Neuroscience Letters, 148 (1992) 101 104 101 ~ 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00

NSL 09152

Partial coexistence of NADPH-diaphorase and somatostatin in the rat hypothalamic paraventricular nucleus

J.R. A l o n s o a, F. Sfinchez b, R. Ardvalo ", J. Carretero b, R. Vfizqtlez b and J. Aij6n ~ ~'Department ~/' Cell Biology and Pathology and bDepartment of Human Anatomy and Histology, Universidad de Salamanca, Salamanca (Spain)

(Received 17 July 1992; Revised version received 18 September 1992: Accepted 18 September 1992)

Key words': Somatostatin; NADPH-diaphorase; Paraventricular nucleus; Hypothalamus; Coexistence: Rat

Coexistence of NADPH-diaphorase (ND) activity and somatostatin (SRIF) immunoreactivity was studied in the paraventricular nucleus (PVN) of the rat hypothalamus by successive incubations of the same sections. ND was found in all PVN subdivisions, mainly in the magnocellular ones. SRIF was practically restricted to the parvicellular periventricular subdivision. Contrary to other brain regions where a wide SRIF-ND coexistence has been observed, the periventricular parvicellular subdivision was the only place of the PVN where some neurons colocalize both markers. The combination of the immunocytochemical and the histochemical labellings allows a further permanent and easy-to-perform parcellation of periventric- ular PVN neurons.

NADPH-diaphorase (ND) is a selective histochemical marker for specific populations of neurons throughout the brain [2-4, 6-11, 15, 17-20, 22 28 among others]. This enzyme has been recently characterized as a nitric oxide synthase [7], providing, therefore, an easy and reli- able method to identify those neurons producing nitric oxide. The magnocellular hypothalamic nuclei, including the paraventricular nucleus (PVN), contain abundant ND-positive neurons scattered or grouped in dense clus- ters [3].

In other brain regions, such as the neostriatum, cere- bral cortex, and olfactory bulb, a wide or general coexis- tence between ND and somatostatin (SRIF) has been found [15, 19, 20, 25, 28]. SRIF is also present in the PVN, especially in the parvicellular periventricular sub- division [12, 16]. Although in some subdivisions of the PVN, especially in the periventricular one, the distribu- tions of ND and SRIF show, therefore, a certain degree of overlapping, whether both markers are expressed by the same neurons or, on the contrary, by two neuronal populations with similar distributions is presently un- known. Thus, we carried out a double-labelling study by successive ND histochemistry and SRIF immunocyto- chemistry of the same sections.

Four adult female Wistar rats (220-250 g b.wt.) were

Correspondence: F. Sfinchez, Dpto. Anatomia e Histologia Humanas, Facultad de Medicina, Avda. Campo Charro s/n, 37007 Salamanca, Spain. Fax: (34) 23 294559.

used. The animals were perfused under deep anaesthesia (Ketamine 50 mg/kg b.wt.) through the ascending aorta with 100 ml Ringer solution followed by 400 ml of a fixative containing 4% paraformaldehyde, and 15% satu- rated picric acid in 0.1 M phosphate buffer, pH 7.25 (PB). After perfusion, the hypothalamic regions were dis- sected out and postfixed at 4°C for a further 2-4 h in the same fixative. Thirty/Jm frontal sections were cut on a cryostat and collected in cold (4°C) PB. Free-floating sections were processed for the demonstration of ND activity following the protocol described by previous pa- pers [2, 3]. Briefly, the sections were incubated in a solu- tion made up of 0.08% Triton X-100, 1 mM reduced fl- NADPH, 0.8 mM nitroblue tetrazolium in 0.1 M Tris- HCI buffer pH 8, at 37°C for 1 3 h. All reagents were obtained from Sigma. The course of the reaction was controlled under the microscope.

When the histochemical reaction was concluded, the sections were rinsed in PB and processed for immunocy- tochemistry. The sections were incubated in anti-SRIF primary antibody [5] diluted 1:1000 in PB for 48 h at 4°C. Thereafter, the sections were processed according to the peroxidase-antiperoxidase method. Following sev- eral rinses, the sections were incubated in 0.025% 3,3'- diaminobenzidine and hydrogen peroxide in 0.1 M Tris buffer (pH 7.6). Sections were then washed in PB, mounted on gelatin-coated slides, dried overnight at room temperature, dehydrated through graded alcohols and xylene, and coverslipped with Entellan.

