Neuroanatomical and biochemical evidence for the involvement of the area postrema in the regulation of vasopressin release in rats

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Brain Research, 447 (1988) 178-182 Elsevier

BRE 22856

Neuroanatomical and biochemical evidence for the involvement of the area postrema in the regulation of vasopressin release in rats Michele Iovino 1, Michele Papa 2, Palmiero Monteleone 1 and Luca Steardo 1'3 t Department of Neurology, 2rid Medical School and 2Department of Anatomy, 1st Medical School, Universityof Naples, Naples (Italy) and 3Department of Pharmacology, 2nd Medical School, University of Rome, Rome (Italy) (Accepted 19 January 1988) Key words: Area postrema; Horseradish peroxidase; Vasopressin; Urine output; Urine osmolality; Urine sodium excretion

Studies were carried out in the rat to determine if the area postrema (AP), a medullary circumventricular organ, might be involved in the control of vasopressin (VP) release. The data from this study demonstrate the existence"of direct neural connections between ihe AP attd the hypotha!ami¢ VPergic neurons of the supraoptic nucleus (SON) as showed by the retrograde tracer horseradish peroxidase (HP,P). Labeled neurons were observed in the AP following HRP injections into the SON. In addition, rats with AP lesions showed an impaired ability to conserve water and concentrate their urine in response to an hypertonic NaC! load. They, also, failed to maintain sodium retention and showed an attenuation of VP release during intracellular dehydration. These findings indicate that AP plays an important role in the regulation of VP release during changes in osmotic environment and suggest that this medullary circumventricular organ is a part of central circuitry subserving salt-water balance.

The area postrema (AP), a medullary circumventricular organ, is located at the transition of the 4th ventricle to the central canal. The AP, a small nucleus containing specialized non-ciliated cells, parenchymal cells, astrocyte-like cells and numerous synapses, is provided with a dense plexus of fenestrated capillaries 11 that are permeable to proteins and peptides that cannot enter other cerebral tissues 16'17. The A P is probably the only brain structure within the vertebral field of irrigation which lacks the bloc, dbrain barrier. Recent lesion experiments indicate that distruction of the AP in the rat causes several p e r m a n e m alterations in water balance control mechanisms 8. Rats with AP lesion become immediately polydipsic and polyuric. They show an elevated intake of salt and fail to concentrate their urine during dehydration as well. Therefore, these data may suggest that the AP might be involved in the control of vasopressin (VP) release during osmotic stimuli, In order t(J investigate this hypothesis, circulating

levels of VP were measured in normal unoperated, sham- and AP-lesioned rats both under ad libitt~m Euid intake conditions and in response to intraceilular dehydration. Additionally, the possibility that the AP may modulate the activity of the magnoceilular neurosecretory system of the anterior hypothalamus via a direct pathway was also investigated using the retrograde tracer horseradish peroxidase (HRP). For this purpose, microinjections of H R P (with tetrame~hyibenzidine (TMB) as the chromogen) were made in rats into the hypothalamic supraoptic nucleus (SON), the most important neurosecretory nucleus, at least quantitatively, in determining the output of VP into the general circulation. Consequently, sections of the A P were examined in order to verify whether cell bodies were labeled following the retrograde axonal transport of the enzyme. Male Wistar rats (initial weight 180 + 10 g) were housed in pairs in metabolic cages, located in a temperature- (22 + i °C), humidity-(55%), illumination(12/12 h light/dark cycle; lights on at 07.00 h)-con-

Correspondence: L. Steardo, Department of Neurology, 2nd Medical School, University of Naples, via S. Pansini 5, 80131 Naples, Italy.

