Dirk H.G. Versteeg

Regional concentrations of noradrenaline and dopamine in rat brain

The concentrations of noradrenaline and lopamine of 92 brain regions have been measured by a radiometric method which enabled discrimination between noradrenaline and adrenaline. Almost all brain regions investigated contained both noradrenaline and lopamine in measurable amount. However, both catecholamines appeared to be unevenly distributed. Very high dopamine concentrations were measured in the olfactory tubercle, the nucleus accumbens, the caudate nucleus and the rostral part of the medial forebrain bundle; the globus pallidus, the nucleus tractus diagonalis and the nucleus septalis lateralis were also very rich in dopamine. Outside the telencephalon the dopamine concentrations were rather low, except in the median eminence and the area tegmentalis ventralis (Tsai), an area corresponding to the A10 region. High noradrenaline concentrations were measured in most hypothalamic nuclei. Relatively high concentrations of this catecholamine were also measured in several mesencephalic (the ventral part of the central gray, the nucleus raphe dorsalis and the nucleus cuneiformis) and pontine (the locus coeruleus and the nuclei parabrachiales) regions. The highest noradrenaline concentrations in the medulla oblongata were observed in the A2 region and the nucleus commissuralis, which contained at least twice as much noradrenaline as did the more rostral part of the nucleus tractus solitarii.

Effect of oxytocin and vasopressin on memory consolidation: sites of action and catecholaminergic correlates after local microinjection into limbic-midbrain structures

The effects of local postlearning microinjections of arginine-vasopressin (AVP) and oxytocin (OXT) on one-trial learning passive avoidance behavior and the influence of AVP on alpha-MPT-induced disappearance of norepinephrine (NE) and dopamine (DA) in discrete brain regions have been studied in the rat. OXT injected bilaterally in the hippocampal dentate gyrus (25-25 pg) or in the midbrain dorsal raphe nucleus (50 pg) significantly attenuated passive avoidance behavior. Facilitation of passive avoidance behavior was observed when the peptide was injected into the dorsal septal nucleus. AVP facilitated passive avoidance behavior when administered into the hippocampal dentate gyrus, dorsal raphe nucleus or dorsal septal nucleus. Injection of either neuropeptides into the central amygdaloid nucleus appeared to be ineffective. One week after the behavioral experiments a repeated injection of AVP into the hippocampal dentate gyrus increased the disappearance of NE in the dentate gyrus and in the nucleus ruber. An injection into the dorsal septal nuclei decreased the NE disappearance in the dorsal septal nucleus itself and increased it in the nucleus ruber. Injection in the dorsal raphe nucleus led to an increase in the disappearance of DA in the locus coeruleus and in the nucleus ruber. It is concluded that memory consolidation can be oppositely influenced by local application of minute amounts of either OXT or AVP into certain limbic-midbrain structures, suggesting an involvement of these brain regions in the memory effects of these peptides. Modulation of catecholamine turnover in specific brain areas after AVP administration may be related to this behavioral effect.