102

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Fig. •.NADPH-diaph•raseands•mat•statinintherathyp•tha•amicparaventricu•arnuc•eus•iv t̀hirdventric•e a:panoramlcviewofthehypotha- lamic paraventricular nucleus. Note the preferential location of ND-active neurons at the level of the posterior magnocellular subdivision (open arrow) and the SRIF-immunoreactive neurons at the level of the parvicellular periventricular subdivision arrowheads~. Some NI)-active neurons can be seen in this latter subdivision tarrows~. Bar = 500 ~m. b: in the ventral part of the parvicellular periventricular subdivision, a predominance of ND-active neurons, as well as scattered SRIF-immunoreactlve neurons ~ open arrowsl and some cells showing both labellings ~arrows were observed Bar = 200/lm. c: high magnification of the dorsal zone of the periventricular subdivision showing a clear predominance of the SR 1F-immunoreactive

neurons. Some isolated N D-active neurons can be seen ~arrowsl. Bar = 100 pro.

After incubat ion without substrate ( f l -NADPH), no residual react ion was observed. Details o f the produc- tion, specificity and character izat ion o f the an t i -SRIF

serum have been reported [5]. Controls o f the specificity o f the immunosta in ing procedure (preabsorpt ion and

substi tut ion tests) were also carried out. Preabsorpt ion o f the pr imary serum with SRIF-28 (10 nmol o f the pep- tide per 100/11 o f the diluted serum; Sigma) abolished totally the immunoreact ion . Preabsorpt ion o f the pri-

mary serum with SRIF-14 (10 nmol o f the peptide per 100/11 o f the diluted serum; Sigma) p roduced a marked decrease (>70%) o f the number o f immunos ta ined neu- rons. Substi tut ion tests with normal rabbit serum or buffer showed no residual immunoreact ivi ty.

In double-labelled sections, the blue coloured reaction o f the N D histochemistry and the b rown D A B reaction produc t o f the immunoperoxidase were clearly distin-

guishabte. The neurons that displayed both stainings showed cell bodies with both colours at the same focus- ing plane.

The number o f reacting cells showing SRIF- im- munoreactivi ty, N D activity, and coexistence was calcu-

lated with an au tomat ic image analyzer system MtP-2

(IMCO-10) . Only the cells in which the nucleus was pre- sent were considered. Numerical data are expressed as the percentage and the mean + S.E.M. The data given incorporate Abercrombie ' s correct ion [1] tbr double count ing errors. Given the scarcity o f SRIF-reac t ing neurons in the rest o f the subdivisions o f the PVN, the number o f reacting cells was exclusively considered in the parvicellular periventricular subdivision.

As in previous papers [12-14], the subdivision o f the P V N proposed by Swanson and Kuypers [2t] has been followed.

I03

ND-act ive neurons were observed in all subdivisions

o f the PVN, In the magnocel lular subdivisions they were

mainly located in the poster ior one (Fig. la). By contrast , in the parvicellular subdivisions only a few ND-s ta ined neurons were detected, especially situated in the anterior

medial and periventricular subdivisions (Fig. la-c) , Most SRIF- immunoreac t ive cells were present in the

parvicellular periventricular subdivision (Fig. la-c) . In the rest o f the subdivisions (magnocellular and parvicel-

lular) only some scattered immunos ta ined neurons were

present (Fig. la). These distributions o f N D and S R I F are superimposable with previous detailed studies for

bo th neuroact ive substances [2, 3, 12, 16]. F r o m the different subdivisions considered in the

PVN, S R I F - N D colocalization was exclusively found at the parvicellular periventricular level (Fig. l b). However,

only a neuronal subpopula t ion represented by a few neu- rons (3.39% of the stained neurons; 25 + 2.73) expressed

both markers. In this same subdivision, other cells ex-

pressing only S R I F (89.68%; 660.5 _+ 22.80) or ND-ac - tivity (6.93%: 51.3 _+ 8.49) were observed. The double- labelled cells were predominant ly located in the ventral

part o f the subdivision (Fig. lb). Thus, the double-la- belling study allows a further parcellat ion o f the neu-

ronal popula t ion o f the PVN in four different and non- homogeneous ly distributed groups: ND-posi t ive , S R I F -

positive, ND- and SRIF-posi t ive (double-labelled) and N D - and SRIF-negat ive .

In other hypotha lamic neurosecretory nuclei such as the supraopt ic and the accessory nuclei, a large g roup o f

ND-act ive neurons was observed. However, no SRIF- immunoreact ive neurons were found.

