Evidence on differential role for alpha 1 and alpha 2 subtypes of AP-2 adaptin in the central nervous system.

Two subtypes of alpha (α)subunits, α1and α2, belonging to AP-2 complex have been described in the central nervous system (CNS). The specific role of each subtype is still unclear. In this study, we evaluated the expression and interaction with cell membranes of both subtypes in the postnatal developing cerebral cortex and cerebellum in two rat strains that display distinct developmental features. We observed that α2 displays higher variations than α1 during development, and at lesser extent in the rats with delayed rate of development. Additionally, by in vitro binding assays we evaluated the interaction of α subunits with bovine brain membranes. Both subtypes displayed clear differences in their performance, maximum binding of α1 was higher and α2 reached it faster than α1. In addition, both subtypes displayed different binding to membranes when bivalent cations or nucleotides were added. We conclude that both subtypes interact differently with membranes and that they may play different roles in clathrin-mediated endocytosis in the CNS.

Hypoxic preconditioning induces an AT2-R/VEGFR-2(Flk-1) interaction in the neonatal brain microvasculature for neuroprotection.

The angiotensin II receptor subtype 2 (AT2-R) has been proposed to mediate protective vascular actions after brain injury. In this study we investigated the participation of this peptide in the tolerance to cellular damage induced by preconditioning in a rat model of neonatal hypoxia-ischemia (HI). We found that injured animals present a decreased number of microvessels in the ipsilateral (IPLT) side of the brain while in the contralateral (CNLT) side the microvessel number is increased. On the contrary, in the preconditioned animals the microvessels maintained the same number as in control animals. However these vessels show a remarkable increase of the fluorescent signal when they are labeled with antiFlk-1 (VEGFR2), while the Flt-1 (VEGFR1) signal faded in both the injured and the preconditioned animals. The pharmacological blockade of the AT2-R by the drug PD123319 (1.69 mM in the lateral ventricle) diminished the resilience of the microvasculature to HI injury provided by preconditioning and also the Flk-1 increase that occurred in these animals. In conclusion these results suggest an interaction of the AT2-R with VEGFR2 in the neonatal brain microvasculature that produces protective effects which are associated with injury tolerance.

c-fos and tyrosine hydroxylase expression after an excitotoxic lesion on the nigrostriatal system: a study on the effects of hypoxia used as a preconditioning stimulus.

Hypoxia-ischemia during the perinatal period causes excitotoxic lesions in sensitive brain areas, such as the striatum. The impact of hypoxia-ischemia on nigral neurons is less well known. Hypoxia alone, a less traumatic event without overt histological sequelae, has neuroprotective properties when used as a preconditioning stimulus. In some pathologies, injured neurons of the nigrostriatal system in the adult may be the result of neurodegenerative processes that originated at early stages of life. The effects of hypoxia on the immunoreactivity to tyrosine hydroxylase of the dopaminergic neurons of the substantia nigra pars compacta and the effects of a period of hypoxia previous to an excitotoxic lesion were examined by means of histological and Western blot methods, at immediate and late periods of the episode. By counting the number of tyrosine hydroxylase-stained neurons and c-fos-positive nuclei a short period after injection of quinolinic acid into the striatum, we observed that hypoxia induced a more marked decrease in the number of tyrosine hydroxylase-stained neurons. On the contrary, c-fos-positive profiles decreased in the substantia nigra pars reticulata of the quinolinic acid-injected animals after the preconditioning hypoxia. Hypoxia alone did not affect the number of tyrosine hydroxylase-positive neurons in the pars compacta nor did hypoxia induce c-fos expression in the pars reticulata. More sensitive Western blot analysis of tissue blocks that included the whole substantia nigra demonstrated the same trend as the immunohistochemical results. We conclude that the responses of the substantia nigra neurons to hypoxia are regionalized and potential neuroprotective effects may depend on the vulnerability of each neuronal type.

Transient increase in rab 3A and synaptobrevin immunoreactivity after mild hypoxia in neonatal rats.

