calculated. 3H-Dextran was included in the perfusate for measurement of CSF secretion rate. After 90-min perfusion, both aspartate and glutamate showed a similar uptake into the whole brain, and this did not change with age (p>0.05). However, clearance from CSF was greater in the adult, as was entry into blood from CSF. Addition of 5 mM excess unlabelled amino acid resulted in reduction in the brain uptake of both 14C-amino acids in the adult rat. In the neonate, addition of aspartate also reduced brain aspartate uptake, whereas addition of glutamate increased brain neonatal [14C]glutamate uptake. The rate of CSF secretion was significantly greater in the adult, 1.26+/-0.18 microl x min(-1) x g(-1), than in the neonate, 0.62+/-0.08 microl x min(-1) x g(-1), and the turnover of CSF was greater in adults (p<0.01). In summary, both aspartate and glutamate showed greater clearances from CSF in the adult than the neonate. This clearance was found to be by carrier-mediated mechanisms.     

Microarray analysis reveals interleukin-6 as a novel secretory product of the hypothalamo-neurohypophyseal system

Physiological activation of the hypothalamo-neurohypophyseal system (HNS) by dehydration results is a massive release of vasopressin (VP) from the posterior pituitary. This is accompanied by a functional remodeling of the HNS. In this study we used cDNA arrays in an attempt to identify genes that exhibit differential expression in the hypothalamus following dehydration. Our study revealed nine candidate genes, including interleukin-6 (IL-6) as a putative novel secretory product of HNS worthy of further analysis. In situ hybridization and immunocytochemistry confirmed that IL-6 is robustly expressed in the supraoptic (SON) and the paraventricular (PVN) nuclei of the hypothalamus. By double staining immunofluorescence we showed that IL-6 is largely co-localized with VP in the SON and PVN. In situ hybridization, immunocytochemistry, and Western blotting all revealed IL-6 up-regulation in the SON and PVN following dehydration, thus validating the array data. The same dehydration stimulus resulted in an increase in IL-6 immunoreactivity in the axons of the internal zone of the median eminence and a marked reduction in IL-6-like material in the posterior pituitary gland. We thus suggest that IL-6 takes the same secretory pathway as VP and is secreted from the posterior pituitary following a physiological stimulus.     

Microarray Analysis of Gene Expression in the Supraoptic Nucleus of Normoosmotic and Hypoosmotic Rats

1. Hypoosmolality produces a dramatic inhibition of vasopressin (VP) and oxytocin (OT) gene expression in the supraoptic nucleus (SON). This study examines the effect of sustained hypoosmolality on global gene expression in the OT and VP magnocellular neurons (MCNs) of the hypothalamo-neurohypophysial system (HNS), in order to detect novel genes in this system that might be involved in osmoregulation in the MCNs.2. For this purpose, we used Affymetrix oligonucleotide arrays to analyze the expression of specific genes in laser microdissected rat SONs, and their changes in expression during chronic hypoosmolality. We identified over 40 genes that had three-fold or more greater expression in the SON versus total hypothalamus, and that also changed more than two fold in expression as a result of the chronic hypoosmolar treatment. These genes contained both novel as well as genes previously known to be present in the SON. All of the raw data for the genes that are expressed in the SON and altered by hypoosmolality can be found on the following NINDS website URL address: http://data.ninds.nih.gov/Gainer/Publications     

Chronic lithium administration attenuates up-regulated brain arachidonic acid metabolism in a rat model of neuroinflammation

Neuroinflammation, caused by a 6-day intracerebroventricular infusion of lipopolysaccharide (LPS) in rats, is associated with the up-regulation of brain arachidonic acid (AA) metabolism markers. Because chronic LiCl down-regulates markers of brain AA metabolism, we hypothesized that it would attenuate increments of these markers in LPS-infused rats. Incorporation coefficients k* of AA from plasma into brain, and other brain AA metabolic markers, were measured in rats that had been fed a LiCl or control diet for 6 weeks, and subjected in the last 6 days on the diet to intracerebroventricular infusion of artificial CSF or of LPS. In rats on the control diet, LPS compared with CSF infusion increased k* significantly in 28 regions, whereas the LiCl diet prevented k* increments in 18 of these regions. LiCl in CSF infused rats increased k* in 14 regions, largely belonging to auditory and visual systems. Brain cytoplasmic phospholipase A(2) activity, and prostaglandin E(2) and thromboxane B(2) concentrations, were increased significantly by LPS infusion in rats fed the control but not the LiCl diet. Chronic LiCl administration attenuates LPS-induced up-regulation of a number of brain AA metabolism markers. To the extent that this up-regulation has neuropathological consequences, lithium might be considered for treating human brain diseases accompanied by neuroinflammation.     

