In vivo GABA release from the medial preoptic area of diestrous and ovariectomized rats

Summary The push-pull cannula technique was used to examine the endogenous release of GABA from the medial preoptic area (MPO) of unanesthetized rats. In diestrous females the mean resting release of GABA was 27.1±2.0 pmol/min. GABA release was significantly elevated by increasing the potassium concentration in the perfusion solution to 50 mM, whereas it was dramatically inhibited by mercaptoproprionic acid (1.0 mM), a glutamic acid decarboxylase inhibitor. A comparison between diestrous females and chronically castrated animals indicated that endogenous GABA release in OVX animals was only 60–70% of that in diestrous animals. A model for the presynaptic inhibition of NE by estrogen receptive GABAergic neurons in the MPO is proposed.Partially supported by the German Research Society (grant No. WU 60/5)Dr. Ondo was supported by a NICHHD Research Career Development Award (HD 00248)     

Effects of electrochemical stimulation of the hypothalamus on serum prolactin and LH in rats

Summary Plasma levels of prolactin and LH were measured by radioimmunoassay following electrochemical stimulation of the medial preoptic area (MPO) or the arcuate nucleus (Arc.N.) in pentobarbital anesthetized proestrous rats. Differences in the secretion pattern of prolactin and LH were observed when stimulated by means of acutely or chronically implanted electrodes. Acute implantation and stimulation of the MPO resulted in no change in serum prolactin levels, whereas stimulation by means of chronically implanted electrodes evoked a marked increase in serum prolactin. The general observation was that electrostimulation in the acute experiments causes a less sharp but more prolonged prolactin and LH release from pituitary than stimulation through chronically implanted electrodes.Aided in part by NIH research grants CA 10771 and AM 4784.     

Effects of neurotransmitters on the release of corticotropin releasing hormone (CRH) by rat hypothalamic tissue in vitro

Summary Effects of neurotransmitters on in vitro release of CRH from rat hypothalamic tissue were investigated. Three whole hypothalami or three mediobasal hypothalami from male rats, adrenalectomized 7 days before, were incubated for 10 min in a medium similar to cerebrospinal fluid at 37 ° C under 95% O₂, 5% CO₂. CRH activity was assayed by radioimmunological measurement of ACTH released by isolated pituitary cells from adrenalectomized rats.Norepinephrine (NE) at a concentration of 0.02×10^–6 M to 2×10^–6 M stimulated dose-related CRH release. At larger concentrations the response decreased again (bell-shaped dose-response relationship). The action of norepinephrine was completely blocked by coincubation with phentolamine. Similar results were obtained whether whole hypothalami or mediobasal hypothalami were incubated with norepinephrine. This suggests that the site of action of NE was within the mediobasal hypothalamus. The possibility that the observed effects were due to the CRH-like activity of vasopressin released from the hypothalamic tissue was excluded by simultaneous measurements of CRH activity and arginine-vasopressin concentrations in the medium. Dopamine at very large concentrations (17.6×10^–6 M) also stimulated CRH release. Acetylcholine, serotonin, histamine and GABA did not influence the basal CRH release at any of the concentrations tested.These results suggest that in the rat, norepinephrine may have a physiological role as stimulator of CRH-release at the level of the mediobasal hypothalamus. However, in view of the conflicting results obtained with similar and other methods, no definite conclusions should yet be drawn.This work was supported by Deutsche Forschungsgemeinschaft. Bonn-Bad Godesberg. SFB 87/B2. K.H. V. is a recipient of a Heisenberg Stipendium     

Effects of electrochemical stimulation of medial preoptic area on prolactin and luteinizing hormone release in old female rats

Summary Serum prolactin and LH levels in old constant estrous rats were higher than in old pseudopregnant rats. Electrochemical stimulation of the medial preoptic area in old constant estrous rats resulted in significant increases in serum concentration of prolactin and LH, and subsequent ovulation. Old pseudopregnant rats showed only a small increase in serum prolactin, no increase in serum LH and no ovulation. The high circulating levels of estrogen in old constant estrous rats, and the relative lack of estrogen and the presence of progesterone in the old pseudopregnant rats, may account for the differences observed in response of the medial preoptic area to electrochemical stimulation.Aided in part by NIH research grants CA 10771 and AM 4784.     