1. In the present work we describe the short term effects of mild neonatal hypoxia on the synapse as assessed by the immunoreactivity (IR) of two synaptic proteins: rab 3A and synaptobrevin (VAMP). 2. Using the sensitive methodology of immunoblotting, we measured rab 3A and VAMP-IR in homogenates from the cerebral cortex, hippocampus, and corpus striatum of control (breathing room air) and hypoxiated (breathing 95.5% N2-6.5% O2 for 70 min) 4-day-old rats at 1, 2, and 6 h after the end of the hypoxia. Immunostaining with examination by light microscopy was performed using the synaptic protein-specific antibodies on fixed brain sections from animals belonging to the same litter and submitted to hypoxia. 3. A transient increase of VAMP-IR was observed in the hippocampus and corpus striatum, and for rab 3A in the striatum, 1 h after initiating reoxygenation. At the following time points the values returned to control levels. This effect was less clearly observed in the immunostained sections. 4. Mild hypoxia has an effect on sensitive brain regions, eliciting an increase in the IR of at least two proteins involved in the synaptic vesicle cycle. The transient nature of this effect possibly indicates the activation of endogenous neuroprotective mechanisms.

Acute and long-lasting effects of neonatal hypoxia on (+)-3-[125I]MK-801 binding to NMDA brain receptors.

The NMDA receptor subtype is the major excitatory mediator for glutamate neurotoxicity. To assess its participation in the noxious effects of postnatal hypoxia, we have characterized the binding of the ionophoric marker of NMDA receptor, dizocilpine (MK-801). Binding of (+)-3-[125I]MK-801 to NMDA brain receptors under nonequilibrium conditions was quantified by in vitro autoradiography in rats exposed to hypoxia induced by 93% N2/6.5% O2 exposure for 70 min on Postnatal Day 4. Acute and long-lasting effects were investigated at 4 h after injury and on Postnatal Day 40. At the acute stage, a transient decrease in binding was found in several specific brain areas, hypothalamus, amygdaloid nuclei, entorhinal cortex, perirhinal cortex, and hippocampus, and no differences were found in temporal cortex, thalamus, and geniculate nucleus, when compared to sham-treated animals. At this early age, there was no increase of binding when slices from both groups were incubated in the presence of glutamate and glycine (Glu/Gly), positive allosteric modulators of MK-801 binding. In the 40-day-old brains, the binding to the NMDA receptors of hypoxiatreated animals was not different with respect to controls in most of the areas studied, but the Glu/Gly stimulation of binding in hypoxic rats showed a reduced, or absent, response to the allosteric modulators. In contrast, control rats showed a remarkable increase of the specific binding induced by the presence of the modulators in the incubation buffer. Binding of (+)-3-[125I]MK-801 was also performed at a higher concentration to clarify whether the altered response to Glu/Gly may be due to differences in the number of channels; however, the density of NMDA receptors at this concentration was similar in both control and hypoxia-treated rats. We conclude that the effect of exposure of newborn rats to hypoxia can generate acute and long-lasting effects on the NMDA receptor. The deleterious action of this kind of noxa on the CNS could be exerted by interference with normal glutamatergic transmission and hence over normal growth and development.

Decrease of (+)-3-[125I]MK-801 binding to NMDA brain receptors revealed at puberty in rats treated neonatally with monosodium glutamate.

Obesity, altered pattern of gonadal hormone secretion, advanced vaginal opening, irregular cycling, altered sexual behavior and infertility are the effects of the neonatal administration of monosodium glutamate (MSG) to rodents. These are the consequences of lesions located mainly in the hypothalamic region. It is believed that the receptors to N-methyl-D-aspartic acid (NMDA) actively participate in the onset and development of such lesions, on the other hand, they may be altered by neuronal dysfunction as well, seriously compromising the glutamatergic pathways that are involved in the neuroendocrine regulation. To clarify the scope of the lesion induced by MSG and its probable effects on the NMDA receptors, we measured them with a very sensitive ligand for autoradiography, (+)-3-[125I]MK-801. Coronal cuts at the level of the arcuate-median eminence of brains from 4-, 8- and 40-day-old rats treated neonatally with MSG (4 mg/g) or saline (controls) were examined. In the normal hypothalamus, NMDA receptor labelling was higher in the young animals than in the 40-day-old animals, and this was observed in both control and treated rats. NMDA receptor labelling of rats at puberty was very low, and no apparent differences were observed between groups. In contrast, in areas where an increase in NMDA binding sites normally occurs with development, a significant impairment of the normal augmentation of MK-801 binding was revealed. In the hippocampal layers, stratum radiatum and stratum oriens and in the cerebral cortex of 40-day-old rats treated with MSG a lower amount of binding was observed, of about 50% fewer sites compared to the untreated controls at the level of CA3 and in the outer layer of the parietal cortex. These results suggest that at an early stage of the MSG lesion the NMDA receptors located in the hypothalamus and other brain areas are apparently expressed normally, but at puberty the effects of the lesion are revealed in the hippocampus and cerebral cortex by a decrease in the density of binding. Thus, the abnormal neuroendocrine and behavioral responses displayed by the MSG-treated rats may be contributed partially by the alteration of the NMDA receptors in these areas.

