In vitro TRH release from hypothalamus slices varies during the diurnal cycle

We have previously described a daily rhythm in thyrotropin releasing hormone (TRH) and TRH mRNA in the rat hypothalamus. To determine whether TRH release fluctuates in a diurnal manner, we have measured basal and potassium stimulated release from hypothalamic slices, and compared it to release from olfactory bulb slices, during the diurnal cycle. Basal TRH release was higher at 7:00 h than at any other time (1:00, 13:00 or 19:00 h) in either hypothalamus or olfactory bulb. The ratio of stimulated over basal release was higher in the hypothalamus at 19:00 h, when TRH content was highest. Potassium stimulated TRH release from olfactory bulb was not different from basal release at any time. TRH release fluctuations were not due to a rhythm of extracellular inactivation: the activity of pyroglutamyl aminopeptidase II, an ectoenzyme responsible for TRH inactivation, was constant throughout the cycle. Our data demonstrate that diurnal variations of TRH release occur in vitro and that the enhanced responsiveness to potassium stimulation in hypothalamus is correlated with increased levels of peptide.     

In vitro study of immunoreactive corticotropin-releasing factor release from the rat hypothalamus

Immunoreactive corticotropin-releasing factor (I-CRF) release from rat hypothalami was studied in vitro utilizing a perifusion of rat hypothalami and a rat CRF RIA. Basal release of I-CRF from the hypothalamus of adrenalectomized or hypophysectomized rats was higher than in that of normal rats. K+-induced I-CRF release was completely suppressed by omission of Ca++ from the medium. Dexamethasone suppressed I-CRF release from hypothalami, but not from median eminence (ME). C-AMP and angiotensin II had mild stimulatory effects on I-CRF release. These results suggest that 1) the feedback mechanism acts mainly on a higher level than ME, and 2) c-AMP and angiotensin II may be involved in CRF-releasing mechanism(s).     

The release of histamine from tissue slices of rat hypothalamus

Abstract— The efflux of endogenous histamine from small slices of rat hypothalamus has been studied. Histamine release was temperature dependent and could be increased 50 % after depolarization of the brain slices by 15 mm potassium. The presence of EDTA in the incubating media inhibited the release of histamine and blocked the effect of potassium. Theophylline and reserpine markedly increased release of histamine, whereas phenoxybenzamine inhibited release and blocked the effects of theophylline and reserpine. Efflux of histidine from hypothalamic tissue slices was not temperature-dependent and was unaffected by the addition of potassium or any of the drugs studied.     

Bombesin-like peptides in rat brain: Localization in synaptosomes and release from hypothalamic slices

The subcellular distribution of bombesin-like immunoreactivity in rat brain was investigated. The number of receptors was significantly greater in the synaptosomal than mitochondrial fraction and quantity of bombesin-like immunoreactivity was greatest in the synaptosomal fraction. Also, the release of bombesin-like peptides from rat hypothalamic slices was investigate K⁺ and veratridine stimulated release of immunoreactivity in a Ca⁺⁺-dependent manner.     

In vitro release of growth hormone-releasing factor from rat hypothalamus: effect of insulin-like growth factor-1

The release of growth hormone-releasing factor (GHRF) from rat hypothalamus was investigated in vitro. After 60 min preincubation the released GHRF from sliced rat hypothalamic fragments during 60 min incubation was detected by a highly specific and sensitive radioimmunoassay for rat GHRF. The release of GHRF was Ca2+-dependent and enhanced by high concentration of K+. Insulin-like growth factor-1 (IGF-1) significantly decreased GHRF release to 65% and 84% of the control at concentrations of 10(-8) M and 10(-7) M, respectively. These results suggest that this in vitro system is useful for the investigation of the mechanism of GHRF release from the hypothalamus and that IGF-1 is probably involved in the feedback inhibition of growth hormone secretion by attenuating GHRF release from the hypothalamus besides countering the effect of GHRF on the pituitary.     