Regional distribution of adrenaline in rat brain

The presence of adrenaline in the mammalian central nervous system has long been recognized 2,12. A first attempt to study the regional distribution in the brain was made by Vogt 11 using a bioassay in combination with a paper chromatographic separation. Vogts conclusion, that the adrenaline concentration in the brain is low relative to that of noradrenaline, was later substantiated by many authors using spectrofluorometric assay methods (for review, see ref. 3). However, these spectrofluorometric methods are not sensitive enough to enable the measurement of adrenaline in small brain regions, unless tissues of many animals are pooled 5. Although the histochemical fluorescence method for the visualization of catecholamines is more sensitive, it lacks the specificity for the discrimination between adrenaline and noradrenaline. Biochemical 9 and immunohistochemical 4 studies have indicated that phenylethanolamine-N-methyltransferase (PNMT), the enzyme which catalyzes the conversion of noradrenaline into adrenaline, shows a distinct regional distribution in the brain. Such findings support the contention that adrenaline may act as a transmitter in certain regions of the central nervous system. Recently, Koslow and Schlumpf 6, using a gas chromatographic-mass spectrometric technique, were able to detect adrenaline in 5 rat brain regions out of the 15 which were investigated. In this communication we present data, obtained with a radiometric method for the simultaneous assay of adrenaline, noradrenaline and dopamine in combination with a micropunch technique for the removal of rat brain nuclei s, indicating that adrenaline is unevenly distributed in rat brain and that, in general, this distribution is consistent with that of PNMT4, 9. The data on noradrenaline and dopamine concentrations will be presented in a separate paper 10. Male rats weighing 180-220 g were used. The animals were killed by decapitation. The brains were rapidly taken out and frozen on dry ice. Serial sections of 300 gm were cut in a cryostat at -10 °C. Individual brain regions were removed with small punches s. Tissue pellets of similar regions of three rats were pooled. Ninety two

Arginine8-vasopressin affects catecholamine metabolism in specific brain nuclei.

Abstract Following the i.c.v. administration of arginine8-vasopressin (30 ng in 1 μl saline) to rats that had been injected i.p with α-MPT 30 min prior to the administration of the peptide catecholamine metabolism was altered in a restricted number of brain nuclei. Noradrenaline disappearance was accelerated as compared to saline treated controls in the dorsal septal nucleus, the anterior hypothalamic nucleus, the medial forebrain bundle, the parafascicular nucleus, the dorsal raphe nucleus, the locus coeruleus, the nucleus tractus solitarii and the Al-region. In the supraoptic nucleus and the nucleus ruber a decreased noradrenaline disappearnce was observed after the administration of the peptide. Dopamine disappearance was accelerated in the caudate nucleus, the median eminence, the dorsal raphe nucleus and the A8-region. These results support the view that vasopressin is participating in the regulation of a variety of physiological processes by modulating neurotransmission in specific brain nuclei.

Phosphorylation of B-50 (GAP43) is correlated with neurotransmitter release in rat hippocampal slices

Abstract: Recent studies have demonstrated that phorbol diesters enhance the release of various neurotransmitters. It is generally accepted that activation of protein kinase C (PKC) is the mechanism by which phorbol diesters act on neurotransmitter release. The action of PKC in neurotransmitter release is very likely mediated by phosphorylation of substrate proteins localized in the presynaptic nerve terminal. An important presynaptic substrate of PKC is B‐50. To investigate whether B‐50 mediates the actions of PKC in neurotransmitter release, we have studied B‐50 phosphorylation in intact rat hippocampal slices under conditions that stimulate or inhibit PKC and neurotransmitter release. The slices were labelled with [32P]orthophosphate. After treatment, the slices were homogenized, B‐50 was immunoprecipitated from the slice homogenate, and the incorporation of 32P into B‐50 was determined. Chemical depolarization (30 μM K+) and the presence of phorbol diesters, conditions that stimulate neurotransmitter release, separately and in combination, also enhance B‐50 phosphorylation. Polymyxin B, an inhibitor of PKC and neurotransmitter release, decreases concentration dependently the depolarization‐induced stimulation of B‐50 phosphorylation. The effects of depolarization are not detectable at low extracellular Ca2+ concentrations. It is concluded that in rat hippocampal slices B‐50 may mediate the action of PKC in neurotransmitter release.

