Push-pull Perfusion Technique Research Articles

Early Stage Alterations in CA1 Extracellular Region Proteins Indicate Dysregulation of IL6 and Iron Homeostasis in the 5XFAD Alzheimer’s Disease Mouse Model

In recent years, an increasing number of research papers revealed that the compositional and volumetric alterations in the extracellular matrix are the consequences of aging and may be related to Alzheimer's disease (AD). In this study, we aimed to demonstrate the alterations in hippocampal extracellular fluid proteins in vivo using the 5XFAD mouse model. Samples were obtained from hippocampi of 5XFAD mice (n = 6) and their non-transgenic littermates by intracerebral push-pull perfusion technique at 3 months of age, representing the pre-pathological stage of the AD. Proteins in the hippocampal perfusates were analyzed by Ultra Performance Liquid Chromatography-Electrospray Ionization Quadrupole Time-of-Flight Mass Spectrometry (UPLC-ESI-qTOF-MS/MS). 178 proteins were identified and 19 proteins of them were found to be statistically significantly altered (p≤0.05, fold change ≥40%, unique peptide count ≥3) in the hippocampal CA1 extracellular fluid of the 5XFAD mouse model. Ingenuity pathway analysis of the protein expression results identified IL6 as an upstream regulator. The upregulation of IL6 was validated by immunohistochemical staining of the hippocampus and cortex of the 5XFAD mice prior to Aβ plaque formation. Furthermore, the iron level in the hippocampus was measured by inductively coupled plasma-mass spectrometry as IL6 is mentioned in several studies to take part in iron homeostasis and inflammation and found to be increased in 5XFAD mice hippocampus. Alterations in extracellular matrix proteins in addition to increasing amount of hippocampal IL6 and iron in the early stages of AD may reveal inflammation-mediated iron dyshomeostasis in the early stages of neurodegeneration.

In Vivo Monitoring of Quantum Dots in the Extracellular Space Using Push−Pull Perfusion Sampling, Online In-Tube Solid Phase Extraction, and Inductively Coupled Plasma Mass Spectrometry

To monitor the dynamic changes of extracellular quantum dots (QDs) in vivo in the livers of anesthetized rats, we developed an automatic online analytical system comprising push-pull perfusion (PPP) sampling, the established in-tube solid phase extraction (SPE) procedure, and inductively coupled plasma mass spectrometry (ICPMS). The method takes advantage of the retention of QDs onto the interior surface of a polytetrafluoroethylene (PTFE) tube as a means of extracting the QDs from complicated push-pull perfusates. For the injected QDs present in the liver extracellular fluid (ECF) at low picomolar levels, a temporal resolution of 10 min was required to collect sufficient amounts of QDs to meet the sensitivity requirements of the ICPMS system. To the best of our knowledge, this study is the first to exploit the PPP technique for the collection of QDs from living animals and PTFE tubing as a SPE adsorbent for the online extraction of QDs and the removal of biological matrix prior to ICPMS analysis of cadmium-containing inorganic nanocrystal. We confirmed the analytical reliability of this method from measurements of the spike recoveries of saline samples; in addition, we demonstrated the systems' applicability through in vivo monitoring of the time-dependent concentration profile of liver extracellular QDs in living rats after intravenous administration.

Enhanced activity of GABA receptors inhibits glutamate release induced by focal cerebral ischemia in rat striatum

Cerebral ischemia causes an excess release of glutamate, which can injure neurons. The striatum is one of the important regions vulnerable to hypoxia and ischemia. Using push-pull perfusion technique, we investigated the regulatory role of gamma-aminobutyric acid (GABA) and its receptors in modifying the amount of glutamate in rat striatum with ischemia. Perfusion with exogenous GABA (1 mM) inhibited cerebral ischemia-induced glutamate release by as much as 47%. We further characterized relative roles of subtype receptors of GABA on glutamate release by using pharmacological tools. While baclofen (500 microM), a GABA(B) receptor agonist, suppressed ischemia-induced glutamate release by 52%, GABA(B) receptor antagonist saclofen (500 microM) failed to produce a significant increase of glutamate release. The GABA(A) receptor agonist muscimol (500 microM) also reduced by 38% the release of glutamate induced by cerebral ischemia but the GABA(A) receptor antagonist bicuculline (500 microM) had very little effect. The present study demonstrates that the excessive release of glutamate or the overly activated glutamate receptor, triggered by cerebral ischemia, can be down-regulated by exogenous GABA or by increased activity of GABA receptors, especially the presynaptic GABA(B) receptors, which might be one of the important mechanisms to protect against striatum neuronal damage from over stimulation by excessive glutamate during ischemia.