Rapid and opposite effects of dexamethasone on in vivo and in vitro hypothalamic somatostatin release

We have previously reported the rapid response of hypothalamic somatostatin (SS) neurons to acute stress. Since it is well known that glucocorticoids (GC) are involved in neuroendocrinal stress regulation, we investigate in this study the effects of acute administration of dexamethasone (Dex) on both in vivo and in vitro SS release. Freely moving animals received stereotaxic implant of a push-pull cannula into the median eminence for 10 days, and then they were perfused with artificial cerebrospinal fluid for 120–150 min. An i.p. injection of Dex (200 or 300 μg/100 g) induced, 15–30 min later, a mean increase in SS hypothalamic output of 62.6±6.2% of basal secretion. By contrast, after 15 min incubation of hypothalamic fragments with either 10⁻⁷ or 10⁻⁶ M Dex, SS release decreased abruptly to 57.3±3.3% (n=16;P<0.001 compared with basal release) and 78.0±9.5% (n=13;P<0.05 compared with basal release) of basal release, respectively. Other Dex concentrations induced no variations, giving the dose-effect curve an abrupt “on-off” effect. The inhibitory effect was blocked by picrotoxin (10⁻⁴ M) and was immediately reversed when Dex was removed from the medium. Specificity was tested by using another steroid, estradiol, and another tissue, cortex. The rapid action of GC whatever the model used and in particular the blocking in vitro effect of picrotoxin could suggest that GCs act at the level of the membrane and could operate physiologically in response to stress. In addition, the opposite in vivo and in vitro effects on SS release would indicate that GCs exert two different controls on SS neurons.     

Autoradiographic localization of 3H-gamma-aminobutyric acid in the medial hypothalamus

Summary Tritium labelled gamma-aminobutyric acid (³H-GABA) was infused into the third ventricle of rats with normal or deafferented hypothalamus and the distribution of the label was studied by light and electron microscopic autoradiography.In control as well as deafferented hypothalamus a few neurones accumulated radioactivity, while the majority was unlabelled. Characteristic clusters and rows of silver grains were observed in the neuropil of several regions probably indicating labelled cell processes and terminal axons. Electron microscopy showed that at least some of the clusters were over axon terminals with synaptic vesicles. ³H-GABA accumulated also in the ependyma and glial elements.The results suggest that in the medial hypothalamus there is a preferential uptake of GABA in some neurones and nerve fibers; at least some of these are hypothalamic interneurones. This supports the hypothesis that some hypothalamic neurones and nerve endings may use GABA as a transmitter.     

Influence of cholinergic agents on dopamine release from medial basal hypothalamus.

The ability of acetylcholine to influence the stimulation-induced release of [3H]dopamine in medial basal hypothalamus was evaluated. A muscarinic facilitation of electrical field-stimulated but not potassium-stimulated release was observed. These data suggest a muscarinic enhancement of dopamine release perhaps via an action at the level of the cell body. These data suggest that observed effects of cholinergic agents on prolactin release may be mediated in part through such a cholinergic-dopaminergic interaction.     

Effects of advancing age on hypothalamic neurotransmitter content and on basal and norepinephrine-stimulated LHRH release

In order to elucidate possible mechanism(s) responsible for the age-related decline in LH secretion, basal and norepinephrine (NE)-stimulated LHRH release was measured from median eminence (ME) fragments of 4-, 11-, 18- and 27-month-old male F344 rats. Serum LH levels declined significantly between 11 and 18 months and were still lower at 27 months of age, while testosterone levels declined continuously between 4 and 27 months. Hypothalamic NE and dopamine (DA) content also declined significantly with age, while serotonin and 5-hydroxy-indoleacetic acid content increased with age. Despite the decline in serum LH levels with age, in vitro basal LHRH release increased gradually with age as did NE-stimulated LHRH release. These data suggest that the age-related reduction in LH secretion by the male rat is due to a reduction in hypothalamic NE metabolism and not to an inability of the LHRH neuron to respond to NE stimulation.     

