Gonadal steroids regulate oxytocin receptors but not vasopressin receptors in the brain of male and female rats. An autoradiographical study

The distribution and the amount of [3H]oxytocin binding were studied in the brain of adult rats of either sex, as well as in male and female castrates, some of which received injections of estradiol or testosterone. Intact males were treated with an aromatase inhibitor. Castration and inhibition of aromatase activity reduced, whereas estradiol and testosterone increased oxytocin binding, particularly in regions of the brain assumed to be involved in reproductive functions, such as the ventrolateral part of the hypothalamic ventromedial nucleus and the islands of Calleja and neighbouring cell groups. Binding of oxytocin to the uterus was also estrogen-dependent. In the same animals, we also studied the distribution of [3H]vasopressin binding sites present in the brain. It was similar in males and females, and was not affected by experimentally manipulating gonadal hormone levels. In immunocytochemical studies we noticed, as others had previously, that the vasopressin content of certain areas of the rat brain was affected by castration, whereas the oxytocin innervation was not. These results are discussed in relation to the possible functions of oxytocin in the brain and of the lack of correspondence between the immunocytochemical and the autoradiographic data.     

Appearance and transient expression of oxytocin receptors in fetal, infant, and peripubertal rat brain studied by autoradiography and electrophysiology

The development of oxytocin (OT) receptors in the rat brain and spinal cord was studied by in vitro light microscopic autoradiography and by electrophysiology. OT receptors were labeled using a monoiodinated OT antagonist in tissue sections from animals aged between embryonic day 12 (E12) and postnatal day 90 (PN90); the response of ongoing spike activity to the addition of OT was assessed in neurons located in the dorsal motor nucleus of the vagus nerve of the neonate. Specific binding was detected first at E14 in a region that later differentiated into the dorsal motor nucleus of the vagus nerve. Many other regions were progressively labeled between E20 and PN5. From PN5 to PN16, the distribution of binding sites remained essentially unchanged but differed markedly from that characteristic of the adult. The change-over from the "infant pattern" to the "adult pattern" occurred in 2 stages: the first change took place between PN16 and PN22, a time corresponding to the preweaning period; the second change occurred after PN35 and thus coincided with the onset of puberty. During the first transition period, binding was reduced or disappeared in several areas intensely labeled at earlier stages, in particular, in the cingulate cortex and the dorsal hippocampus. At the same time, binding sites appeared in the ventral hippocampus. At puberty, high densities of OT binding sites appeared in the ventromedial hypothalamic nucleus and the olfactory tubercle. Electrophysiological activity was recorded from vagal neurons in slices obtained from animals sacrificed at PN1-PN12. OT and a selective OT agonist reversibly increased the firing rate of these neurons in a concentration-dependent manner. The neuronal responsiveness was similar to that reported previously in the adult. These results suggest that OT binding sites detected by autoradiography in the developing rat brain represent, at least in some areas, functional neuronal receptors.     

Interaction of a vasopressin antagonist with vasopressin receptors in the septum of the rat brain

The ability of d(CH2)5-Tyr(Me)-arginine-8-vasopressin, an antagonist of peripheral pressoric (V1-type) vasopressin receptors, to label vasopressin binding sites in the septum of the rat brain was evaluated. Using crude membrane preparations from the septum, 3H-arginine-8-vasopressin (AVP) specifically labels a single class of binding sites with a Kd of 2.9 nM and maximum binding site concentration of 19.8 fmole/mg protein. 3H-Antag also labels a single class of membrane sites but with higher affinity (Kd = 0.47 nM) and lower capacity (10.1 fmole/mg protein) than 3H-AVP. The rank order of potency of various competitor peptides for 3H-AVP and 3H-Antag binding was similar. Oxytocin was 100-1,000 fold less potent than AVP in competing for binding with both ligands. 3H-AVP and 3H-Antag showed similar labeling patterns when incubated with septal tissue slices. Unlabeled Antag also effectively antagonized vasopressin-stimulated phosphatidylinositol hydrolysis in septal tissue slices.     

Chronic treatment with vasopressin analogues alters affinity of vasopressin receptors in the septum and amygdala of the rat brain.