179 trolled room. Commercial rat chow and tap water were available ad libitum, except when specifically noted. Electrolytic lesions were performed under sodium pentobarbital anesthesia (Nembutal, i.p. 40 mg/kg) using a Stoelting stereotaxic apparatus; the tip of a thorium electrode was lowered at level of the AP according to the coordinates of Pellegrino and Cushman Atlas n~ (12.4 mm posterior to bregma, and 6 mm ventral to the dorsal surface). Radiofrequency lesions were generated using a Grass LM-4 radiofrequency generator (5 mA/5 s; voltage 75 V; frequency 100 kHz). The animals were allowed a minimum of 5 days postoperative recovery before experiments were started. Urine volume, urine osmolality, urine Na + concentration, and plasma VP levels were determined also on the day preceding the beginning of the experiment in order to ascertain the baseline conditions in animal groups. On the day of experiment, groups of 8 rats received orally 2 ml/kg of a 10% NaCI water solution at 09.00 h and water was immediately withheld for 3 h. During this period, urine volume, osmolality and Na + concentration were determined in normal controls, sham- and AP-!esioned rats. In the same experimental groups, plasma VP concentration was measured at the end of the experimental procedure (12.00 h), and compared to rats receiving an isotonic NaCI solution (0.9%). Urine volume was collected in graduate cylinders; urine osmolality was measured with a Vogel osmometer OM 801 (Sedas, Italy); and urine Na ÷ concentration was determined by an Elvi 660 photometer (Elvi, Italy). Plasma VP concentrations were measured by radioimmunoassay as described elsewhere 4. An aliquot of stored plasma diluted in assay buffer (0.1 M phosphate, 0.3 NaCI and 0.1% bovine serum albumin (pH 7.6) was extracted using acetone and petroleum according to a modification of the method of Husain et al.l, The recovery of standard VP added to the plasma was 73 + 9.1%. The rabbit anti-VP antibody showed 0.081 and 0.7% cross-reactivity with oxytocin and ArgS-vasotocin, respectively. The inter- and intra-assay coefficients of variation were 7.2% and 8.4%. For HRP procedure, 16 male Wistar rats (250 + 30 g) were employed. In different experiments, injections of 0.25-10% solutions of HRP-conjugated wheat germ agglutinin ( W G A - H R P ; Sigma, St.

Louis, MO) in phosphate-buffered saline were placed stereotaxicaUy into the main portion of SON with reference to the Atlas of Pellegrino and Cushman n~ (1.4 mm anterior to bregma, 2 mm lateral to the midline and 9.5 rnm ventral to the dorsal surface). Volumes of 15-20 nl were delivered over 20 min via pressure from a micropipette syringe. Following a survival period of 24-30 h, animals were anesthetized and perfused transcardially with room temperature solutions of 0.9% saline (100 ml for 2 min), followed by 2% paraformaldehyde-0.1% glutaraldehyde in 0.1 M sodium phosphate buffer (PB) (pH 7.4) (500 mi for 20 rain), and finally with cold 10% sucrose in PB (500 ml for 20 rain). After perfusion," brains were removed and placed into a solution of cold 30% sucrose in PB at 4 °C for 12-16 h. Brains were then cut into 40~tm sections on a freezing microtome. Mesulam's TMB procedure 7 was ;ed to reveal the presence of HRP, as modified by Rye et al. 12 for stabilizing the reaction product. Labeled perikarya were examined by a light microscope under brightfield illuminations. The effects induced by AP lesions on urine volume, osmolality and Na + concentration in rats treated with a load of 10% NaCI solution are presented in Fig. 1. Salt-loaded lesioned rats excreted an increased urine volume (11 + 1.2 ml/kg), a reduced urine osmolality (1180 + 105 mOsm/kg) and increased Na + excretion (2.0 + 0.1 mEq/kg) when compared to urine volume (6.2 + 0.7 and 5.9 + 0.6 ml/kg), urine osmolality (1812 + 125 and 1821 + 122 mOsm/kg) and Na ~" excretion (1.2 + 0.2 and 1.3 + 0.15 mEq/kg) of normal controls and sham-lesioned rats. Plasma VP concentrations following the administ~'ation of an isotonic (0.9%) and an hypertonic (10%) NaCI solution in experimental animals are presented in Fig. 2. No significant difference of circulating radioimmunoassayable VP was found between the 3 experimental groups following the administration of the isotonic NaCI solution. On the other hand, after the hypertonic NaCI load, sham-lesioned and normal unoperated animals significantly increased plasma VP concentration from 2.5 + 0.4 ttU/ml and 2.3 _ 0.4 ~tU/ml to 11.3 _ 1.2 !tU/ml and 10.8 + 1.2 FtU/ml, respectively. Rats with AP lesion showed a significant (P < 0.01) attenuation of the increase in plasma VP concentration stimulated by NaCI chal-