Adrenaline involvement in the presynaptic beta-adrenoceptor-mediated mechanism of dopamine release from slices of the rat hypothalamus

Slices of rat hypothalamus were superfused and endogenous release of dopamine (DA) was measured by high performance liquid chromatography combined with electrochemical detection. The K+ (20 mM)-evoked release in the presence of tetrodotoxin was Ca2+-dependent. The evoked release was facilitated by a beta-agonist, isoproterenol and this effect was completely abolished by a beta-antagonist, 1-propranolol. Isoproterenol also concentration-dependently facilitated the electrically (at 5Hz) evoked release of DA. The pretreatment with 1-propranolol, beta 1-antagonist, atenolol and beta 2-antagonist, butoxamine shifted the concentration-effect curve of isoproterenol to the right. On the other hand, beta 1-agonist, tazolol, beta 2-agonist, salbutamol and low concentration (10(-9) M) of adrenaline also facilitated the release. 1-Propranolol alone reduced the electrically (at 2 Hz) evoked release, and this effect was completely abolished when the adrenaline content in the brain was drastically reduced by use of a potent PNMT inhibitor, DCMB. These findings suggest that in the rat hypothalamus adrenaline released from adrenaline-containing nerve terminals probably modulates DA release via presynaptic beta 1- and beta 2-adrenoceptors on DA nerve terminals.     

In vitro synaptic reconsolidation in amygdala slices prepared from rat brains

The retrieval of consolidated fear memory causes it to be labile or deconsolidated, and the deconsolidated fear memory is reconsolidated over time in a protein synthesis-dependent manner. We have recently developed an ex vivo model where during fear memory deconsolidation and reconsolidation the synaptic state can be monitored at thalamic input synapses onto the lateral amygdala (T-LA synapses), a storage site for auditory fear memory. In this ex vivo model, the deconsolidation and reconsolidation processes of auditory fear memory in the intact brain were prevented following brain slicing; therefore, we could monitor the synaptic state for memory deconsolidation and reconsolidation at the time of brain slicing. However, why the synaptic reconsolidation process stopped after brain slicing in the ex vivo model is not known. One possibility is that brain slicing severs neuromodulatory innervations, which are required for memory reconsolidation, from other brain regions (e.g., noradrenergic innervation). In the present study, we supplemented amygdala slices with exogenous norepinephrine as a substitute for the severed noradrenergic innervations. DHPG (a group I metabotropic glutamate receptor agonist)-induced depotentiation (mGluRI-depotentiation), a marker for consolidated synapses, was observed following norepinephrine application to slices prepared immediately after tone presentation (fear memory retrieval) to rats that had been pre-conditioned to a tone paired with a shock. These results suggest that noradrenergic activation initiates synaptic reconsolidation. In contrast, mGluRI-depotentiation was absent following norepinephrine application to slices that were prepared immediately after the tone presentation (no fear memory retrieval) to rats when a tone and a shock were unpaired, ruling out the possibility that noradrenergic activation somehow facilitates a subsequent synaptic depression induced by DHPG irrespective of synaptic reconsolidation. Furthermore, the restored mGluRI-depotentiation following application of exogenous norepinephrine was dependent on de novo protein synthesis, as is memory reconsolidation. Thus, our findings suggest that T-LA synapses from acute slice preparations can undergo a reconsolidation process, thereby providing an optimal preparation to study a fear memory reconsolidation process in vitro.     

In vitro release of growth hormone-releasing factor (GRF) from the hypothalamus: somatostatin inhibits GRF release.

The manner of release of growth hormone-releasing factor (GRF) from the rat hypothalamus was studied in a perifusion system using a highly sensitive radioimmunoassay for rat GRF. The recovery of GRF in this system was 50-60%. The release of GRF from the rat hypothalamic blocks was almost stable for 20-240 min after the start of the perifusion and was stimulated by depolarization induced by high K+ concentration. The release of GRF was inhibited by somatostatin at concentrations of 10(-11) to 10(-8) M with maximum inhibition to 52.5% of the basal release at a concentration of 10(-9) M. These results suggest that this system is useful in studying the regulatory mechanism of GRF release and that, in addition to its action on the pituitary, somatostatin appears to act at the level of the hypothalamus in inhibiting GRF release in the regulation of GH secretion.     

Galanin stimulates immunoreactive growth hormone-releasing factor secretion from rat hypothalamic slices perifused in vitro.

The effect of galanin (GAL) on the release of GH-releasing factor (GRF) and somatostatin (SRIF) was examined in an in vitro perifusion system of rat hypothalamic slices. GAL at doses of 10(-7) and 10(-6)M stimulated the release of immunoreactive GRF while it failed to affect SRIF release. Therefore, in vivo stimulation of GH release by GAL may be explained in part by the GRF-releasing effect of this peptide.     

