Miklós Palkovits

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

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

Evidence for pituitary-brain transport of a behaviorally potent acth analog

Abstract Following intrapituitary injection of 3 H-ACTH 4–9 analog, the radioactivity of various brain regions was determined in intact rats and in rats with the pituitary stalk cut one or eight days previously. The regional distribution of radioactivity in the brain was also investigated after intravenous and intrasellar administration. Intrasellar and intrapituitary administration resulted in significantly higher radioactivity levels in the brain than did intravenous injection of an equimolar dose of labeled peptide. Intrapituitary injection resulted in an uptake with clear regional differences and which was highest in the hypothalamus. Twenty four hours after stalk section the uptake of radioactivity in the hypothalamus, but not in other brain regions was markedly depressed. Hypothalamic uptake, however, was restored at eight days after stalk section. The results suggest a significant flow of radioactivity from the pituitary to the brain, particularly to the hypothalamus. Transport to the hypothalamus is presumably partly vascular via the stalk. Transport to other brain areas may occur via the cerebrospinal fluid, but a neural route cannot be excluded.

Elevated adrenaline content in nuclei of the medulla oblongata and the hypothalamus during the development of spontaneous hypertension

We have previously investigated catecholamine (CA) levels in brain nuclei involved in blood pressure regulation in various models of hypertension 15,17. The adrenaline (A) content in several nuclei of older spontaneously hypertensive rats (SH rats) was found to be higher than that of normotensive Wistar/Kyoto rats (WK rats). A markedly elevated content of this catecholamine was found in the nucleus tractus solitarius (NTS), in the medulla oblongata and in the nucleus periventricularis (NPE) and the nucleus paraventricularis (NPV) in the hypothalamus. There were no such changes however in rats with renalor DOCA/salt-hypertension at a similar phase of hypertension development. On the basis of these findings it was postulated that the Acontaining neurons in the medulla oblongata and in the hypothalamus might play a role in the development of genetic hypertension 15. The present study was undertaken to investigate how the differences in the A content of the individual nuclei are related to the early development of spontaneous hypertension. Male rats, SHR-NIH-Cpb, F32 and WK rats (Wistar/Kyoto Cpb, F6) were used, with the latter as controls. Rats were obtained from the Central Proefdieren Bedrijf, TNO, Zeist, The Netherlands. They were caged in groups of 6 and kept in the laboratory for l week. Blood pressure measurements were carried out with a tailsphygmographic method 6. All rats were given rat chow and water ad libitum. They were kept at a 14 h light, 10 h dark schedule, with lights on from 5.00 a.m. to 7.00 p.m. On the day of the experiment the rats were decapitated between 9.00 a.m. and 10.30 a.m. and the brains rapidly taken out and frozen on dry ice. Serial sections (300 #m) were cut in a cryostat at -10 °C. From the frozen sections, the following 6 nuclei were punched out according to the method of Palkovits s : the paraventricular nucleus and the periventricular nucleus from the hypothalamus; the Al-catecholaminergic cell groups (lateral reticular formation); 3 parts of the nucleus tractus solitarii complex: the nucleus tractus solitarius proper (rostral to the obex) (NTS), the Az-catecholamin-

Hypertension after localized transection of brainstem fibres.

Abstract Transections in the brainstem of the rat just lateral to the nucleus tractus solitarii (NTS) caused severe hypertension. The results of this study suggest that the fibres involved are entering the NTS laterally and that deafferentation of the cells of this nucleus results in hypertension by removing an inhibitory central control.

Distribution of a behaviorally highly potent ACTH4-9 analog in rat brain after intraventricular administration.

Distribution within the brain of a 3-fold modified ACTH4-9 analog with a remarkably potentiated behavioral activity, 4-MET (O2), 8-d-Lys, 9-Phe-ACTH4-9, was investigated. The radioactive labeled [7-3H-Phe]ACTH4-9 analog was administered intraventricularly in urethane anesthetized rats in a dose of approximately 170 ng. Total radioactivity in CSF, measured in samples drawn from the cisterna magna, decreased over the period of 0.5-4 h after injection from 51 to 2% of the injected dose. Intraventricular injection of the ACTH4-9 analog resulted in high intact peptide levels in the brain. At 2 h after injection the distribution of radioactivity over 2500 micronm and 300 micronm frontal cut brain slices was rather homogenous. Data from distribution studies over topographically defined gross brain structures indicated that the septal area, which is involved in eliciting behavioral activities of ACTH-like neuropeptides, accumulated most of the injected radioactivity per gram wet weight. The distribution profiles within the brain of the [3H]ACTH4-9 analog and [3H]Phe showed considerable differences. Uptake studies in various brain nuclei after intraventricular administration of the [3H]ACTH4-9 analog demonstrated that the greatest part of the investigated nuclei exhibited relative low or medium uptake of radioactivity. This was also true for hippocampal and thalamic nuclei, which have been suggested as effected sites of action for ACTH peptides. Very high accumulation of radioactivity occurred only in the septal nuclei, particularly the dorsal and fimbrial septal nuclei. The results indicate selective uptake of the ACTH4-9 analog in the septal area, suggesting a possible significance of this area as a site of action of ACTH neuro-peptides.