Accurel devices were used to administer AVP or AVP-antagonist into the lateral ventricle of the rat brain for 7 days. Neither AVP nor antagonist altered total binding site concentration in either septum or amygdala. However, treatment with antagonist caused a marked decrease in the affinity of the receptor for agonist in both the septum and amygdala.     

An electrophysiological study of the ontogenesis of adenosine receptors in the CA1 area of rat hippocampus

The depressant effect of adenosine (Ad) was studied electrophysiologically in hippocampal slices from 5-, 10-, 15-, 20-, 30- and 120-day-old rats. Ad (10 microM) depressed the field EPSP in CA1 to the same extent in all age groups. Caffeine (Caf), an Ad receptor antagonist, enhanced and nitrobenzylthioinosine (NBI), an Ad uptake blocker, depressed the field EPSP. Both these effects were, however, less prominent in slices from younger animals, a finding consistent with lower extracellular levels of endogenous Ad in neonatal rats.     

Early postnatal appearance of enhanced noradrenaline content in the brain of vasopressin-deficient brattleboro rat; normal adrenoceptor densities and aberrant influences of vasopressin treatment

The course of postnatal development of noradrenaline (NA) and its unconjugated free metabolite 3-methoxy-4-hydroxyphenylglycol (MHPG), as well as the influence on early chronic vasopressin treatment, were investigated in various brain regions of the hereditary vasopressin-deficient (homozygous di/di) Brattleboro rat. In addition, the densities of the adrenergic receptor subtypes were measured in adult brain.Brain NA levels of di/di pups appeared enhanced already at 7 days of age when compared with data of heterozygous (+/di) controls. This was also seen in areas not known to receive a vasopressinergic input, e.g. the frontal cortex. Levels of MHPG also differed between genotypes, but changes were slight and either a decrease or increase, depending on age and region tested. Saturation analyses of α1-, α2-, and β-adrenoceptor binding on crude membrane preparations of some brain regions revealed no differences in adulthood.Chronic treatment with vasopressin between 6 and 13 days of age reduced the enhanced NA brain levels throughout the brain of the di/di Brattleboro pups. The known vasopressin-mediated enhancement of NA turnover in adult brain was also measurable in +/di pups of this neonatal period (MHPG/NA ratios), indicating the early maturation of the interaction of vasopressinergic and NAergic systems. However, the dose-response in the di/di Brattleboro rat was biphasic with a decrease at a low dose of vasopressin.Since changes were found throughout the brain, it was concluded that vasopressin deficiency had altered the maturation of NA neurons of the locus coeruleus which may be due to the absence of a presumed inhibitory control of vasopressin on synthesis and storage mechanisms at the perikaryal level.     

An autoradiographical study of the projections from the feline sensorimotor cortex to the brain stem

Following an autoradiographical study on the projections from the feline sensorimotor cortex (representation of the limbs) to the brain stem, new projections to nucleus pretectalis posterior, the coerulear nuclei, nuclei corporis pontobulbares, nucleus intertrigeminalis, nucleus f, x and z of the vestibular nuclear complex, nucleus parvocellularis compactus, nucleus parasolitarius, nuclei insulae cuneati laterales, the nuclei of the raphé, nucleus reticularis lateralis and nucleus fastigii were found besides the well known projections to the tectal nuclei, the reticular formation, nucleus ruber, griseum pontis, the sensory trigeminal nuclei and the dorsal column nuclei. There was no evidence of projections to nucleus tractus solitarii. The amounts of radioactivity within the nuclei seem to be in favour of EM autoradiographical studies on the synaptology of these projections, apart from faintly labelled structures like most of the reticular nuclei, the sensory trigeminal nuclei, the coerulear nuclei and nucleus fastigii.     