Effects of opioid peptides on immunoreactive corticotropin-releasing factor release from the rat hypothalamus in vitro

Effects of opioid peptides on immunoreactive corticotropin-releasing factor (I-CRF) release from the rat hypothalamus were examined using a rat hypothalamic perifusion system and a rat CRF RIA in vitro. beta-Endorphin (0.3 - 30 nM), dynorphin (0.3 - 30 nM) and FK 33-824 (1 - 10 microM) suppressed basal I-CRF release in a dose-dependent fashion. At 2.2 nM concentrations of these peptides, mean percent inhibition was 56% for beta-endorphin; less than 5% for alpha-endorphin; 44% for dynorphin; 23% for leucine-enkephalin; 6% for methionine-enkephalin; less than 5% for FK 33-824; and less than 5% for D-ala2, D-leu5-enkephalin. The inhibitory effects of beta-endorphin and enkephalins were completely blocked by naloxone, but those of dynorphin were only partially blocked. These results suggest that opioid peptides act through opioid receptors and inhibit I-CRF release from the hypothalamus under our conditions. Therefore, endogenious opioid peptides may have a physiological role in the CRF-releasing mechanism of the hypothalamus.     

Inhibitory effect of norepinephrine on immunoreactive corticotropin-releasing factor release from the rat hypothalamus in vitro

Effects of catecholamines on immunoreactive corticotropin-releasing factor (I-CRF) release from the rat hypothalamus were examined using a rat hypothalamic perifusion system and a rat CRF RIA in vitro. Norepinephrine had a potent inhibitory effect on I-CRF release in a dose-dependent manner at 0.1 nM-1 microM concentrations, but dopamine did not. This inhibitory effect of norepinephrine was completely blocked by propranolol, but only partially blocked by phentolamine. Isoproterenol also had a potent inhibitory effect at 0.01-100 nM concentrations, and a high dose of phenylephrine (10 nM) inhibited I-CRF release. Clonidine did not influence I-CRF release. These results suggest that norepinephrine inhibits I-CRF release mainly through the beta-adrenergic receptor and partially through the alpha 1-receptor.     

Stimulatory effect of acetylcholine on immunoreactive corticotropin-releasing factor release from the rat hypothalamus in vitro

Effects of acetylcholine (Ach) and gamma-aminobutyric acid (GABA) on immunoreactive corticotropin-releasing factor (CRF) release from the rat hypothalamus were examined using a rat hypothalamic perifusion system and a rat CRF RIA in vitro. Ach stimulated CRF release in a dose-dependent manner (1 pM-1 nM). One nM Ach-induced CRF release was inhibited by atropine in a dose-dependent manner (1-100 nM), but was inhibited by only a high concentration (100 nM) of hexamethonium. In addition, such Ach-induced CRF release was inhibited by norepinephrine. GABA did not influence basal CRF release. These results suggest that Ach stimulates CRF release mainly through muscarinic receptors at least under our conditions.     

Progesterone stimulation of in vitro GnRH release from the hypothalamus of the estrogen-primed, ovariectomized rat: serotoninergic role

The ability of ovarian steroids to affect luteinizing hormone secretion is closely related to the influence of these steroids on the activities of several neurotransmitter systems within specific areas of the hypothalamus and associated brain areas. The purpose of this study was to characterize in vitro progestagenic effects on serotonin (5-hydroxytryptamine, 5-HT) and gonadotropin-releasing hormone (GnRH) release from hypothalamic slices from estrogen-primed, ovariectomized rats. Results of this study show that (1) progesterone can stimulate in vitro GnRH and 5-HT release from hypothalamic tissue slices of ovariectomized rats primed with estrogen and (2) the 5-HT receptor antagonist mianserin blocks the ability of progesterone to augment in vitro GnRH release from these tissue slices. This suggests that the influence of progesterone on the estrogen-induced LH surge is, at least in part, via progestagenic release of 5-HT and the subsequent effect of this neurotransmitter on the release of GnRH within the hypothalamus.     

Depolarization-evoked release of gamma-hydroxybutyrate from rat brain slices

The release of gamma-hydroxybutyrate from preloaded rat brain striatal slices was investigated. K+-induced depolarization caused an efflux of gamma-hydroxybutyrate of about 50 fmol min-1 mg-1 (wet weight), but in a Ca2+-free medium containing Mg2+, the evoked release was reduced by 50-60%. The release was higher when 100 microM veratridine was used as a depolarizing agent. The efflux of gamma-hydroxybutyrate is related to veratridine and K+ concentration, and is strongly inhibited by 10 microM tetrodotoxin. The Ca2+ channel blocker verapamil induces a large decrease in the efflux of gamma-hydroxybutyrate after both K+- and veratridine-induced depolarization. These results are in favour of a possible transmitter function for gamma-hydroxybutyrate in rat striatum.     

