Localization of vasopressin in the rat brain

Brain Research, 197 (1980) 75 81 ,~3 Elsewer/North-Holland Blomedtcal Press

75

L O C A L I Z A T I O N OF VASOPRESSIN IN THE RAT B R A I N

JANET HAWTHORN, VINCENT T Y. ANG and JOHN S JENKINS Department of Medtcme, St. Geolge's Hospttal Medic al School, Cranmel Tert ace, London S WI 7 ORE (U.K)

(Accepted March 13th, 1980) Key words, vasopressm --cerebral dlstrtbut~on -- radto~mmunoassay-- hypothalamus

SUMMARY The distribution ofargmlne vasopressm (AVP) m the rat brain was studied using a sensltwe radloJmmunoassay. The highest concentration of AVP was found m the hypothalamus. Individually, the supraoptic, paraventncular and suprachiasmatlc nucle~ contained in the order of 10~o of the total hypothalamic content. Vasopressln was also found in the thalamus, medulla, cerebellum, amygdala, substantm nigra and hlppocampus. Much lower levels were detected m 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 ~s of a similar nature to that of hypcthalamo-hypophyslal origin. This study supports previous reports of extrahypothalam~c localization of vasopressm by immuno-histochemical methods. It ~s clear that AVP ~s not confined to the hypothalamo-hypophysml axis, and the posslbihtJes that th~s may reflect an involvement m brain function are discussed.

INTRODUCTION There ~s evidence to suggest that vasopressln (AVP) has functions outside its classic role in salt-water homeostasis, m particular, attention has focused on the fact that vasopressin may affect brain function: ~t has been ~mphcated in learning and memory processes 15 and alterations m cerebro-spmal fluid (CSF) vasopressin that do not parallel the levels m the systemic circulation have been found m certain pathological conditions 6. A role of vasopressin in cerebral function would suggest that the hormone may be found in brain regions other than the hypothalamo-hypophysial system, and these studies have prompted investigations into the locahzatlon of vasopressm.

76 lmmunohistochem]cal methods have provided much information, but .so far there have been few reports of quantitative evaluations of vasopressm. Summy-Long ct al. 1'~ have measured AVP in the sub-fornlcal organ and hlppocampus-tbrnlx, and Dogterom et al. 3 have quoted levels of AVP in the llmb~c areas, parafascicular nucleus, cortex, choroJd plexus and medulla, but other authors were unable to detect extrahypothalamlc vasopressm 5. Vasopressm has been found in the CSF of several species ~ and measurements ofneurophysln m cerebrospinal fired suggest that this hormone has not entered via the peripheral circulation 1~ We have developed a very sensitive radlolmmunoassay for arglnlne vasopressln and have investigated the distribution of this hormone in the rat brain with a view to obtaining information on the AVP content of extra-hypothalamic areas t~l"the brain Thin layer chromatography has been used to establish the similarity of brain vasopressin with that of pituitary origin MATERIALS AND METHODS Tissue samples were obtained from male Wistar Furth rats (150--200 g). The ammals were stunned, decapitated and the brains removed into chdled petri-dishes. Dissection was carried out immediately at 4 C, taking care to remove the hypothalamus in a separate procedure due to possible contamination from the extremely high levels of AVP expected m this region. In the case of smaller regions of the brain, such as specific nuclei, thin sections of tissue were cut free-hand and dissected according to the atlas of K6mg and Khppel v using a stereomlcroscope, external landmarks, most notably the optic chiasma, were used to ensure that each brain was sectioned m a similar manner, and reproducibility of dissection was monitored by estimation of total protein, subsequently discarding any samples which had protein levels outside the limits of ~- 20°o of the average. The limited size of tissue samples from some areas necessitated tissue from 3-4 animals being pooled at this stage (n thus equals number of samples). All samples were homogemzed in cold 1 M acetic acid, centrifuged, and the supernatant removed for assay. Details of the extraction and assay procedure are as pubhshed previously ~I.Specifically, the acetic acid extracts were diluted 1:10 with 0.05 M Tns buffer, pH 7.5, and neutralized if necessary: the vasopressm was then absorbed onto Flonsd (Koch-Light or Sigma) which had previously been carefully washed and heat activated. The Florisfl was washed, acidified with HCI and the hormone eluted with 90 o~,,acetone; samples were then dried at 4 0 ' C under a stream of nitrogen. This serves to concentrate the hormone and avoid non-specific interference in the assay Dried samples were re-dissolved in Tris buffer for assay. Standard vasopressin gave 70-85 °o recovery in this procedure. Using our own antiserum in a dilution of I '60,000 the sensitivity of the assay was 0.5 pg/ml, the mtra-assay co-efficient of variation in the range 2-10 pglml was 7.5°,, and the inter-assay variation 13 o . The antiserum was highly specific for vasopressln, the cross-reaetw~ty w~th oxytocm and vasotocm being less than 0.01 ",,, Protein was determined by the method of Lowry et al. l~. Thin layer chromato-