In vivo brain-derived neurotrophic factor release and tyrosine kinase B receptor expression in the supraoptic nucleus after osmotic stress stimulus in rats

Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family involved in plasticity and neuroprotective processes. In recent years, we have reported the presence of BDNF mRNA in the supraoptic nucleus (SON) as well its sensitivity to osmotic stress. The rat SON is a relatively homogenous nucleus mainly consisting of magnocellular soma with their dendritic processes. BDNF may be released from dendrites to the extracellular space to stimulate tyrosine kinase (Trk) B receptors which are hypothetically present on these subcellular SON compartments. The main goal of this work was thus to study the presence and the in vivo BDNF-IR release from SON using the push–pull perfusion technique following systemic (i.p.) or local (within the SON) osmotic stimulation. BDNF was detected by immunocytochemistry and its release was measured by immunological assay (ELISA). Likewise, TrkB receptor localization in the SON—mRNA and their respective proteins—were studied by in situ hybridization and immunohistofluorescence techniques, respectively. Phosphorylation of CREB was detected by immunohistofluorescence.We present here direct evidence of in vivo dendritic BDNF release from SON which is highly sensitive to osmotic stress. The osmotic response latency period clearly depends on the mode of stimulus application (210 min for i.p. route vs. 15 min for intra-SON administration). The fact that BDNF is released as a very rapid peak when osmotic stimulation is locally applied is strong evidence in favor of an intra-SON origin of this secretion. Osmotic stress also increased phosphorylated cAMP response element binding protein immunoreactivity in the SON. In addition, we show in control rats that truncated forms of tyrosine kinase B receptor 2 mRNA represent the most abundant messenger in the SON as compared with brain-derived neurotrophic factor full-length catalytic receptor or truncated forms of tyrosine kinase B receptor 1 mRNA. In conclusion, it is likely that BDNF and their receptors are involved in neuronal plasticity changes induced by osmotic stress in the SON.

Gonadotrophin‐Releasing Hormone (GnRH) Release in Marmosets I: In Vivo Measurement in Ovary‐Intact and Ovariectomised Females

In vivo hypothalamic gonadotrophin-releasing hormone (GnRH) release was characterised for the first time in a New World primate. A nonterminal and repeatable push-pull perfusion (PPP) technique reliably measured GnRH in conscious common marmoset monkeys. Nineteen adult females (n = 8 ovary-intact in the mid-follicular phase; n = 11 ovariectomised) were fitted with long-term cranial pedestals, and a push-pull cannula was temporarily placed in unique locations within the pituitary stalk-median eminence (S-ME) 2 days prior to each PPP session. Marmosets underwent 1-3 PPPs (32 PPPs in total) lasting up to 12 h. Plasma cortisol levels were not elevated in these habituated marmosets during PPP, and PPP did not disrupt ovulatory cyclicity or subsequent fertility in ovary-intact females. GnRH displayed an organised pattern of release, with pulses occurring every 50.0 +/- 2.6 min and lasting 25.4 +/- 1.3 min. GnRH pulse frequency was consistent within individual marmosets across multiple PPPs. GnRH mean concentration, baseline concentration and pulse amplitude varied predictably with anatomical location of the cannula tip within the S-ME. GnRH release increased characteristically in response to a norepinephrine infusion and decreased abruptly during the evening transition to lights off. Ovary-intact (mid-follicular phase) and ovariectomised marmosets did not differ significantly on any parameter of GnRH release. Overall, these results indicate that PPP can be used to reliably assess in vivo GnRH release in marmosets and will be a useful tool for future studies of reproductive neuroendocrinology in this small primate.