These results in the hypo tha lamus contras t with those reported in the olfactory bulb, neost r ia tum and cerebral

cortex [15, 19, 20, 25, 28], where a wide or general coexis- tence between S R I F and N D has been reported. How-

ever, there are other regions such as the midbrain and

pontine reticular fo rmat ion where ND-act ive neurons do not contain this neuropept ide [26, 28]. Thus, we can con- clude that N D - S R I F coexistence is not a general feature

for all the brain, but a regional-specific characteristic and, with the exception o f nitric oxide and citrulline - - a

side produc t in the nitric oxide synthesis - - [7], the over-

all distr ibution o f N D active neurons does not match that o f any neuroactive substance hitherto described.

However, a recent study [8] demonst ra tes that ND-act ive neurons are postsynapt ic to cort icotropin-releasing fac- tor.

To date, the distr ibution o f N D has been compared

with those o f calcium-binding proteins such as calbindin D-28k [2], with classical neurotransmit ters such as ace- tylcholine (acetylcholinesterase [4]; choline acetyltrans- ferase [18, 26-28] and G A B A [6, 10, 22]), and with neu-

ropeptides: neuropept ide Y, c-pon and avian pancreatic

polypept ide [19, 23 25], enkephalins [10], and SR1F [15,

19, 20, 25, 28]. In the hypotha lamus , the degree o f coexistence o f N D

with SRIF, as is the case for calbindin D-28k [2], vaso-

pressin and oxytocin (S~inchez et al., in preparat ion), is only partial, indicating that they are different popula-

tions; however, the number o f ND-ac t ive cells expressing

also S R I F is much lower than those colocalizing these other neuroactive substances.

The authors want to express their grati tude to Miss E.L. Shorten for kindly revising the English version of

the manuscript . This research was supported by grants

f rom the University o f Salamanca ( 'Acciones Concer- tadas ' to J .R.A.) and the D G I C y T (PB91-0424).

1 Abercrombie, M., Estimation of nuclear population from mi- crotome sections, Anat. Rec., 94 (1946) 239-247.

2 Alonso, J.R., Sanchez, F., Ardvalo, R., Carretero, J., Aij6n, J. and V~izquez, R., CaBP D-28k and NADPH-diaphorase coexistence in the magnocellular neurosecretory nuclei, NeuroReport, 3 (1992) 249 252.

3 Ardvalo, R., S~_nchez, F., Alonso, J.R., Carretero, J., Vfizquez, R. and Aij6n, J., NADPH-diaphorase activity in the hypothalamic magnocellular neurosecretory nuclei of the rat, Brain Res. Bull., 28 (1992) 599 603.

4 Caballero-Bleda, M., Fernfindez, B. and Puelles, L., Comparative mapping of acetylcholinesterase and reduced nicotinamide adenine dinucleotide diaphorase in the rabbit dorsal thalamus, Acta Anat., 140 (1991) 224-235.

5 De los Frailes, M.T., Cacicedo, L., Lorenzo, M.J., Ferntindez, G. and Sfinchez-Franco, F., Thyroid hormone action on biosynthesis of somatostatin by fetal rat brain cells in culture, Endocrinology, 123 (1988) 898-904.

6 Hedlich, A., Lfith, H.-J., Werner, L., Bfir, B., Hanisch, U. and Winkelmann, E., GABAerge NADPH-diaphorase-positive Marti- nottizellen im visuellen Cortex der Ratte, J. Hirnforsch., 31 (1990) 681 -687.

7 Hope, B.T.. Michael, G.J., Knigge, K.M. and Vincent, S.R., Neu- ronal NADPH diaphorase is a nitric oxide synthase, Proc. Natl. Acad. Sci. USA, 88 (1991) 2811 2814.

8 Knigge, K.M. and Schock, D.. Neurons in rat CNS containing NADPH-diaphorase are post-synaptic to CRF, Neurosci. Res. Commun.,10 (1992) lll 113.

9 Kuonen, D.R., Kemp, M.C. and Roberts, P.J., Demonstration and biochemical characterisation of rat brain NADPH-dependent dia- phorase, J. Neurochem., 50 (1988) 1017 1025.

10 Roberts, R.C. and Difiglia, M.. Localization of immunoreactive GABA and enkephalin and NADPH-diaphorase-positive neurons in fetal striatal grafts in the quinolinic-acid-lesioned rat neostria- turn, J. Comp. Neurol., 274 (1988) 406~,21.

11 Sagar, S.M., NADPH diaphorase histochemistry in the rabbit ret- ina, Brain Res,, 373 (1986) 153 158.

12 Sfinchez, F., Carretero, J, Riesco, J.M., Blanco, E., Juanes, J.A., Gonzfilez, R. and Vfizquez, R., Role of the paraventricular nucleus in the hypothalamic control of the release of corticotropin hormone, Anales de Anatomia, 36 (1990) 199 209.