Localization of vasopressin in the rat brain

The distribution of arginine vasopressin (AVP) in the rat brain was studied using a sensitive radioimmunoassay. The highest concentration of AVP was found in the hypothalamus. Individually, the supraoptic, paraventricular and suprachiasmatic nuclei contained in the order of 10% of the total hypothalamic content. Vasopressin was also found in the thalamus, medulla, cerebellum, amygdala, substantia nigra and hippocampus. Much lower levels were detected in the pons, spinal cord, frontal and occipital lobes and caudate putamen. No AVP could be detected in any other regions of the cortex or corpus callosum. Chromatographically the vasopressin found outside the hypothalamus is of a similar nature to that of hypothalamo-hypophysial origin. This study supports previous reports of extrahypothalamic localization of vasopressin by immuno-histochemical methods. It is clear that AVP is not confined to the hypothalamo-hypophysial axis, and the possibilities that this may reflect an involvement in brain function are discussed.     

Ontogeny of mineralocorticoid (type 1) receptors in brain and pituitary: an in vivo autoradiographical study

The ontogeny of high affinity [3H]corticosterone uptake and retention in brain and pituitary of 24-h adrenalectomized rats was examined using autoradiography of in vivo labeled brain sections. Our data indicate: (1) There is specific uptake of radiolabeled steroid in both brain and pituitary already at 2 days of age, following administration of a tracer (2 microCi/g body wt.) dose of [3H]corticosterone. This uptake is maximum around 4-8 days of age and decreases towards adult values around postnatal day 16. (2) High affinity uptake, at least in the brain, probably represents mostly binding to the mineralocorticoid receptor (MR) and not to the glucocorticoid receptor (GR), as it was not displaced by an excess dose of a GR antagonist, RU 38486, and its location in the hippocampus resembled that of MRs in the adult animal. The tracer amounts of [3H]corticosterone circulating after injection in the rat pups resulted in steroid levels comparable to basal levels of non-adrenalectomized animals of equivalent age. Thus, MRs may be the receptors mainly responsible for mediating physiological effects of glucocorticoids during early ontogeny.     

Late consequences of cryogenic brain lesion in rat; an electrophysiological study.

The left somatosensory cortex of PVG/C rats was subjected to a transcranial cold lesion, spontaneous and evoked cortical activity was studied 2-3 weeks after the lesion. The spontaneous activity underwent a depression at the focus of the lesion. Close to the epicentre of the lesion, the amplitude of the potentials evoked by electrical stimulation of the contralateral whisker pad was considerably smaller than in the controls and an almost complete reduction of the initial positive phase was found. Perifocally, 3-4 mm from the epicentre, evoked potentials with enhanced amplitudes were recorded in all experiments. It seems that considerable damage took place at the epicentre of the lesion, while perifocally an excitatory (and/or inhibitory?) mechanism was activated.     

Role of arginine vasopressin V1 and V2 receptors for brain damage after transient focal cerebral ischemia.

Brain edema formation is one of the most important mechanisms responsible for brain damage after ischemic stroke. Despite considerable efforts, no specific therapy is available yet. Arginine vasopressin (AVP) regulates cerebral water homeostasis and has been involved in brain edema formation. In the current study, we investigated the role of AVP V1 and V2 receptors on brain damage, brain edema formation, and functional outcome after transient focal cerebral ischemia, a condition comparable with that of stroke patients undergoing thrombolysis. C57/BL6 mice were subjected to 60-min middle cerebral artery occlusion (MCAO) followed by 23 h of reperfusion. Five minutes after MCAO, 100 or 500 ng of [deamino-Pen(1), O-Me-Tyr(2), Arg(8)]-vasopressin (AVP V1 receptor antagonist) or [adamantaneacetyl(1), O-Et-D-Tyr(2), Val(4), Abu(6), Arg(8,9)]-vasopressin (AVP V2 receptor antagonist) were injected into the left ventricle. Inhibition of AVP V1 receptors reduced infarct volume in a dose-dependent manner by 54% and 70% (to 29+/-13 and 19+/-10 mm3 versus 63+/-17 mm3 in controls; P<0.001), brain edema formation by 67% (to 80.4%+/-1.0% versus 82.7%+/-1.2% in controls; P<0.001), blood-brain barrier disruption by 75% (P<0.001), and functional deficits 24 h after ischemia, while V2 receptor inhibition had no effect. The current findings indicate that AVP V1 but not V2 receptors are involved in the pathophysiology of secondary brain damage after focal cerebral ischemia. Although further studies are needed to clarify the mechanisms of neuroprotection, AVP V1 receptors seem to be promising targets for the treatment of ischemic stroke.     