Role of the paraventricular nucleus in renal excretory responses to acute volume expansion: role of nitric oxide.

Acute volume expansion (VE) produces a suppression of renal sympathetic nerve discharge (RSND) resulting in diuresis and natriuresis. Recently, we have demonstrated that the endogenous nitric oxide (NO) system within the paraventricular nucleus (PVN) produces a decrease in RSND. We hypothesized that endogenous NO in the PVN is involved in the suppression of RSND leading to diuretic and natriuretic responses to acute VE. To test this hypothesis, we first measured the VE-induced increase in renal sodium excretion and urine flow with and without blockade of NO, with microinjection of NG-monomethyl-L-arginine (L-NMMA; 200 pmol in 200 nl), within the PVN of Inactin-anesthetized male Sprague-Dawley rats. Acute VE produced significant increases in urine flow and sodium excretion, which were diminished in rats treated with L-NMMA within the PVN. This effect of NO blockade within the PVN on VE-induced diuresis and natriuresis was abolished by renal denervation. Consistent with these data, acute VE induced a decrease in RSND (52% of the baseline level), which was significantly blunted by prior administration of L-NMMA into the PVN (28% of the baseline level) induced by a comparable level of acute VE. Using the push-pull perfusion technique, we found that acute VE induced a significant increase in NOx concentration in the perfusate from the PVN region. Taken together, these results suggest that acute VE induces an increase in NO production within the PVN that leads to renal sympathoinhibition, resulting in diuresis and natriuresis. We conclude that NO within the PVN plays an important role in regulation of sodium and water excretions in the volume reflex via modulating renal sympathetic outflow.

Leptin directly acts within the hypothalamus to stimulate gonadotropin-releasing hormone secretion in vivo in rats.

It is still not known whether leptin, an adipocyte-derived hormone, acts directly within the hypothalamus to stimulate the gonadotropin-releasing hormone (GnRH)-luteinizing hormone (LH) system. In order to address this question, the present study examined the effects of direct intrahypothalamic perfusions with leptin on the in vivo release of GnRH in ovarian steroid-primed ovariectomized rats utilizing the push-pull perfusion technique. Both alpha-melanocyte-stimulating hormone (alpha-MSH) and neuropeptide Y were also measured in the hypothalamic perfusates. In normally fed animals, the leptin infusion was without effect on the release of these three hypothalamic peptides and also without effect on plasma LH and prolactin (PRL), whether leptin was infused into the medial preoptic area (where the majority of GnRH neuronal cell bodies exist) or the median eminence-arcuate nucleus complex (where axon terminals of GnRH neurons are located). In contrast, in 3-day fasted rats leptin was effective in stimulating the secretion of GnRH, alpha-MSH, and LH, regardless of the site of perfusion. These three hormones were increased in a temporal order of alpha-MSH, GnRH and LH. Irrespective of the site of perfusion, leptin was without effect on the release of neuropeptide Y. Only when leptin was infused into the median eminence-arcuate nucleus complex was PRL secretion also stimulated, although its onset was 1 h behind that of LH. The leptin-induced elevations of GnRH, alpha-MSH, LH and PRL were all dose-dependently stimulated by subnormal (1.0 ng ml(-1)) and normal (3.0 ng ml(-1)) concentrations of leptin, but at higher concentrations (10 ng ml(-1)) it did not produce additional effects. Leptin infusion into the anterior hypothalamic area, a control site equidistant from both the medial preoptic area and the median eminence-arcuate nucleus complex, did not produce a significant change in any of the hormones in either the fed or fasted rats. These results demonstrate for the first time that leptin can act at both the cell bodies and axon terminals of GnRH neurons to stimulate the release of the neurohormone in vivo, and they also suggest that alpha-MSH may play a significant intermediary role in linking leptin and GnRH secretion.