Inhibition of serotonin release by bombesin-like peptides in rat hypothalamus in vitro

We investigated the activity of bombesin (BN), neuromedin-C (NM-C) and neuromedin-B (NM-B) on serotonin (5-HT) release and reuptake in rat hypothalamus (HYP) in vitro. BN and NM-C but not NM-B (all 1 microM) decreased K+ evoked 3H-5-HT release from superfused HYP slices by 25%. Bacitracin (BCN, 2 micrograms/ml), a nonspecific peptidase inhibitor, reversed the inhibitory effect of BN on K+ evoked 3H-5-HT release. Phosphoramidon (PAN, 10 microM) an endopeptidase 24.11 inhibitor, abolished the inhibitory effect of BN, but not NM-C, on K+ evoked 3H-5-HT release. The peptidyl dipeptidase A inhibitor enalaprilat (ENP, 10 microM), enhanced both BN and NM-C inhibition of 3H-5-HT release. Bestatin (BST, 10 microM) had no effect on BN or NM-C inhibitory activity on 3H-5-HT release. Neither BN, NM-C nor NM-B affected reuptake of 3H-5-HT into HYP synaptosomes alone or in combination with any of the peptidase inhibitors, nor did these peptides alter the ability of fluoxetine to inhibit 3H-5-HT uptake. These data suggest: a) that BN-like peptides may alter neurotransmission in the HYP by acting presynaptically on the 5-HT release mechanism; b) a similarity in the structural requirements for the BN induced inhibition of 5-HT release and BN evoked thermoregulatory disturbances; and c) that peptidases may selectively augment or reduce pharmacologic activity of BN-like peptides upon CNS administration.     

Uptake and release of glutamate in cerebral-cortex slices from the rat

1. Cerebral-cortex slices from rat brain, loaded with labelled l-glutamate as a result of aerobic incubation with labelled glucose, lost less than 15% of this glutamate on subsequent incubation in the presence of unlabelled glucose and l-glutamate. This indicates that very little exchange occurs between extracellular l-glutamate and glutamate accumulated in the neurons as a result of glucose metabolism. 2. Slices, loaded with labelled l-glutamate as a result of aerobic incubation in a medium containing unlabelled glucose and labelled l-glutamate, lost more than half of this glutamate on subsequent incubation in the presence of unlabelled l-glutamate. This indicates that exchange occurs between extracellular glutamate and glutamate accumulated in brain slices as a result of its uptake from the incubation medium. 3. Evidence was obtained suggesting that only a part of the glutamate, accumulated in brain slices as a result of its uptake from an incubation medium containing both glucose and l-glutamate, entered the neurons; apparently almost all the rest entered the glia. 4. It is concluded that the slices contain a pool of glutamate, derived from glucose and located in the neurons, which is poorly exchangeable with extracellular glutamate, and another pool of glutamate, derived from extracellular glutamate and located in the glia, which is freely exchangeable with extracellular glutamate.     

Interleukin-1 inhibits serotonin release from the hypothalamus in vitro.

During a 60-min incubation period, the in vitro release of serotonin (5-HT) from the hypothalami of control male rats decreased by 12.3 +/- 3.1%. In contrast, the presence of 25 ng of interleukin-1 beta (IL-1 beta) in the incubation medium more than doubled this decrease to 29.3 +/- 3.3% (P < 0.001), and the presence of 50 ng of IL-1 beta more than quadrupled this decrease to 53.7 +/- 7.4% (P < 0.001). The decrease produced by the higher dose of IL-1 beta was significantly greater than that produced by the lower dose (P < 0.01), indicating a dose response. During the next two 60-min periods when the hypothalami of the control as well as treatment groups were incubated without IL-beta, 5-HT release continued to decrease and then became stabilized in the control group. In contrast, 5-HT release in the treatment groups rebounded before becoming stabilized at levels that were not significantly different from those in the control group. It is concluded that IL-1 beta inhibits the release of serotonin from the hypothalamus in vitro.