Proteins synthesized in medial hypothalamus and transported to midbrain in estrogen-treated female rats

Summary Results on the anatomy of ventromedial hypothalamic outputs, together with the temporal aspects of electrophysiological studies, predict that for reproductive behavior control sex steroids alter hypothalamic protein synthesis and transport of proteins to the dorsal midbrain. We have studied labeled proteins arriving in the dorsal midbrain after local microinjection of tritiated amino acids to the ventromedial hypothalamus. Estrogen treated and control ovariectomized female rats are significantly different in this respect, and in particular some proteins appear to be synthesized or transported in greater amounts in the estrogen treated animals. Physical characterization of these proteins and comparisons under a variety of endocrine conditions will suggest whether their synthesis could be part of the mechanism by which ovarian steroids affect behavior.     

Effect of an inhibitor of aromatization, 1,4,6 androstatriene-3,17-dione (ATD) on LH release and steroid binding in hypothalamus of adult female rats

Summary Prevention of testosterone aromatization in the female rat pups by perinatal treatment with 1,4,6 androstatriene-3,17-dione (ATD) induces an important defeminization as shown by a reduction of fluctuations of LH release after castration and estradiol implantation. The fact that, under our in vitro experimental conditions, ATD is able to displace testosterone binding in the hypothalamus whereas estradiol does not, confirms the hypothesis that ATD acts on aromatase. The most attractive explanation for the defeminization effect of ATD is then an estrogen-like action of ATD.     

The effect of prolactin on dopamine release from rat striatum and medial basal hypothalamus

Electrical stimulation of supervised rat striatal slices or medial basal hypothalamus evokes a calcium-dependent release of dopamine; the release is blocked by tetrodotoxin. Prolactin (0.5–5 μg/ml) enhanced the evoked release of dopamine from both these tissues.It is well known that dopamine will inhibit the release and synthesis of prolactin in the pituitary. Our results suggest a possible presynaptic effect of prolactin on dopamine release.     

Vasopressin release from rat medial basal hypothalamus in vitro after adrenalectomy or lesions of the paraventricular nuclei

Rat medial basal hypothalami (MBH) or neurointermediate lobes of the hypophysis (NIL) were superfused in vitro and stimulated electrically. The evoked release of vasopressin from the MBH was enhanced to 700% of controls when the tissue was taken from adrenalectomized rats. By contrast, the evoked release of vasopressin from the NIL was not changed after adrenalectomy. After bilateral lesions of the paraventricular nuclei, the evoked release of vasopressin from the MBH was reduced in subsequent in vitro experiments. The marked changes in vasopressin release occurred in spite of no or only small changes in the total tissue content of vasopressin. These data support the view that vasopressin may be released from the external layer of the median eminence into the hypophysial portal blood after activation of the hypothalamo-pituitary-adrenal axis.     

A stereotaxic X-ray map of the hypothalamus of the marmoset monkey Callithrix jacchus

Summary Precise hypothalamic surgery in the marmoset monkey Callithrix jacchus cannot be performed by means of the Horsley-Clarke stereotaxic procedure. Thus, a simple stereotaxic X-ray technique was developed. Technical requirements are a stereotaxic frame for small animals and a dental X-ray source.A sagittal map shows hypothalamic structures in relation to surrounding bony landmarks. Corresponding coronal plates allow target determination in that plane. Intracerebral coordinates based on the position of the optic chiasma can be transformed into individual Horsley-Clarke coordinates. The error of the atlas is estimated as ± 0.25 mm anteroposteriorly, ± 0.1 mm coronally, and ± 0.5 mm vertically.List of Abbreviations AP-O Anteroposterior zero plane of Horsley-Clarke - BO Bulbus olfactorius - CA Commissura anterior - CC Corpus callosum - CM Corpora mamillaria - CO Chiasma opticum - CSM Commissura supramamillaris - FC Flocculus - FS Fissura lateralis (Sylvii) - FX Fornix - GA Decussatio supraopticus dorsalis (Ganser) - H₁ Field H₁ - H₂ Field H₂ - HH Horizontal plane of Horsley-Clarke - HHL Hypophysis, posterior lobe - HVL Hypophysis, anterior lobe - HY Hypophysis - INF Infundibulum - LMI Lamina medullaris interna - LPC Lobus posterior cerebelli - N II Foramen nervi optici - N III Fissura orbitalis superior (with Nervus oculomotorius) - PI Hypophysis, pars intermedia - PT Nuclei posteriores tuberis - PV Nucleus paraventricularis - SC Sulcus calcarinus - SO Nucleus supraopticus - ST Nucleus subthalamicus - STS Sulcus temporalis superior - TH Thalamus - THP Tractus habenulopeduncularis - TL Nucleus tuberis lateralis - TMT Tractus mamillothalamicus - TO Tractus opticus - VMH Nucleus ventromedialis hypothalami     