Effect of acupuncture on monoamines and adenosine triphosphatase activity in lateral hypothaiamic area of obese rats

Objective: To study the mechanism of acupuncture in treating simple obesity.Methods: Central nerve push-pull perfusion and biochemical technique were used to observe the effect of acupuncture on the obese parameters, changes of monoamine transmitters and activity of ATPase in the lateral hypothalamic area (LHA) of obese rats.Results: Noradrenaline (NA) level in LHA of obese rats was higher but serotonin (5-HT) level and ATPase activity were lower than those in normal rats. After acupuncture treatment, in the same time of reducing body weight, NA level in LHA of rats was reduced, and 5-HT level and ATPase activity in it were increased. (P<0.05 and P< 0.01).Conclusion : The effective regulation on LHA of obese rats is possibly one of the key factors in anti-obesity effect of acupuncture.

A Comparative Study of the Effects of Nitric Oxide and Carbon Monoxide on the in vivo Release of Gonadotropin-Releasing Hormone and Neuropeptide Y from Rat Hypothalamus during the Estradiol-Induced Luteinizing Hormone Surge: Estimation by Push-Pull Perfusion

Recent evidence suggests that nitric oxide (NO), a free radical gas, plays an important role in regulating the function of a variety of neuroendocrine systems. With respect to the hypothalamo-pituitary-gonadal axis, a stimulatory effect of NO on the release of gonadotropin-releasing hormone (GnRH) from rat hypothalamus has been demonstrated in vitro. However, no previous study has reported NO-stimulated secretion of GnRH from in vivo hypothalamus, and also the precise cellular site of action of NO within the GnRH neuronal system remains to be elucidated. In the present study, utilizing the push-pull perfusion technique of rat hypothalamus, we examined the effect of L-arginine (L-Arg), an NO donor, on the release of GnRH, neuropeptide Y and cyclic GMP (c-GMP), which is a pivotal second messenger molecule of the NO system. For comparison, we also examined the effect of carbon monoxide (CO), another putative gaseous neurotransmitter, using hematin, a CO donor. During the period of 11.00–18.00 h, we collected blood and hypothalamic perfusates from ovariectomized adult rats that had been implanted with an estradiol capsule 2 days before. During the entire period of observation, L-Arg (1.0 or 10 mM), hematin (10 or 100 µM) or artificial cerebrospinal fluid alone (as the control) was infused into the medial preoptic area (MPOA) where there are cell bodies of GnRH neurons, or the median eminence-arcuate nucleus complex (ME-ARC) where axon terminals of GnRH neurons are localized. Although 10 mM of L-Arg significantly stimulated GnRH and c-GMP, but not neuropeptide Y, levels in both the MPOA and ME-ARC, GnRH and c-GMP in the ME-ARC were already increased by 1.0 mM of L-Arg. By contrast, both concentrations of hematin were without effect at either site of the hypothalamus. This study is the first to demonstrate that NO is capable of stimulating GnRH release from rat hypothalamus in vivo. Our data also suggests that both cell bodies and axon terminals of GnRH neurons may be sites of action of NO. Our data do not support a previous study by other investigators that reported a stimulatory effect of CO on the GnRH release.

Temporal profiles of interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha in the plasma and hypothalamic paraventricular nucleus after intravenous or intraperitoneal administration of lipopolysaccharide in the rat: estimation by push-pull perfusion.