Stimulation of angiotensin II AT1 receptors in rat median eminence increases phosphoinositide hydrolysis.

The aim of our study was to determine the second messenger systems for angiotensin II in the rat median eminence. Angiotensin II AT1 receptors are highly expressed in the median eminence and binding is selectively inhibited by the guanine nucleotide GTP gamma S, indicating possible coupling to G-proteins. In male rats, angiotensin II increased phosphatidylinositol hydrolysis about 45% over basal values, with an EC50 of about 2.7 nM. This effect was antagonized by 10 microM losartan, the selective AT1 antagonist, but not by the AT2 competitor PD 123319. Conversely, angiotensin II, 1 microM, did not alter basal or forskolin-stimulated cAMP production, and failed to influence cGMP production. These results support a role for angiotensin II, through stimulation of AT1 receptors and increased phosphatidylinositol hydrolysis, in the median eminence. Angiotensin II increased the phosphatidylinositol hydrolysis not only in male rats but also in ovariectomized rats, with or without estrogen-progesterone replacement. However, angiotensin II (up to 1 microM) failed to increase the phosphatidylinositol hydrolysis in randomly selected intact female rats. Estrogen treatment did not alter the number or affinity of median eminence AT1 receptors in ovariectomized rats. The increase in phosphatidylinositol hydrolysis resulting from stimulation of median eminence AT1 receptors appears to be sexually dimorphic, but hormonal manipulations failed to point to a role for reproductive hormones in this phenomenon.

Regulation of luteinizing hormone-releasing hormone and luteinizing hormone secretion by hypothalamic amino acids.

1. The present review discusses the proposed roles of the amino acids glutamate and GABA in the central regulation of luteinizing hormone-releasing hormone (LHRH) and in luteinizing hormone (LH) secretion. 2. Descriptions of the mechanisms of action of these neurotransmitters have focused on two diencephalic areas, namely, the preoptic-anterior hypothalamic area where the cell bodies of LHRH neurons are located, and the medial basal hypothalamus which contains the nerve endings of the LHRH system. Increasing endogenous GABA concentration by drugs, GABA agonists, or blockade of glutamatergic neurotransmission by selective antagonists in rats and non-human primates prevents ovulation and pulsatile LH release, and blunts the LH surges induced by estrogen or an estrogen-progesterone combination. In contrast, glutamate and different glutamate agonists such as NMDA, AMPA and kainate, can increase LHRH/LH secretion. 3. The simultaneous enhancement of glutamatergic activity and a decrease of GABAergic tone may positively influence the maturation of the pituitary-gonadal system in rats and non-human primates. Administration of glutamate receptor agonists has been shown to significantly advance the onset of puberty. Conversely, glutamate antagonists or increased endogenous GABA levels may delay the onset of puberty. The physiological regulation of LHRH/LH secretion may thus involve a GABA-glutamate interaction and a cooperative action of the various types of ionotropic glutamate receptors. 4. The inhibitory actions of GABA on LH release and ovulation may be exerted at the level of afferent nerve terminals that regulate LHRH secretion. A likely candidate is noradrenaline, as suggested by the synaptic connections between noradrenergic nerve terminals and GABAergic interneurons in the preoptic area. Recent experiments have provided complementary evidence for the physiological balance between inhibitory and excitatory transmission resulting in modulation of the action of noradrenaline to evoke LHRH release.