Immunocytochemically-stained vasopressin binding sites in rat brain. Ventricular application of vasopressin/Accurel in the Brattleboro rat

An immunocytochemical procedure was developed to localize binding sites for vasopressin (VP) in the brain of Brattleboro (di/di) rats after 2 weeks of continuous ventricular administration of the peptide. Accurel-polypropylene tubing loaded with 0.15, 1.5 or 15 micrograms vasopressin was implanted into the lateral ventricle. Subsequently, bound VP was detected immunocytochemically in 2 distinct patterns: in perineuronal structures and dots between cells, in the lateral septum (dorsorostral part), striatum, cingulate cortex, granular cells of the dentate gyrus of the hippocampus, pyramidal cells of CA1 and CA3 hippocampal areas and around cerebellar Purkinje cells. The high dose (15 micrograms) loaded implants revealed the most intense staining; in the cytoplasm of neuronal cell bodies in the lateral and medial septum, striatum, cingulate cortex, bed nucleus of the stria terminalis, organum vasculosum of the laminae terminalis and locus coeruleus. The most intense staining in cell bodies was observed in brains which had low-loaded implants (0.15-1.5 microgram). A variety of controls, proved that no aspecific uptake was involved in the present procedure. The distribution of VP binding sites was only partly coincident with known sites of VP fiber innervation, and largely agrees with data obtained by autoradiographic techniques for [3H]VP binding. The present immunocytochemical technique gave a higher resolution than the currently used autoradiographic techniques. The differences in pattern and intensity of staining due to increasing the dosage rate of the in vivo vasopressin treatment, might mean that the current procedure retains preferentially either low or high affinity populations of binding sites depending on the implanted dose.     

Differential expression of PACAP receptors in postnatal rat brain

Pituitary Adenylate Cyclase Activating Polypeptide (PACAP) is a multi-functional neuropeptide that acts through activation of three common G-protein coupled receptors (VPAC1, VPAC2 and PAC1). In this study, we have investigated the gene expression profile of PAC1 isoforms (Hop1, Hip, Hip-Hop) and VPAC1, VPAC2 receptors in distinct brain regions during different stages of rat postnatal development. Using quantitative real time PCR approach we found that PAC1 isoforms were highly expressed in the cortex of newborns with marked decrease in expression during later stages of development. In contrast, mRNA levels of VPAC1, VPAC2 receptors were markedly lower in newborns in comparison to later developmental stages. Expression of PAC1 isoforms predominated in the hippocampus, while expression of VPAC1 was more prominent in the cortex and VPAC2 in the striatum and hippocampus. In addition we found that during early stages of postnatal development the expression of PAC1 receptor in the hippocampus was significantly higher in females than in males. No sex dependent differences in expression were observed for the VPAC1 and VPAC2 receptors. In summary, differential expression of PAC1, VPAC1 and VPAC2 receptors during postnatal development as well as gender dependent differences of PAC1 receptor expression in the hippocampus, will contribute to our understanding of the role of PACAP/VIP signaling system in normal brain development and function.     

5-HT3-like receptors in the rat medial prefrontal cortex: an electrophysiological study.