Lipopolysaccharide (LPS) is known to stimulate the synthesis and secretion of various proinflammatory cytokines in both the peripheral immune cells and the brain. Yet, the relative contribution of peripheral and central cytokines to the LPS-induced activation of the hypothalamo-pituitary-adrenal axis is still poorly understood. In this study, utilizing the push-pull perfusion technique of the rat brain, we attempted to characterize in detail the temporal profiles of interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-alpha after intravenous (i.v.) or intraperitoneal (i.p.) administration of LPS in both the general circulation and the hypothalamic paraventricular nucleus (PVN), which is the primary source of corticotropin releasing hormone (CRH). Temporal changes in plasma adrenocorticotropic hormone (ACTH) and CRH levels in the PVN were also monitored. We collected blood and perfusates every 30 min from 11:00 to 17:00 h. At 12:00 h, 1.0 or 2.5 mg/kg body weight of LPS was given via an i.v. or i.p. route, respectively. Peak ACTH response occurred 30 min after i.v. LPS and 1.5 h after ip LPS. Of the three cytokines measured in the plasma, TNF-alpha showed the fastest rise in synchrony with peak ACTH secretion after both i.v. and i.p. LPS. Although plasma IL-6 also showed a robust rise, its peak level occurred later than the ACTH peak. Elevation of plasma IL-1beta was the smallest among the three cytokines. CRH levels in the PVN reached their peaks 1 and 2.5 h after the ACTH peak following i.p. and i.v. LPS, respectively. Irrespective of the route of LPS administration, IL-6 and TNF-alpha levels in the PVN showed significant rises 1-2 h after the ACTH peak, but IL-1beta in the PVN did not significantly change during the entire period of observation. The results of the present study suggest that circulating TNF-alpha may play the most important role in triggering the early, peak phase of ACTH secretion after both i.v. and i.p. LPS. Although it is possible that brain TNF-alpha, IL-6, and circulating IL-6, may be involved in the later, protracted phase of ACTH secretion induced by LPS, IL-1beta in both the brain and peripheral circulation seems to play the smallest role in ACTH secretion. This is the first study to characterize the LPS-induced temporal changes in IL-1beta, IL-6, and TNF-alpha in both plasma and PVN simultaneously in conscious, freely moving rats.

Neuropeptide Y Release in the Paraventricular Nucleus of Long–Evans Rats Treated with Leptin

Neuropeptide Y (NPY) and leptin are actually two of the most potent peptides involved in the regulation of food intake with their respective stimulatory and inhibitory actions. The infusion of each peptide has a significant influence on the mRNA expression of the other in the adipose tissue for leptin and in the arcuate nucleus of the hypothalamus for NPY. To confirm this functional interaction, we measured the in vivorelease of hypothalamic neuropeptide Y in awake fasted and refed Long–Evans rats after intraperitoneal (I.P.) injection of leptin. For this purpose, we used the push–pull perfusion technique with the cannula placed above the right paraventricular nucleus. I.P. leptin significantly inhibited food intake during the two hours of food access (−50%; p<0.02). NPY release was not modified by leptin alone. But, when food was present, it slightly but significantly increased (p<0.03 or less) and remained at a sustained level in the leptin-treated rats whereas it decreased in the control saline-injected group (p<0.04). Thus, leptin did not acutely regulate NPY release and other food-related factors are probably involved as mediators of its anorexigenic effects.

N-Methyl- d-aspartate receptor involvement in dexamethasone and stress-induced hypothalamic somatostatin release in rats

The median eminence (ME) push-pull perfusion technique was used in this work and the results clearly showed that i.p. administration of MK-801 (4 mg/kg), a specific N-methyl- d-aspartate (NMDA) receptor antagonist, totally abolished dexamethasone (Dex) (300 μg/100 g i.p. injected) and immobilization stress-induced hypothalamic somatostatin release in adult male rats. We also observed that glutamate from median eminence-hypothalamic medio basal (ME-MBH) complex, measured by high performance liquid chromatography (HPLC), exhibited a conspicuous secretory pattern, with the total amount released not modified by Dex administration. This indicates that Dex and stress-induced somatostatin (SS) secretion is not mediated by endogenous glutamate variations but likely by activation of NMDA receptors.