Catecholamine content of individual brain regions of spontaneously hypertensive rats (SH-rats)

Many reports have contributed to the notion that catecholamine neurons in the brain stem participate in the central regulation of arterial blood pressure (for reviews see refs. 2, 6). In recent years considerable effort has been made to relate the increase in blood pressure of spontaneously hypertensive rats (SH-rats 5) to catecholamine metabolism in the brain. Initially, a lower noradrenaline level and aromatic amino acid decarboxylase activity 2a, and also a decreased noradrenaline synthesis 9, were observed in the brain stem of SH-rats compared to that of normotensive controls. However, in later studies, in which SH-rats were compared with rats of the genetically related normotensive Wistar-Kyoto strain (W/K-rats), these findings were not corroborated2L Moreover, considerable differences in the activity of catecholamine synthesizing enzymes occurred in the brain stem of various strains of rats with substantial differences in bloodpressure level 10, whereas no correlation was evident between the activity of any of these enzymes and blood pressure 10. Although the latter observations seem to argue against the existence of obvious correlates, it might be that the occurrence of changes in catecholamine content and metabolism in relatively small brain regions escaped detection in these studies, a possibility which was also suggested by Yamabe et al.2L In fact, it was observed in our laboratory that noradrenaline levels were slightly elevated in the pons-medulla of recent generation SH-rats compared to those of W/K-rats 7 and 10 weeks after birthL The present study was undertaken to investigate this phenomenon in more detail. Using a sensitive radiometric method for the simultaneous assay of noradrenaline, dopamine and adrenaline 20, we measured the catecholamine content of individual nuclei and brain regions of SH-rats and W/K-rats. Male Wistar-Kyoto-NIH Cpb (F6) and SHR-NIH Cpb (F32) were obtained from TNO Zeist (The Netherlands) at an age of 14 weeks (for genealogy see ref. 5). Systolic blood pressure measurements were carried out with a tail-plethysmographic method on trained conscious rats s. The rats were killed by decapitation at an age of

Decreased serotonin turnover in the dorsal hippocampus of rat brain shortly after adrenalectomy : Selective normalization after corticosterone substitution

Pargyline-induced accumulation of serotonin (5-HT) was used as an index of 5-HT turnover rate in the dorsal hippocampus. One hour after bilateral removal of the adrenals, 5-HT turnover was significantly reduced when compared to that of the sham-operated controls. A low dose of corticosterone given immediately after adrenalectomy restored the 5-HT response, while the same dose of dexamethasone was ineffective. Pretreatment with dexamethasone blocked the 5-HT response to corticosterone in the acutely adrenalectomized rat. The specificity of the 5-HT response in the hippocampus corresponds to the properties of the glucocorticoid receptor system in rat hippocampal neurons.

Facilitation of memory consolidation by vasopressin: Mediation by terminals of the dorsal noradrenergic bundle?

Administration of arginine-vasopressin (AVP, 5 micrograms, s.c.) immediately after the learning trial results in a long-term facilitation of a one-trial learning passive avoidance response. This effect of AVP is absent in animals with prior destruction of the ascending dorsal noradrenergic bundle by bilateral microinjection of 6-hydroxydopamine (6-OHDA). Postlearning local microinjection of a minute amount of AVP via chronically implanted cannulae into the locus coeruleus did not influence passive avoidance behavior. Upon injection into the midbrain dorsal raphe nucleus, however AVP facilitated passive avoidance behavior. This effect, however, was absent in rats receiving previous microinjection of 5,6-dihydroxytryptamine (5,6-DHT) or of 6-OHDA into the dorsal raphe nucleus. Bilateral 6-OHDA-induced lesions of the nucleus accumbens or 5,6-DHT-induced destruction of the dorsal raphe nucleus did not prevent the effect of AVP administered subcutaneously. The data suggest that vasopressin facilitates memory consolidation processes by modulating noradrenergic neurotransmission in terminals of the dorsal noradrenergic bundle. The serotoninergic neuronal network originating from the dorsal raphe nucleus has a secondary--norepinephrine-mediated--influence upon these processes.