In this study, we have identified and characterized 5-HT₃-like receptors in the rat medial prefrontal cortex (mPFc), an area with a moderate density of 5-HT₃ binding sites, using the techniques of single unit recording and microiontophoresis. The microiontophoresis of the 5-HT₃ receptor agonist 3-methylserotonin (2-Me-5HT), similar to the action of 5-HT), produced a current-dependent (10–80 nA) suppression of the firing rate of both spontaneously active and glutamate (GLU)-activated (quiescent) mPFc cells. Phenylbiguanide (PBG), another 5-HT₃ receptor agonist, suppressed the firing rate of mPFc cells but was less effective compared to 2-Me-5HT. The continuous iontophoresis (10–20 min) of 1 M magnesium chloride markedly attenuated the suppressant effect produced by electrical stimulation of the ascending 5-HT pathway, but did not alter 2-Me-5HT's action, suggesting that the action of 2-Me-5HT is a direct one. The suppressant action of 2-Me-5HT on mPFc cells was blocked by a number of structurally diverse and selective 5-HT₃ antagonists, with a rank order of effectiveness as follows: JCS 205930=(±)-zacopride>granisetron=ondansetron= LY 278584 >MDL72222. Furthermore, the intravenous administration of(±)-zacopride antagonized the action of 2-Me-5HT and PBG on mPFc cells. In contrast to the effects of the 5-HT₃ receptors antagonists, other receptor antagonists such as metergoline (5-HT_1A,1B,1C,2), (±)-pindolol (5-HT_1A,1B,β), SCH 23390 (5-HT_1C,2, D1), 1-sulpiride (D2) or SR 95103 (GABA_A) failed to block 2-Me-5HT's action. These results combined suggest that 2-Me-5HT's suppressive action on mPFc cells is mediated directly by 5-HT₃-like receptors.     

The effect of vincristine on nerve regeneration in the rat. An electrophysiological study

Weekly injections of vincristine to produce a dose-dependent delay in regeneration following sciatic nerve crush. With 20 micrograms/kg/wk recovery was similar to that in control animals. With 50 and 100 micrograms/kg/wk electrophysiological evidence of reinnervation of the foot muscles was significantly delayed and muscle action potential amplitude increased at a slower rate. However, once begun the increase in motor nerve conduction velocity was closer to that in control animals. With 200 micrograms/kg/wk no evidence of reinnervation of the foot muscles was found even after 6 months. These doses produced no abnormality of muscle action potential amplitude or of nerve conduction velocity on the opposite non-crushed side.     

Differential expression of orexin receptors 1 and 2 in the rat brain

Orexins (hypocretins) are neuropeptides synthesized in the central nervous system exclusively by neurons of the lateral hypothalamus. Orexin-containing neurons have widespread projections and have been implicated in complex physiological functions including feeding behavior, sleep states, neuroendocrine function, and autonomic control. Two orexin receptors (OX(1)R and OX(2)R) have been identified, with distinct expression patterns throughout the brain, but a systematic examination of orexin receptor expression in the brain has not appeared. We used in situ hybridization histochemistry to examine the patterns of expression of mRNA for both orexin receptors throughout the brain. OX(1)R mRNA was observed in many brain regions including the prefrontal and infralimbic cortex, hippocampus, paraventricular thalamic nucleus, ventromedial hypothalamic nucleus, dorsal raphe nucleus, and locus coeruleus. OX(2)R mRNA was prominent in a complementary distribution including the cerebral cortex, septal nuclei, hippocampus, medial thalamic groups, raphe nuclei, and many hypothalamic nuclei including the tuberomammillary nucleus, dorsomedial nucleus, paraventricular nucleus, and ventral premammillary nucleus. The differential distribution of orexin receptors is consistent with the proposed multifaceted roles of orexin in regulating homeostasis and may explain the unique role of the OX(2)R receptor in regulating sleep state stability.     

An electrophysiological study of amygdalohypothalamic projections to the ventromedial nucleus of the rat.