Hypothalamic Site‐Dependent Effects of Neuropeptide Y on Gonadotropin‐Releasing Hormone Secretion in Rhesus Macaques

In rodents and rabbits, neuropeptide Y (NPY) has a bimodal effect on gonadotropin-releasing hormone (GnRH) secretion. Intracerebroventricular (icv) administration or direct infusion of NPY into the median eminence (ime) suppresses GnRH release in ovariectomized (OVX) animals, but stimulates GnRH release in intact or OVX animals treated with ovarian steroids. Specific ovarian steroid-dependent NPY effects are, however, not obvious in non-human primates. In OVX rhesus monkeys, icv administration of NPY has been shown to suppress luteinizing hormone (LH) secretion whereas ime infusion of NPY stimulates GnRH pulses. In such animals, estrogen replacement does not reverse the inhibitory NPY effect on LH release, although estrogen enhances the stimulatory NPY effect on GnRH secretion. These observations led us to speculate that the bimodal NPY effects in non-human primates may depend on either the site of NPY action or the nature of the steroid milieu. This study utilized the push-pull perfusion (PPP) technique to examine the effects of either ime or icv infusion of NPY on GnRH release in OVX monkeys treated with or without both ovarian steroids. Without exception, ime infusion of NPY increased GnRH concentrations in push-pull perfusates regardless of the steroid status of the animals. In contrast, GnRH levels were reduced during icv infusion of NPY in both untreated and estrogen/progesterone-treated, OVX monkeys. These results indicate that, unlike other mammalian species, in the rhesus monkey the stimulatory and inhibitory effects of NPY on GnRH release depend on the site of NPY infusion within the brain rather than the ovarian steroidal environment.

Central interleukin-1 beta stimulation of vasopressin release into the rat brain: activation of an antipyretic pathway.

1. Arg8-vasopressin (AVP)-containing neurones of the bed nucleus of the stria terminalis (BST), which terminate in the ventral septal area (VSA) of the rat brain, provide a pathway which controls body temperature during fever. The present study was conducted to test the hypothesis that interleukin-1 beta (IL-1 beta) may trigger the antipyretic response by evoking AVP release from BST neurones projecting into the VSA. 2. The push-pull perfusion technique and radioimmunoassay were utilized to determine the AVP concentrations of retrieved VSA perfusion fluid in urethane-anaesthetized rats following BST infusion of vehicle or IL-1 beta (125 or 500 pg (2 microliters)-1). 3. Ventral septal AVP levels significantly increased from basal levels, in a dose-related manner, in response to IL-1 beta (0-500 pg). Electrical stimulation of the same areas of the BST also evoked AVP release into the VSA. 4. IL-1 beta infusions and electrical stimulation of the BST resulted in significant increases in rectal temperature. In IL-1 beta-treated animals (500 pg), the change in body temperature and VSA AVP release were negatively correlated (P < 0.001). However, external heating of the animals to approximately the same levels as electrically stimulated or IL-1 beta treated rats did not affect basal AVP release. 5. These data show that IL-1 beta is a potent stimulus for AVP release from BST neurones and supports BST involvement in neuro-immune interactions. We propose, that in addition to febrogenesis, IL-1 beta is also a key component in the process of endogenous antipyresis by activating vasopressinergic BST neurones to release AVP during fever.

Release of gonadotropin-releasing hormone (GnRH) from the medio-basal hypothalamus induced by electroacupuncture in conscious female rabbits.

In electroacupuncture (EA) treatment, successful induction of ovulation is usually accompanied with the facilitation of pulsatile secretion of gonadotropin. The present work studied the release of GnRH from the mediobasal hypothalamus (MBH) using push-pull perfusion technique in conscious New Zealand female rabbits before, during and after EA stimulation. EA (4Hz, 5mA, 30 min) of Guanyuan (Ren 4), Zhongji (Ren 3), bilateral Sanyinjiao (SP 6) and Zigong (Extra 16) was given at the end of the 1st, 3rd and 5th hour during 6 hours' perfusion (EA group, n = 6). Cupric acetate (CuAC) was given (1%, 0.9 ml, i.v.) at the end of the 1st hour perfusion (CuAC group, n = 5) as a control since it is known as a stimulant for the release of GnRH. Rabbits in the control group (n = 4) were under the same perfusion condition except with no CuAC injection or EA stimulation. It was found that GnRH level was elevated immediately and significantly (4.64 +/- 0.98 v.s. 2.08 +/- 0.01 pg/ml, P < 0.05) after EA stimulation. In the CuAC group, GnRH level was also significantly elevated (3.84 +/- 0.37 v.s. 2.08 +/- 0.001 pg/ml, P < 0.05) one hour after CuAC injection. In addition, corpus lutein were observed both in the EA group (2/6) and the CuAC group (5/5). No similar changes were found in the control group. The data suggest that EA is capable of facilitating GnRH release from the MBH.