Regulation of luteinizing hormone-releasing hormone and luteinizing hormone secretion by hypothalamic amino acids

1. The present review discusses the proposed roles of the amino acids glutamate and GABA in the central regulation of luteinizing hormone-releasing hormone (LHRH) and in luteinizing hormone (LH) secretion. 2. Descriptions of the mechanisms of action of these neurotransmitters have focused on two diencephalic areas, namely, the preoptic-anterior hypothalamic area where the cell bodies of LHRH neurons are located, and the medial basal hypothalamus which contains the nerve endings of the LHRH system. Increasing endogenous GABA concentration by drugs, GABA agonists, or blockade of glutamatergic neurotransmission by selective antagonists in rats and non-human primates prevents ovulation and pulsatile LH release, and blunts the LH surges induced by estrogen or an estrogen-progesterone combination. In contrast, glutamate and different glutamate agonists such as NMDA, AMPA and kainate, can increase LHRH/LH secretion. 3. The simultaneous enhancement of glutamatergic activity and a decrease of GABAergic tone may positively influence the maturation of the pituitary-gonadal system in rats and non-human primates. Administration of glutamate receptor agonists has been shown to significantly advance the onset of puberty. Conversely, glutamate antagonists or increased endogenous GABA levels may delay the onset of puberty. The physiological regulation of LHRH/LH secretion may thus involve a GABA-glutamate interaction and a cooperative action of the various types of ionotropic glutamate receptors. 4. The inhibitory actions of GABA on LH release and ovulation may be exerted at the level of afferent nerve terminals that regulate LHRH secretion. A likely candidate is noradrenaline, as suggested by the synaptic connections between noradrenergic nerve terminals and GABAergic interneurons in the preoptic area. Recent experiments have provided complementary evidence for the physiological balance between inhibitory and excitatory transmission resulting in modulation of the action of noradrenaline to evoke LHRH release.

Glomerular angiotensin II receptor subtypes during development of rat kidney.

We used quantitative autoradiography to investigate distribution of angiotensin II (ANG II) receptor subtypes during development of the kidney in the rat. In fetal, newborn, and 3-day-old rats, immature glomeruli in the form of comma and S-shaped bodies, located in the nephrogenic zone of the renal cortex, expressed only the angiotensin AT2 receptor subtype. Conversely, the juxtamedullary glomeruli, in more advanced developmental stages, expressed only the AT1 subtype. Similarly, maturing and fully developed glomeruli, present in 1-, 2-, and 8-wk-old rats, expressed only AT1 receptors. In the kidney medulla, there was a similar change in ANG II receptor subtype expression, with the AT2 subtype expressed earlier and the AT1 subtype later during development. Our results demonstrate a selective expression of ANG II receptor subtypes during kidney development. We have found glomerular and medullary AT1 receptors only at developmental stages when kidney function has matured. Conversely, AT2 receptors are expressed only in immature structures, suggesting that they may have a role during kidney organogenesis.

Restraining action of GABA on estradiol-induced LH surge in the rat: GABA activity in brain nuclei and effects of GABA mimetics in the medial preoptic nucleus.

The relationship between GABA dynamics and LH release was studied on day 2 after subcutaneous estrogen implant in short-term ovariectomized rats. GABA accumulation, used as an index of GABA turnover, was determined in the medial preoptic nucleus (MPN), medial (MS) and lateral (LS) septal nuclei, median eminence-mediobasal hypothalamus (MBH) and locus ceruleus (LC). Measurements of GABA were performed at two different times of day (11.00 and 15.00 h), 3 h after intraperitoneal administration of gamma-vinyl-GABA (GVG), an irreversible inhibitor of GABA transaminase. Either morning or afternoon ovariectomized rats (OVX) showed a significant increase in GABA accumulation after GVG treatment in all the areas studied. Estrogen-treated OVX rats showed in the morning a lower GABA accumulation in the MPN, MBH and LC, and GABA levels remained unchanged in the LS and MS. In the afternoon, the MPN and LS showed a lower rate of GABA accumulation whereas in the MBH and LC the GABA increase was not observed. In contrast the MS showed a rate of GABA accumulation similar as in the OVX rats. Local administration in the MPN of 20 micrograms GVG, or GABA-A receptor stimulation by muscimol (50 ng), prior to the increase in plasma LH levels, prevented the occurrence of the estradiol-induced LH surge. The effect of muscimol was reversed by bicuculline (30 ng), a GABA-A receptor antagonist. Bicuculline in low doses lacked effect by itself. In conclusion, these results strongly suggest that a decreased GABAergic activity in MPN, MBH and LC precedes the estradiol-evoked LH surges in ovariectomized rats. Moreover, that in septal nuclei, a low GABAergic activity takes place well before the occurrence of plasma LH increase.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of ovariectomy and ovarian steroids on binding of 3H-mepyramine, an H1-histamine antagonist, in rat hypothalamus.