Participation of neuronal NO-synthase in regulation of hypothalamus vasopressinergic neurons of rat pups at early stages of postnatal ontogeny

To reveal character of interaction of catecholamines (CA) and NO in regulation of development and of the functional state of vasopressinergic (VP-ergic) neurons of supraoptic (SON) and paraventricular (PVN) nuclei, the female rats were injected intraperitoneally with the inhibitor of CA synthesis α-methyl-p-tyrosine, daily, from the 13th to the 20th days of pregnancy. Rat pups born by the females administered with saline at the same period of pregnancy as well as intact pups and adult rats were used as control. Expression of neuronal NO-synthase (nNOS) in neurons of SON and PVN of rat pups at early stages of postnatal development was found to be significantly higher than the definitive level, which allows suggesting participation of NO in development of hypothalamic VP-ergic neurons. The revealed differences of periods of the maximal nNOS expression in the SON and PVN neurons have permitted suggesting development of SON to be completed earlier than that of PVN. The pups exposed to stress at the last third of embryonic development had a long-lasting effect on the state of VP-ergic neurons of the pups after birth. The nNOS expression in neurons does not change, which suggests that NO is not involved in regulation of VP-ergic neurons after exposure to stress at early stages of ontogenesis. A decrease of CA level in the brain at the last third of embryogenesis led to a long preserved decrease of the functional activity of VP-ergic neurons. The nNOS expression in VP-ergic neurons of SON and PVN rose substantially under effect of a compensatory enhancement of tyrosine hydroxylase (TH) expression in neurons of SON and of an increase of the level of CA-ergic innervation of PVN. Thus, we have shown that a decrease of CA level in the embryonic brain leads to an increase of nNOS expression of hypothalamic VP-ergic neurons of rat pups after birth and that the character of NO action on function of VP-ergic neurons does not differ from that of adult animals as soon as at early stages of ontogenesis.     

Immunocytochemistry and ultrastructure of the neuropil located ventral to the rat supraoptic nucleus

Summary The neuropil located ventral to the SON was investigated by the use of immunoperoxidase staining for neurophysins, oxytocin and vasopressin, and electron miroscopy. The study was performed in six groups of rats: 1) control; 2) infusion of isotonic saline into the CSF; 3) infusion of hypertonic saline into the CSF; 4) drinking hypertonic saline for 4 days; 5) same as group 4 but injection of colchicine into the CSF on second day of dehydration; 6) salt loading for 3 months. In the control rats the ventral neuropil contained a few immunoreactive processes, the general morphology of which was completely different from that of the neurosecretory axons emerging from the SON at its dorsal aspect. In rats of groups 3 to 6 the ventral processes (VP) became loaded with neurosecretory granules, whereas the perikarya and axons were depleted. Based on their general morphology and reactivity pattern it is suggested that the VP are dendrites. Most of these dendrites were embedded in a glial cushion formed by the processes of a particular type of marginal glia. Some of these dendrites enveloped an arteriole penetrating the optic tract. All VP were rich in synaptic contacts. The possibility that the VP of neurosecretory cells may be functionally related to the subarachnoid CSF and the arteriolar blood flow is discussed.Supported by Grant RS-82-18 from Direccíon de Investigaciones, Universidad Austral de Chile     

In vivo imaging detects a transient increase in brain arachidonic acid metabolism: a potential marker of neuroinflammation

In a rat model of neuroinflammation produced by an intracerebral ventricular infusion of bacterial lipopolysaccaride (LPS), we measured the coefficients of incorporation (k*) of arachidonic acid (AA, 20 : 4n-6) from plasma into each of 80 brain regions, using quantitative autoradiography and intravenously injected [1-(14)C]AA. Compared with control rats infused with artificial cerebrospinal fluid (aCSF), k* was increased significantly in 25 brain areas, many of them close to the CSF compartments, following 6-days of LPS infusion. The increases, ranging from 31 to 76%, occurred in frontal, motor, somatosensory, and olfactory cortex, thalamus, hypothalamus, and septal nuclei, and basal ganglia. Following 28 days of LPS infusion, k* was increased significantly in only two brain regions. Direct analyses of microwaved brain showed that 93 +/- 3 (SD) and 94 +/- 4% of brain radioactivity was in the organic extract as radiolabeled AA in the 6-day control and LPS-infused animals, respectively, compared with 91 +/- 3 and 87 +/- 6% in the 28-day control and LPS-infused animals. These results confirm that brain AA metabolism is disturbed after 6 days of LPS exposure, show this increase is transient, and that these changes can be detected and localized using in vivo imaging with radiolabeled AA.     

Genomic Effects of Cold and Isolation Stress on Magnocellular Vasopressin mRNA-Containing Cells in the Hypothalamus of the Rat

We assessed the effects of cold and isolation stress on arginine vasopressin (AVP) mRNA in the paraventricular (PVN) and supraoptic (SON) nuclei of the hypothalamus. Vasopressin mRNA levels were determined by in situ hybridization histochemistry at the cellular level. In posterior magnocellular neurons of the PVN isolation stress for 7 or 14 days increased vasopressin mRNA levels 28 and 29%, respectively, compared to group-housed controls. No significant alterations in vasopressin gene expression were observed in the SON after 7 or 14 days of isolation stress. Scattered magnocellular AVP mRNA-expressing cells of the medial parvocellular PVN showed increases of 19 and 34% after 7 and 14 days of isolation, respectively. We also studied the effect of cold or combined cold and isolation stress on vasopressin gene expression in the PVN and SON. Cold stress for 3 h daily for 4 consecutive days increased AVP mRNA levels in the posterior magnocellular PVN by 15%. Cold-isolated animals showed an increase of 21%. No significant effect on AVP mRNA levels in the SON was observed. In contrast to the posterior magnocellular PVN, cold or cold-isolation stress increased AVP mRNA in magnocellular neurons of the medial parvocellular region of the PVN by 25 and 43%, respectively, relative to control rats. These results suggest that psychological and metabolic stress may be added to the list of stressors that activate the hypothalamo-neurohypophysial system.