Effects of intravenous administration of interleukin-1-beta on the release of prostaglandin E2, corticotropin-releasing factor, and arginine vasopressin in several hypothalamic areas of freely moving rats: estimation by push-pull perfusion.

Utilizing the push-pull perfusion technique, we examined the effect of an intravenous bolus injection of recombinant human interleukin (IL)-1 beta on the release of prostaglandin E2 (PGE2), CRF, and AVP in several hypothalamic areas of freely moving rats, simultaneously monitoring plasma ACTH levels. Perfused hypothalamic areas were the median eminence (ME), the paraventricular nucleus (PVN), and the medial preoptic area (MPOA). During the period 12:00-15:00 h, perfusates and blood samples were collected every 10 min (between 13:00 and 13:40 h) or 20 min (between 12:00 and 13:00 h, and between 13:40 and 15:00 h). IL-1 beta (1.0 micrograms/rat) or vehicle only (in control groups) was injected at 13:00 h. In both the ME and the PVN but not in the MPOA, the outputs of CRF and AVP were significantly stimulated by IL-1 beta, prior to the rise in plasma ACTH. A significant stimulation of PGE2 by IL-1 beta was observed only in the PVN, and its temporal profile was very similar to those of CRF and AVP in the PVN. These results suggest that PGE2 may be a trigger in the PVN for the activation of CRF and AVP neurons, and thereby ACTH secretion, which follows IL-1 beta injection.

Interleukin-1β increases 5-hydroxyindoleacetic acid release in the hypothalamus in vivo

Push-pull perfusion technique was used to infuse interleukin-1β (IL-1β) into and collect perfusate from the medial basal hypothalamus (MBH) of conscious, freely moving rats. The serotonin metabolite, 5-hydroxyindoleacetic acid (5-HIAA), was measured in the perfusate by high performance liquid chromatography. Infusion of the vehicle, PBS-0.1%BSA, had no significant effect on 5-HIAA release except near the end of the perfusion period (325 min) when the release was below the pretreatment level ( p < 0.05). Infusion of 25 ng of IL-1β prevented this decrease, whereas infusion of 50 ng produced an increase of more than 50% ( p < 0.05) at 25 min and maintained it at that level during the remaining posttreatment period. In the animals infused with 100 ng of IL-1β, 5-HIAA release increased by more than 70% at 25 min and was more than 120% ( p < 0.05) above the pretreatment level at the end of the posttreatment period. We concluded that IL-1β affects the metabolism of serotonergic system in the hypothalamus and that this is a component of the mechanism by which IL-1 produces its central actions.

Evidence that neuropeptide Y secretion in the median eminence increases prior to the luteinizing hormone surge in ovariectomized steroid-primed rats: estimation by push-pull perfusion.

Utilizing the push-pull perfusion technique, we examined the secretory profiles of neuropeptide Y (NPY) and luteinizing hormone (LH)-releasing hormone (LHRH) in the median eminence (ME) of ovariectomized adult rats which were primed with estrogen and progesterone to provoke LH and prolactin (PRL) surges. The ME was perfused with artificial cerebrospinal fluid between 13.00 and 18.00 h, and perfusates and blood samples were collected every 20 min. NPY and LHRH in the ME started to significantly increase 40 min earlier than the initial significant rise in the plasma LH, and the highest ME levels of the neuropeptides clearly preceded the occurrence of the LH surge. Regarding the PRL surge, however, such temporal relationship was not apparent. These in vivo data appear to support the putative facilitatory role of NPY in the generation of the steroid-induced LH surge. This is the first study to characterize the temporal profile of in vivo release of NPY in rat ME in terms of its relationship to LH and PRL surges.