77 graphy was performed on silica plates usmg a solvent system o f butanol:acetic a c l d : p y n d m e : w a t e r (15:3:10:6) II. The dried chromatograms were sprayed with fluorescamme and visualized under ultra-violet light 13. Areas showingfluorescence were scraped from the plate, eluted with 90°0 acetone and assayed for A V P along with several control (non-fluorescing) regions o f each plate. RESULTS The distribution of vasopressm m the brain areas studied is shown in Table I. The hypothalamus displayed the highest concentration o f this h o r m o n e and the supraoptic, paraventricular and suprachiasmatic nuclei individually contained appreciable quantities o f vasopressm, as was to be expected. The most important part of this investigation, however, was the finding o f appreciable quantities o f vasopressm in the thalamus, cerebellum, amygdala, substantia mgra, hlppocampus and medulla oblongata. Small amounts were found m the pons, spxnal cord, caudate putamen, frontal and occipital lobes. The corpus callosum and other areas o f cortical tissue did not contain detectable amounts o f AVP. Pooled extracts o f hypothalamus, temporal lobe and pituitary glands showed good parallelism with standard A V P (Fig. 1) when assayed using serial dilutions. The identity o f the vasopressin found in several brain areas was established using thin layer c h r o m a t o g r a p h y followed by radiolmmunoassay. Table II gives the Rf values for standard argmme vasopressin ( M R C ) and extracts from the hlppocampus, temporal lobe, hypothalamus, cerebellum and pituitary gland. TABLE I Dt~ trtbutton o f A VP m the rat btain

Values are given :- S.E.M. and the number of observations in parentheses Area o f rat brain

pg A VP/mg protetn

Hypothalamus (whole) Supraoptlc nucleus Paraventrlcular nucleus Suprachlasmatlc nucleus Thalamus Cexebellum Medulla Amygdala Substantla mgra Hippocampus Pons Spinal cord Occipitallobe Caudate putamen Frontal lobe Corpus callosum Cerebral cortex (anterior) Cerebral cortex (posterior)

2066.6 ~k 197 3 (6) 253.0 ± 11.3 (6) 163.7 i 27.0 (7) 100.5 ~- 26 7 (6) 53.5 ± 40 (5) 45.0 ± 6.1 (6) 40 8 -k 6.8 (6) 31.4 + 1.9 (4) 30.5 ~ 6 8 (7) 27.0 :k 2 5 (7) 12 l :k 3.2 (8) 7 8 zk 1.2 (5) 6.8 ~- 1.5 (5) 5 7 ~k 24 (9) 5.7 j_ 0 7 (6) -- 2 0 (8) ~. 2.0 (5) --- 2.0 (5)

78 9o 80 70

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lo

3'~

6'2

~5

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log AVP/m I Fig. 1. Parallelism of tissue extracts with standard vasopressm. [~-"~I]AVP bound ~s calculated as a percentage of the maximum binding capacity of the antiserum. Standard arginine vasopressin (Q) from 1.5 to 100 pg/ml is plotted on a logarithmxc scale The graph shows serial dilutions of pituitary ( ~ ) , hypothalamus (A), and temporal lobe (V) extracts

All material showing immunoreactlvlty with our AVP antiserum ~s of a smnlar nature chromatographically and identifies with standard AVP. Control (non-fluorescing) areas scraped from the plates gave no detectable actlwty m the AVP assay. Other fluorescent spots were occasionally noted but these d~d not react m the assay. [t would appear that some unidentified peptxdes are absorbed by the Flonsfl extrachon procedure, since however they do not cross-react with our antiserum their presence does not seem to be important. DISCUSSION

lmmunocytochem~cal studies have located large amounts of vasopressm m the TABLE II Thin layer eh~ omatography oJ tissue e ~:tta( t~

Values are gwen ~_ S.E.M. and the number of observations in parentheses. Sample

Rj value

Standard AVP Cerebellum Hippocampus Temporal lobe Hypothalamus Pituitary

0.638 0 62l 0.639 0.622 0.631 0 625

t: 0.007 (7) 2_ 0 027 (5) :{- 0.015 (4) :~ 0 013 (5) _L 0.018 (5) ?• 0 014 (4)