A second look at the barriers of the medial basal hypothalamus

The cell bodies of hypothalamic secretory neurons are localized in areas protected by the blood-brain barrier (BBB), whereas their axon terminals are localized in the median eminence, which lacks a BBB. This implies a complex barrier system, allowing neurons of the central nervous system to secrete into the blood stream without making the BBB leaky. In the present study, three experimental protocols were applied to clarify certain relevant aspects of the barriers operating in the medial basal hypothalamus of the rat. We established that the milieu of the arcuate nucleus is exposed to both the ventricular and the subarachnoidal cerebrospinal fluid (CSF).The median eminence milieu, the perivascular space of the portal vessels, and the subarachnoid space appear to be in open communication; also, beta2-tanycytes establish an efficient barrier between the median eminence milieu and the ventricular CSF. Similarly, beta1-tanycytes establish a lateral barrier, separating the intercellular space of the median eminence from that of the arcuate nucleus. We also found that the glucose transporter I (GLUT I), a BBB marker, is localized throughout the whole plasma membrane of beta1-tanycytes, but is missing from beta2-tanycytes. Expression of GLUT I by tanycytes progressively develops during the first postnatal weeks; while the degree of damage of the arcuate nucleus by administration of monosodium glutamate, at different postnatal intervals, parallels that of the GLUT I immunoreactivity of beta1-tanycytes. An explanation is offered for the selective destruction of the arcuate neurons by the parenteral administration of monosodium glutamate to infant rats.     

Differential effects of flurothyl- and electro-convulsive shock on sexual maturation and prolactin release in the rat

Summary The effects of single and repeated seizures on luteinizing hormone (LH), follicle stimulating hormone (FSH) and prolactin secretion and on the onset of sexual maturation in rats are described. In addition, the influence of convulsions generated electrically (electroconvulsive shock, ECS) and chemically (using flurothyl) are compared. Repeated flurothyl convulsions and ECS (one daily convulsion from age 24 days) significantly delay vaginal opening in female rats. The incidence of first ovulation at maturation is reduced to 20% compared with 70–100% for untreated groups. Body and adrenal weights in immature rats are not modified by flurothyl convulsions. Repeated ECS does not influence adrenal weight although somatic growth is inhibited. In an effort to clarify the mechanism of action of convulsions on puberty onset, we examined acute changes in LH, FSH and prolactin secretion and the surge response of LH/FSH to gonadal steroid priming. A single flurothyl convulsion potently inhibits prolactin secretion. In contrast, an ECS acutely stimulates prolactin release in male and female rats. Convulsive seizures do not consistently alter tonic gonadotropin output. However, both flurothyl convulsions and ECS attenuate estradiol benzoate/progesterone-induced LH and FSH surges in ovariectomized rats though this is apparently not mediated by dopamine/prolactin since bromocriptine treatment delays sexual maturation without preventing ovulation at first estrus. Similarly, bromocriptine does not disrupt LH/FSH surges induced by gonadal steroid treatment. One component in the neural response to convulsions may be hypothalamic endogenous opiate peptides since the ability of naloxone to influence LH and prolactin secretion is potentiated by repeated ECS. On the other hand, dopamine is clearly implicated in the flurothyl-induced inhibition of prolactin secretion, since haloperidol reverses this effect.     

In vivo electrochemical studies of monoamine release in the medial prefrontal cortex of the rat

The magnitude and duration of release of monoamines evoked by local applications of potassium were measured in vivo in the medial prefrontal cortex using high-speed chronoamperometry. Typical electrochemical signals reflecting released of electroactive species ranging from 0.5 to 3.0 microM and lasting 90-120 s were detected at a variety of dorsal-ventral and anterior-posterior electrode placements in the medial prefrontal cortex. The magnitude of the reduction current measured following the oxidation reaction suggests a contribution of both serotonin and dopamine to the electrochemical signal, dopamine serving as the predominant monoamine in the medial prefrontal cortex proper and serotonin appearing to predominant in the more posterior regions of the frontal cortex. This conclusion was reinforced by the fact that unilateral 6-hydroxydopamine lesions of ascending dopamine fibers almost completely abolished electrochemical signals in the ipsilateral but not in the contralateral medial prefrontal cortex. The present study provides an in vivo characterization of monoamine release in the mesocortical dopamine terminal field, where it has been suggested that psychomotor stimulants may produce some of their positive reinforcing effects.     