Decreased norepinephrine release in anterior hypothalamus of NaCl-sensitive spontaneously hypertensive rats during high NaCl intake

We previously demonstrated that dietary NaCl supplementation reduces endogenous norepinephrine stores and turnover in the anterior hypothalamic area (AHA) of male NaCl sensitive spontaneously hypertensive rats (SHR-S) but not in NaCl resistant control rats and have implicated this mechanism in the pathogenesis of NaCl sensitive hypertension. In the current study, we tested directly the hypothesis that dietary NaCl supplementation decreases the release of norepinephrine from nerve terminals in the AHA of SHR-S using the push-pull perfusion technique. Conscious, freely moving SHR-S and control Wistar-Kyoto (WKY) rats were studied after 2–3 weeks of 8% or 1% NaCl feeding. In the 1% NaCl fed SHR-S, 3-methoxy-4-hydroxyphenylglycol (MOPEG, the major metabolite of norepinephrine in brain) levels averaged 272 ± 32 pg/10 min; norepinephrine levels, 17 ± 2 pg/10 min; in the 8% NaCl fed SHR-S, MOPEG levels averaged 72 ± 7 pg/10 min; norepinephrine levels were 6 ± 1 pg/10 min. There was a positive linear correlation ( r = 0.77; P < 0.01) between MOPEG and norepinephrine levels in AHA perfusates, indicating that perfusate MOPEG levels provide a useful index of norepinephrine release from AHA nerve terminals. In contrast, MOPEG levels in AHA perfusates were not affected by dietary NaCl intake in control WKY, and in control posterior hypothalamic perfusates, were not affected by dietary NaCl intake in SHR-S. These results give the first direct evidence that dietary NaCl supplementation is associated with greatly reduced norepinephrine release from nerve terminals in the AHA in SHR-S, but not in WKY controls, supporting the hypothesis that NaCl sensitive hypertension in this model is mediated by a NaCl induced reduction in AHA mediated inhibition of sympathetic outflow.

Prostaglandin E 2 mediates the stimulatory effect of methoxamine on in vivo luteinizing hormone-releasing hormone (LH-RH) release in the ovariectomized female rhesus monkey

Previously, we found that noradrenergic input through α 1-receptors modulates pulsatile release of luteinizing hormone-releasing hormone (LH-RH) in ovariectomized rhesus monkeys in the absence of estrogen. In the present study, the role of prostaglandin E 2 (PGE 2) in mediating α-adrenergic stimulation of LH-RH release is investigated. In the first experiment the effects of the α 1-adrenergic agonists methoxamine (MTX) on LH-RH and PGE 2 release were examined. Push-pull perfusion of the stalk-median eminence (S-ME) was performed in conscious, ovariectomized monkeys, and perfusate samples were collected on ice. MTX (10 −5 M) was infused into the S-ME through the push cannula for 10 min at 90-min intervals, and LH-RH and PGE 2 in aliquots of the same perfusate samples were measured by radioimmunoassay. Infusion of MTX significantly stimulated LH-RH release ( n = 12; P < 0.01) and PGE 2 release ( P < 0.05). In the second experiment, the effect of PGE 2 infusion on LH-RH release was tested. PGE 2 (10 −7 M) was infused using the same protocol as above, and LH-RH was measured in the perfusates. Infusion of PGE 2 through the push cannula significantly stimulated LH-RH release ( n = 23; P < 0.05). These results suggest that the stimulatory effect of MTX on LH-RH release is at least partly mediated by PGE 2, since MTX stimulated not only LH-RH but also PGE 2 release, and since PGE 2 itself stimulated LH-RH release. Therefore, PGE 2 may be an important endogenous mediator of α 1-adrenergic input stimulating pulsatile PH-RH release. Moreover, the stimulatory effects of MTX and PGE 2 can be observed in the absence of estrogen in the rhesus monkey, unlike in rhodents. Our results also demonstrate the usefulness of the push-pull perfusion technique for studies of cellular mechanisms in neuroendocrine research.