Aldosterone blocks the response to corticosterone in the raphe-hippocampal serotonin system

The accumulation of serotonin induced by the monoamino oxidase inhibitor pargyline was used as an index for 5-HT turnover in the dorsal hippocampus and raphe area. A low dose of corticosterone administered s.c. immediately after adrenalectomy significantly increased serotonin turnover in both regions over the subsequent 1 h interval. The same dose of aldosterone was ineffective, but pretreatment with aldosterone blocked the serotonin response to corticosterone in the acutely adrenalectomized rat. [3H]Corticosterone administered to adrenalectomized rats was not retained by cell nuclei of the raphe area in a limited capacity manner as occurred in the hippocampus. Pretreatment with aldosterone blocked the uptake of [3H]corticosterone in hippocampal cell nuclei. It is concluded that corticosterone triggers a serotonin response and that the specificity of the corticosterone action suggests involvement of the steroid receptor system located postsynaptically to the raphe-hippocampal serotonin projection.

Melanocortins and cardiovascular regulation.

The melanocortins form a family of pro-opiomelanocortin-derived peptides that have the melanocyte-stimulating hormone (MSH) core sequence, His-Phe-Arg-Trp, in common. Melanocortins have been described as having a variety of cardiovascular effects. We review here what is known about the sites and mechanisms of action of the melanocortins with respect to their effects on cardiovascular function, with special attention to the effects of the gamma-melanocyte-stimulating hormones (gamma-MSHs). This is done in the context of present knowledge about agonist selectivity and localisation of the five melanocortin receptor subtypes cloned so far. gamma2-MSH, its des-Gly12 analog (= gamma1-MSH) and Lys-gamma2-MSH are 5-10 times more potent than adrenocorticotropic hormone-(4-10)(ACTH-(4-10)) to induce a pressor and tachycardiac effect following intravenous administration. The Arg-Phe sequence near the C-terminal seems to be important for full in vivo intrinsic activity. Related peptides with a C-terminal extension with (gamma3-MSH) or without the Arg-Phe sequence (alpha-MSH, as well as the potent alpha-MSH analog, [Nle4,D-Phe7]alpha-MSH), are, however, devoid of these effects. In contrast, ACTH-(1-24) has a depressor effect combined with a tachycardiac effect, effects which are not dependent on the presence of the adrenals. Although the melanocortin MC3 receptor is the only melanocortin receptor subtype for which gamma2-MSH is selective, in vivo and in vitro structure-activity data indicate that it is not via this receptor that this peptide and related peptides exert either their pressor and tachycardiac effects or their extra- and intracranial blood flow increasing effect. We review evidence that the pressor and tachycardiac effects of the gamma-MSHs are due to an increase of sympathetic outflow to the vasculature and the heart, secondary to activation of centrally located receptors. These receptors are most likely localised in the anteroventral third ventricle (AV3V) region, a brain region situated outside the blood-brain barrier, and to which circulating peptides have access. These receptors might be melanocortin receptors of a subtype yet to be identified. Alternatively, they might be related to other receptors for which peptides with a C-terminal Arg-Phe sequence have affinity, such as the neuropeptide FF receptor and the recently discovered FMRFamide receptor. Melanocortin MC4 receptors and still unidentified receptors are part of the circuitry in the medulla oblongata which is involved in the depressor and bradycardiac effect of the melanocortins, probably via interference with autonomic outflow. Regarding the effects of the gamma-MSHs on cortical cerebral blood flow, it is not yet clear whether they involve activation of the sympathetic nervous system or activation of melanocortin receptors located on the cerebral vasculature. The depressor effect observed following intravenous administration of ACTH-(1-24) is thought to be due to activation of melanocortin MC2 receptors whose location may be within the peripheral vasculature. Melanocortins have been observed to improve cardiovascular function and survival time in experimental hemorrhagic shock in various species. Though ACTH-(1-24) is the most potent melanocortin in this model, alpha-MSH and [Nle4,D-Phe7]alpha-MSH and ACTH-(4-10) are quite effective as well. As ACTH-(4-10) is a rather weak agonist of all melanocortin receptors, it is difficult to determine via which of the melanocortin receptors the melanocortins bring about this effect. Research into the nature of the receptors involved in the various cardiovascular effects of the melanocortins would greatly benefit from the availability of selective melanocortin receptor antagonists.