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Fig. 1. Effect of hypertonic NaCI (10%) solution on urine volume, urine osmolality and urine Na÷ excretion in normal unoperated (O ©), sham- (O---------~) and AP-lesioned (A A ) rats. *P < 0.01 vs normal controls and sham-lesioned rats

lenge, with the control level of 2.6 + 0.5 pU/ml rising to only 6.6 + 0.7pU/ml. For the localization of the retrogradely labeled neurons, serial sections from the frontal pole of the brain to the cervical medulla were examined in all animals. The results indicate that the SON receives afferents from neurons located in the limbic system, in the circumventricular organs and in mesencephalic, pontine and bulbar regions. Since the aim of the present neurohistocbemical study was to investigate

sI° I"l !l 0.9' 10% I NaCl I normal controls

0.9~ 10% 0.9% IO% I NaCl I I NaCI _.j sham.lesioned AP-lesioned animals animals

Fig. 2. Effect of isotonic (0.9~) or hypertonic NaCI (10%) solution on plasma VP concentration in normal controls, shamand AP-iesioned rats. *P < 0.01 vs normal controls and shamlesioned animals.

whether a neural projection between AP and SON exists in rat brain, here the presence of many labeled neurons in AP following HRP injection into th.e SON is reported (Fig. 3). The histochemical evidence provided from the present investigation demonstrates the existence of direct neural connections between the AP and the hypothalamic peptidergic neurons secreting VP of the SON as showed by the retrograde tracer HRP. These neuroanatomical findings, confirming previous morphological studies 13'15 showing that efferent projections of the AP directly connect magnocel|ular neurosecretory neurons of the anterior hypothalamus, suggest that this pathway may be part of the apparatus controlling body fluid homeostasis in rats. Therefore, the present study, testing the hypothesis that AP may affect VP release, shows that AP lesions induce a clear..cut decrease of plasma VP levels in rats during osmotic stimuli. The ablation of AP attenuates VP release into the systemic circulation from the neurohypophysis by inhibiting the synthesis and/or release of VP via the interruption of information carried along the pro!, ction from the AP to the SON. The observation that VP release is blunted only during osmotic stress, but not in resting conditions~ might indicate that this medullary circumventricular organ plays an impc~rtant role during dehydration in the control of salt-water balance. Under the present experimental conditions, lesioned animals have been unable, in response to an acute intra-

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Fig. 3. Frame A (40x) and B (100x) illustrate the region of the AP at the transition from the 4th ventricle to the central canal and the presence of many labeled neurons in the body of AP. Frame C (400x) shows a stained neuronal soma in the AP with dendrites and axofl.

cellular dehydration challenge, to concentrate their urine and reduce volume losses. In addition, lesioned animals have shown an impaired ability to maintain sodium retention in response to NaCI load. These findings are well related to the blunted release of VP from the neurohypophyseal system into the systemic circulation and indicate that renal VP receptors are inadequately activated during changes in osmotic environment. O t h e r circumventricular organs ( C V O s ) , such as the subfornical organ and the o r g a n u m vasculosum laminae terminalis~ have been implicated in the regulation of VP synthesis and/or release 2'3'5'6'~']4 and have been candidated as o s m o r e c e p t o r areas for the regulation of water balance. R e m a r k a b l e anatomical evidence d e m o n s t r a t e s that A P ~'17 is e m b e d d e d with-

in a close network of C V O interconnections and suggests that the activity of A P may be closely coordinated with that of the forebrain CVOs. The existence of such a circumventricular system may have potent implications for the neurosecretory processes. Indeed, CVOs may play a pivotal role in carrying information from the periphery to specific central structures such as the SON, which is involved in the synthesis and release of VP, that is not able to directly sense changes in osmotic ~:,nvironment. The biochemical resuhs reported here support the hypothesis that A P may modulate VP release from magnocellular neurons within SON. The neurohistochemical evidence of a direct input from A F to SON provides a neural substrate for the A P homeostatic f,,mction in body fluid balance in rats.

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