The effects of ovariectomy and ovarian steroids on 3H-mepyramine binding, a histamine antagonist considered as a suitable ligand for H1-histamine receptors, were studied. Saturation binding assays and Scatchard plot analyses were performed in membranes from hypothalamus of adult female rats. In the hypothalamus of ovariectomized (OVX) rats, a decrease of the Bmax and a lowering of Kd values as compared to diestrous rats, were found. Estradiol administration to OVX rats as a single SC injection (25 micrograms), or by means of SC Silastic capsules (500 micrograms/ml estradiol benzoate; 10 mm/100 g b.wt.), reversed the effects of OVX, increasing both Bmax and Kd. The addition of progesterone (25 mg) blunted the effects of estradiol (50 micrograms). The present results suggest that hypothalamic H1-histamine receptors may be modulated by the ovarian steroids and provide further evidence of a possible role of histamine in female gonadotropin regulation.

Restraint stress stimulation of prolactin and ACTH secretion: role of brain histamine.

The possible role of brain histamine in the release of prolactin, ACTH and corticosterone following acute restraint, was pharmacologically evaluated in adult male rats. Fifteen min of restraint caused marked increases in the plasma levels of these hormones. alpha-Fluoromethyl histidine (FH), a histidine decarboxylase inhibitor which depleted hypothalamic histamine, inhibited the enhancement of plasma prolactin levels. In contrast, plasma ACTH levels were not modified. FH treatment decreased plasma corticosterone concentrations in animals submitted to stress or in rest; this suggests a direct action of FH on the adrenal. Intraventricular (IVT) injection of ranitidine (H2 antagonist) blunted the prolactin response to restraint stress whereas its systemic administration had no effect. On the contrary, pyrilamine (H1 antagonist) given systemically decreased slightly, but significantly, the prolactin rise but when injected IVT it was ineffective. Pyrilamine was also unable to affect the ranitidine action. ACTH and corticosterone levels in plasma of restrained rats were not modified by the histamine antagonists. It is concluded that histamine is involved, mainly through central H2 receptors, in the enhancement of plasma prolactin levels produced by an acute stress. The failure of both antihistaminic compounds and a histamine depletor to alter the ACTH stimulation suggest that histamine has no participation in the hypophysio-corticoadrenal response to acute restraint.

Histamine-induced prolactin release and activity of tuberoinfundibular dopaminergic neurons in male rats.

The role of tuberoinfundibular dopamine (DA) in the histamine-induced rise of plasma prolactin was studied in male rats. Right lateral ventricle (icv) histamine injection (30 micrograms/rat) caused a significant rise of plasma prolactin at 15 and 30 min; at 60 min values returned to basal levels. Histamine does not modified steady-state DA concentrations. For turnover evaluation, histamine was icv injected immediately or 30 min after the tyrosine hydroxylase inhibitor alpha-methyltyrosine (250 mg/kg i.p.) and DA concentration in the median eminence was measured at 0, 1 and 2 hours after the inhibitor injection. Rate constants of DA decline and DA synthesis rates were found similar in both controls (cerebrospinal fluid) and histamine-injected rats. These results indicate that the stimulatory action of histamine on prolactin release might not be associated to DA. It would be due mainly to its action on prolactin-releasing factors and to a minor extent, to changes in the dopaminergic system.