79 supraoptic, suprachmsmatic and paraventricular nuclei of the rat hypothalamusl, 2,1a, ~0 and at is generally accepted that these areas are the major sates of synthesis of the p~tuitary pool of this pept~de, as these nuclea show direct mnervat~on to the neurohypophys~s and thus prowde a route for transport of the hormone. The abundance of pept~de m these areas has also been shown using rad~olmmunoassay a,a and as further confirmed m this study. Absolute values m these two studies dafter, but the dlscrepanc~es may be attributable to very different techmques employed m obtaining t~ssue and extracting the hormone for assay as well as d~fferences m a m m a l s used. Reports of vasopressm outside the hypothalamo-hypophysial system, however, have been very limited. George and Jacobow~tz 5 were not able to detect any extrahypotbalam~c AVP m the rat brain, but it is hkely that these results would perhaps stem from the bruits of senslt~wty of their assay system rather than an absence of hormone. Using a more sensitive assay, vasopressm has been found m the sub-formcal organ, and adjacent structures of the hippocampal-formx, anterior commissure and fornix 1.~. The hormone has also been measured m the septum, organum vasculosum of the lamina termmahs, amygdala, hippocampus, cortex, choroad plexus and medulla oblongata 3. Other reports of AVP existing outside the hypothalamo-neurohypophysml complex use only hlstochemlcal techniques. Several studies have mapped the distribution of neurophysin, which as it as the career protein for vasopressin or oxytocm, would denote the presence of AVP. Neurophysm has been found m the ependymal tanycytes of the third ventNcle 1~ and neurophysm-contaimng fibres have been shown in the triangular nucleus of the septum, coursing medially from the hypothalamus m the stria termmalis to the central nucleus of the amygdala, and in the brain stem and spinal cord. Fine fibres were found ascending from the suprachiasmat~c nucleus to the medial dorsal thalamus a4. More specifically vasopressm itself has been shown to have a wide distribution m the rat brain. F~bre pathways have been demonstrated extending from the hypothalamus to the stria termmahs and stria lateralis and AVP has been located m neurosecretory fibres under the ependyma of the lateral ventricles". Vasopressm and oxytocm pathways have been shown to emanate from the paraventrlcular nucleus towards the dorsal and ventral hlppocampus, the nuclei of the amygdala, substantla mgra and substantla grJsea, nucleus tractus solitarlus, nucleus amblguus and to the substantia gelatmosa of the spinal cord. A pathway has also been shown from the suprach~asmatJc nucleus to the lateral habenular nucleus I. The high sensitivity of our assay has enabled us to provide quantitative measurements of vasopressm m some of these areas and our findings are in good agreement with the distribution of the fibre pathways. It is perhaps surprising that we have found qmte high levels of AVP ~n the cerebellum. This structure is in general relatively devoid of peptides as compared to the rest of the brain, and fibre pathways have not been reported m this region, although intense foci staining for AVP have

80 been found in the cerebellum of sheep with scraple 8. Other authors were unable to detect AVP m the cerebellum using radioimmunoassay 3 but they were using a punched sample from one hemisphere, and it may be that the hormone ~s located ~n a discrete region of this structure, and is thus only detectable ffthe whole structure Is extracted as in the present study. It is not clear if vasopressin is actively synthesized in several regions of the brain or transported from the hypothalamic area. Clearly, fibre pathways are numerous. However, there is also the possibdity that AVP is transported in the CSF. A blood-CSF barrier exists for AVP 6A7 which would preclude its ready transport within the brain via the plasma. Furthermore, behavioural effects of AVP can be demonstrated when it ~s admmistered directly into the ventncular system of the brain in far smaller doses than when ~t is given systemically1% Locahzatlon o f A V P m the tanycytes close to cerebral venmcles shows a possible pathway for entry of vasopressin into the CSF and CSF vasopressm content has been reported for several species 4,6,10. The levels of hormone m the bram areas studied however, are far above those of the CSF and would suggest that if de novo synthesis or axonal transport does not occur, there must be a selective uptake system by certain areas. The reason for th~s accumulation ofvasopressin outside the hypothalamo-hypophysial system is not clear but it may be of significance that not all areas of AVP in the brain respond to alteraticns m fired balancelL Vasopressin has been implicated in learning and memory processes is, and thus it would not be surprising that appreciable quantmes of this hormone have been found m the limbic system and m particular the hlppocampus which has long been considered to be an area involved in the memory process. Our results would lend support to th~s view that vasopressm has a role m brain function, perhaps acting like other peptides as a neurotransmltter substance, or serwng to modify neuronal function. ACKNOWLEDGEMENTS We wish to thank the Wellcome Trust for a research grant m support of J.H. and are most grateful to Dr. B. Donovan of Ferring Pharmaceuticals for supplying arginine vasopressin.

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