Responses of cat preoptic neurons to stimulation of the medial frontal cortex and the medial basal hypothalamus

Summary Responses of single preoptic neurons to electrical stimulation of the medial frontal cortex, the mediobasal hypothalamus (MBH) and the medial forebrain bundle (MFB) were recorded in anaesthetised cats. Single pulse stimulation of the medial frontal cortex orthodromically drove 96 otherwise quiescent preoptic neurons, which were found more frequently in the dorsal preoptic region, inhibited 53% of the spontaneously active preoptic neurons and excited 16%. Testing of cortically influenced preoptic neurons with MBH or MFB stimulation resulted in antidromic invasion of 6% (MBH) and 9% (MFB). Convergence of orthodromic inputs from medial frontal cortex and MBH was detected in 78% of spontaneously active preoptic neurons, and three-way convergence including input from MFB was noted in 17% of neurons tested with all stimulators. Some cortex-responsive neurons were found to also respond to vaginal or anal probing, paw squeezing and haemorrhage. The role of this input to the preoptic region from medial frontal cortex remains to be elucidated, but may include neuroendocrine, behavioural and homeostatic functions.     

In vivo release of somatostatin from rat median eminence after local K infusion or delivery of nociceptive stress

The effects of local infusion of a 16 mM K⁺ solution or of a nociceptive stress on the release of somatostatin (SRIF) from the hypothalamus was measured in unanesthetized male rats implanted with a push-pull cannula in the median eminence. Although the baseline secretion rate of SRIF was increased in animals displaying agitation as a result of handling stress, both treatments induced fast doubling of SRIF release lasting for 15–30 min. Neither an equimolar Na⁺ infusion into the median eminence nor a similar K⁺ infusion into the 3rd ventricle had any affect on this release. The possible role of SRIF release in the mechanism of growth hormone inhibition following nociceptive stress is discussed.     

Immunohistochemical identification of the oxytocin and vasopressin neurons in the hypothalamus of the monkey (Macaca fuscata)

Summary The hypothalamic oxytocin and vasopressin neurons of the monkey, Macaca fuscata, were demonstrated in Golgi-like images by a modified immunoperoxidase method. The magnocellular oxytocin and vasopressin neurons were distributed mainly in the supraoptic and paraventricular nuclei. In addition to these main nucleic, both types of magnocellular neurons were found in the accessory supraoptic nucleus, the periventricular and perifornical areas, the nucleus of the stria terminalis, the lateral hypothalamic area, and the pars interna of the globus pallidus. Magnocellular oxytocin neurons were seen immediately ventral to the anterior commissure, and parvocellular vasopressin neurons were localized in the medial portion of the suprachiasmatic nucleus. The preferential distribution of the oxytocin and vasopressin neurons was recognized not only in the supraoptic and paraventricular nuclei, but also in other areas. In all areas observed, the cytological difference between the oxytocin and vasopressin neurons could be identified. The area, of the perikarya of the vasopressin neurons was determined to be larger than that of the oxytocin neurons. Most of the axons of the oxytocin neurons issued from the perikarya, while the axons of the vasopressin neurons originated in most cases from the thick proximal dendrites. These results show that the oxytocin and vasopressin neurons are distributed in areas much broader than has hitherto been assumed, and that these two peptidergic neurons can be definitely differentiated morphologically as well as functionally.Supported by grants (No. 56440022, 56770037) from the Ministry of Education, Science, and Culture, Japan     

Changes in the physiological roles of neurotransmitters during individual development

The classical neurotransmitters (acetylcholine and biogenic monoamines) are multifunctional substances involved in intra- and intercellular signaling at all stages of ontogenesis in multicellular animals. A cyclical scheme is proposed to describe age-related changes in neuro-transmitter functions at different stages of development from oocyte maturation to neuron formation. This may reflect not only the temporospatial organization of neurotransmitter processes, but also the origin of the functions of acetylcholine and biogenic monoamines from the protosynapses of the cleaved embryo to neuronal synapses. Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 83, No. 10, pp. 1–15, October, 1997.