Acute and Chronic Hypertension after Lesions and Transections of the Rat Brain Stem

Publisher Summary In this chapter, the effect of damage to the nucleus tractus solitarii (NTS) on blood pressure, heart rate, and baroreceptor reflex responses is studied under acute and chronic conditions. When located at the level of the area postrema or just rostral from it, bilateral electrolytic lesions of the NTS of normotensive Wistar rats acutely cause hypertension. A slight tachycardia is observed in the first group. Extensive lesions also cause hypertension, but no change of heart rate is found. Lesions located more caudally in the NTS and in the nucleus commissuralis are ineffective. A similar type of acute hypertension occurs after bilateral transections just lateral to the NTS at the level of the area postrema. Effective transections are found to extend into the reticular formation between the NTS and the nucleus tractus spinalis n. trigemini. Bilateral transection of the tractus failed to cause hypertension. Removal of the area postrema does not affect either blood pressure or heart rate. Electrical stimulation of the NTS causes frequency dependent hypotension and bradycardia. These data may indicate that the cells of the NTS mediate a tonic inhibitory influence on blood pressure. The effect of the lesions located at the level of the area postrema is also studied in the chapter under chronic conditions.

Effect of Various Lesions in the Nucleus Tractus Solitarii of the Rat on Blood Pressure, Heart Rate and Cardiovascular Reflex Responses

The inhibitory role of the nucleus tractus solitarii (NTS) in cardiovascular control was studied in the rat. Bilateral electrolytic lesions of the dorsal medulla oblongata, located in the NTS, acutely caused hypertension and tachycardia. Effective sites were at the level of the obex and just rostral and caudal of this level. More rostrally or more caudally located lesions were ineffective in changing blood pressure and heart rate. The ascending or descending pathways from the NTS structures in the dorsal midline appear not to be essential. Frontal transections rostral of the obex or lesions and transections in the commissural part of the NTS did not block the hypertension caused by lesions at the level of the obex. The latter lesions completely blocked the reflex-induced changes of heart rate caused by administration of bradykinin, angiotensin II and the diving reflex bradycardia. The hypertension was associated with a marked increase in plasma renin activity and plasma adrenaline concentration. Adrenalec...

The spontaneously hypertensive rat: catecholamine levels in individual brain regions.

Publisher Summary This chapter summarizes the results of a study comparing catecholamine levels in punched regions from the brains of 16-week-old spontaneously hypertensive (SH) and Wistar/Kyoto strain (W/K)-rats. A sensitive radiometric method is used for the simultaneous assay of noradrenaline, dopamine, and adrenaline. The assay involves the conversion of the catecholamines to their respective tritiated methoxy derivatives by incubating them for 60 min at 37°C with S-adenosyl- l -[methyl- 3 H]-methionine in the presence of the enzyme catechol-O-methyltransferase. Subsequently, the labeled methoxy derivatives are extracted and separated by descending paper chromatography. The assay is linear up to 2 ng; the sensitivity is approximately 10 pg for all the three catecholamines. Major differences in brain noradrenaline concentrations between SH- and W/K-rats are found in the brain stem regions. In all noradrenergic cell body regions except for the locus coeruleus, the noradrenaline concentrations are found to be elevated. In addition, elevated noradrenaline levels are measured in the reticular formation and in the nucleus raphe magnus. It is of interest to note that no changes are apparent in the rostral nucleus tractus solitarii (NTS).

Hypothalamic Control of Aldosterone Production in Sodium-Deficient Rats

The effect of small bilateral hypothalamic lesions on the rate of aldosterone production by adrenal glands from sodium-deficient rats was studied in vitro. Lesions located in the me