The influence of the amygdala on the activity of single neurons within the hypothalamic ventromedial nucleus (HVM) was studied in pentobarbital or urethane anesthetized rats. The results are summarized as follows: (1) Stimulation of different amygdaloid nuclei or of the stria terminalis (ST) evoked a prominent field potential within HVM and altered the spike discharge patterns of the majority of HVM neurons. (2) More than 80% of 428 HVM neurons tested with single amygdala shocks exhibited excitation or excitation-inhibition sequences; the remainder displayed inhibitory responses of 100-150 msec duration at latencies slightly longer than for most of the observed excitatory responses. ST stimulation also evoked excitation or excitation-inhibition sequences from 85% of 240 HVM neurons tested; of the remainder, those with spontaneous activity displayed inhibitory responses with durations of 100-150 msec at latencies slightly longer than for most observed excitatory responses. (3) Evoked potential interaction studies suggested that stimulation of either ST or the amygdala activated the same population of HVM neurons. Single cells tested with both amygdala and ST stimulation displayed similar patterns of response. HVM field potentials and single unit responses to amygdala stimulation were markedly diminished by lesions of ST. Thus, in the rat, only one pathway, i.e., the stria terminalis, contains amygdalofugal fibres to the ventromedial hypothalamic nucleus. (4) The orthodromic responses of HVM neurons were dependent on the frequency of amygdala stimulation. Less than 50% of HVM neurons responded to amygdala stimuli at frequencies greater than 33Hz. Many cells could not be activated at stimulation frequencies greater than 10 Hz, and the spontaneous discharges from certain HVM neurons were effectively abolished at this stimulation frequency. (5) Evidence of prominent postsynaptic inhibition was present throughout HVM. Seventeen HVM neurons displayed amygdala evoked unitary activity different from that of the majority of HVM neurons, and these cells were considered to represent possible inhibitory neurons. In contrast to most HVM neurons activated via probable monosynaptic amygdalohypothalamic pathways, these putative inhibitory neurons were apparently activated via polysynaptic pathways. (6) In summary, these results suggest that the amygdala exerts a prominent monosynaptic influence on the activity of many HVM neurons, coupled with polysynaptic activation of powerful local postsynaptic inhibitory mechanism. In the rat, these amygdala evoked events depend on the integrity of the stria terminalis.     

Major difference in the expression of delta- and mu-opioid receptors between turtle and rat brain.

The reptilian turtle brain has a remarkably higher endurance for anoxia than mammalian brains. Since the response to O(2) deprivation is dependent in a major way on the expression and regulation of membrane proteins, differences in such proteins may play a role in the species-related differences in hypoxic responses. Because opioid system is involved in the regulation of hypoxic responses, we asked whether there are differences between rat and turtle brains in terms of opioid receptor expression. In this work, we compared the expression and distribution of delta-and mu-opioid receptors in the turtle and rat brains. Our results show that (1) the dissociation constant (K(d)) for delta-receptor binding was approximately four times lower and B(max) was more than double in the turtle brain homogenates than in rat ones; (2) the delta-receptor binding density was heterogeneously distributed in the turtle brain, with a higher density in the rostral regions than in the brainstem and spinal cord, and was generally much higher than in rat brains from the cortex to spinal cord; (3) the delta-opioid receptors in the rat brains were mostly located in the cortex, caudate putamen, and amygdala with an extremely low density in most subcortical (e.g., hippocampus and thalamus) and almost all brainstem regions; and (4) in sharp contrast to delta-opioid receptors, mu-opioid receptor density was much lower in all turtle brain regions compared with the rat ones. Our results demonstrate that the turtle brain is actually an organ of delta-opioid receptors, whereas the rat brain has predominantly mu-opioid receptors. Because we have recently found that delta-opioid receptors protect neurons against glutamate and hypoxic stress, we speculate that the unique pattern of delta-receptor receptor expression and distribution plays a critical role in the tolerance of turtle brain to stressful situations characterized by glutamate excitotoxicity.     

Neuropathy in the spontaneously hypertensive rat: An electrophysiological and histological study

Introduction: Hypertension is identified as a risk factor for development of polyneuropathy. In this study we examined nerve conduction and morphological alteration of peripheral nerves in spontaneously hypertensive rats (SHR). Methods: Motor nerve conduction velocity (MNCV) in the sciatic–tibial nerve and sensory nerve conduction velocity (SNCV) in the sural nerve were measured. Pathological investigations included spinal cord, dorsal root ganglion, and hindlimb nerves in SHR and Wistar–Kyoto rats (WKY) aged 4–64 weeks. Results: Blood pressure was significantly higher in SHR than WKY animals at 4 weeks and elevated further with aging. MNCV and SNCV were significantly slower in SHR compared with WKY after age 24 weeks. Prominent morphological changes in SHR nerves included axonal atrophy and myelin splitting. SHR also had endoneurial microangiopathy with reduplication of basement membrane. Conclusions: SHR showed slowed nerve conduction velocity and pathological abnormalities of hindlimb nerves. Sustained severe hypertension may cause axonal atrophy and endoneurial microangiopathy. Muscle Nerve 54 : 756–762, 2016