13 Sfinchez, F., Carretero, J., S~inchez-Franco, F.. Riesco, J.M.,

] 04

Blanco, E., Juanes, J.A. and VMquez, R., Morphometric changes of specific located vasopressin-reacting parvicellular neurons in the paraventricular nucleus of the rat after adrenalectomy, Neuropep- tides, 17 (1990) 127 134.

14 Sfinchez, F., Alonso, J.R., Ar6valo, R., Carretero, J., Wizquez, R. and Aijdn, J,, Calbindin D-28K- and parvalbumin-reacting neurons in the hypothalamic magnocellular neurosecretory nuclei of the rat, Brain Res. Bull., 28 (1992) 39-46.

15 Sandell, J.H., Graybiel, A.M. and Chesselet, M.F., A new enzyme marker for striatal compartmentalization: NADPH diaphorase ac- tivity in the caudate nucleus and putamen of the rat, J. Comp. Nen- rol., 243 (1986) 326 334.

16 Sawchenko, RE. and Swanson, L.W., Immunohistochemical iden- tification of neurons in the paraventricular nucleus of the hypothal- amus that project to the medulla or to the spinal cord in the rat, J. Comp. Neurol., 205 (1982) 260-272.

17 Scherer-Singler, U., Vincent, S.R., Kimura, H. and McGeer, E.G., Demonstration of a unique population of neurons with NADPH- diaphorase histochemistry, J. Neurosci. Methods, 9 (1983) 229-234.

18 Schober, A., Brauer, K. and Luppa, H., Alternate coexistence of NADPH-diaphorase with choline acetyltransferase or somatostatin in the rat neostriatum and basal forebrain, Acta Histochem. Cyto- chem., 22 (1989) 669--674.

19 Scott, J.W., McDonald, J.K. and Pemberton, J.L., Short axon cells of the rat olfactory bulb display NADPH-diaphorase activity, neu- ropeptide Y-like immunoreactivity, and somatostatin-like im- munoreactivity, J. Comp. Neurol., 260 (1987) 378--391.

20 Sharp, F.R., Gonz,'ilez, M.F. and Sagar, S., Fetal frontal cortex transplanted to injured motor/sensory cortex of adult rats. II. VIE somatostatin, and NPY-immunoreactive neurons, J. Neurosci., 7 (1987) 3002-3015.

21 Swanson, L.W. and Kuypers, H.G.J.M., The paraventricular nu- cleus of the hypothalamus: Cytoarchitectonic subdivisions and or-

ganization of projections to the pituitary, dorsal vagal complex and spinal cord as demonstrated by retrograde fluorescence double-la- belling methods, J. Comp. Neurol., 194 (1980) 555 570.

22 Vaney, D.I. and Young, H.M, GABA-Iike immunoreactivity in NADPH-diaphorase amacrine cells of the rabbit retina, Brain Res,, 474 (1988) 380-385.

23 Villalba, R.M., Martinez-Murillo, R., Blasco, l., Alvarez, F.J. and Rodrigo, J., C-PON containing neurons in the rat striatum are also positive for NADPH-diaphorase activity. A light microscopic study, Brain Res., 462 (1988) 359 362.

24 Villalba, R.M., Rodrigo, J., Alvarez, F.J., Achaval, M. and Martinez-Murillo, R., Localization of C-PON immunoreactivity in the rat main olfactory bulb. Demonstration that the population of neurons containing endogenous C-PON display NADPH-diapho- rase activity, Neuroscience, 33 (1989) 373-382.

25 Vincent, S.R., Johansson, O., H6kfelt, T., Skirboll, L., Elde, R.R, Terenius, L., Kimmel, J. and Goldstein, M., NADPH-diaphorase: a selective histochemical marker for striatal neurons containing both somatostatin- and avian pancreatic polypeptide (APP)-like im- munoreactivities, J. Comp. Neurol., 217 (1983) 252-263.

26 Vincent, S.R., Satoh, K., Armstrong, D.M. and FiNger, H.C., NADPH-diaphorase: a selective histochemical marker for the cholinergic neurons of the pontine reticular formation, Neurosci. Lett., 43 (1983) 31 36.

27 Vincent, S.R., Steines, W.A. and FiNger, H.C., Histochemical dem- onstration of separate populations of somatostatin and cholinergic neurons in rat striatum, Neurosci. Lett., 35 (1983) l 11 114.

28 Vincent, S.R., NADPH-diaphorase histochemistry and neurotrans- mitter coexistence. In R Panula, H. P~iiv~rinta and S. Soinila (Eds.), Neurohistochemistry: Modern Methods and Applications, Alan R. Liss, New York, 1986, pp. 375 396.