Serotonin and NADPH-diaphorase in the dorsal raphe nucleus of the adult rat

ELSEVIER

Neuroscience Letters 173 (1994) 31 36

NEUROSCIENCE LEIIERI

Serotonin and NADPH-diaphorase in the dorsal raphe nucleus of the adult rat Glen Wotherspoon, Maya Albert, Marcus Rattray, John V. Priestley* Departments of Pl y~'iolo~y and Biochemistry, United Medical and Dental Schools qf Guy's and St. Thomas's Hospitals. Lamhelh P , lace Road. London SE1 7EH, UK

Received I February 1994; Accepted 24 March 1994

Abstract

NADPH-diaphorase histochemistry, employed as a marker for nitric oxide synthase (NOS), was combined with serotonin (5-HT) immunofluorescence to investigate the relationship between NOS and 5-HT in the rat dorsal raphe nucleus. Many NADPHdiaphorase labelled cells and varicose axons were observed in the nucleus. Coexistence between NADPH-diaphorase and 5-HT occurs in cells of the dorsomedial and ventromedial subgroups but not in the lateral subgroups. Coexistence was not observed in axons, but NADPH-diaphorase labelled axons contact 5-HT/NADPH-diaphorase containing cell bodies. These findings have implications for the role of nitric oxide in 5-HT pathways and for the mechanism of action of 5-HT neurotoxins. Key wor&. NADPH-diaphorase; Serotonin: Nitric oxide synthase; Dorsal raphe nucleus; Coexistence

It has long been known that neurones with high amounts of nicotinamide adenine dinucleotide phosphate diaphorase (NADPH-diaphorase) activity can be detected using a simple histochemical method [28]. The recent identification of NADPH-diaphorase as a nitric oxide synthase [10,18] means that this method allows investigations into the precise anatomical localization of cells synthesizing the putative messenger molecule nitric oxide (NO). Nitric oxide synthase is activated by increases in intracellular calcium, for example, triggered by activation of the N M D A receptor, and catalyses the synthesis of NO from k-arginine and molecular oxygen [7]. NO is rapidly diffusible and its major action is to activate soluble guanyl cyclase and thus cause an increase in cyclic G M P (cGMP) which may then regulate activity of protein kinases, phosphodiesterases and ion channels. However, NO can also react with superoxide anions (02-) to form peroxynitrite anions ( O N O O ) . These decompose to yield highly damaging hydroxyl free radicals and nitrogen dioxide. For this reason NO has been implicated in certain types of neurotoxicity [14,26]. Inhibition of NOS activity by k-NG-nitroarginine re-

*Corresponding author. Fax: (44) (71) 928-0729. 0304-3940/94157.00~ 1994 Elsevier Science Ireland Ltd. All rights reserved S S D I 0304-3940(94)00255-9

duces the neurotoxic effects on serotonin (5-hydroxytryptamine; 5-HT) neurones of certain amphetamine derivatives [2]. The locus and mode of action of NOS in this case is not known but Johnson and Ma [13] have recently reported that 5-HT and NADPH-diaphorase coexist in some raphe nuclei cell bodies. Because different subgroups within the dorsal raphe nucleus have been reported to be differentially sensitive to 5-HT neurotoxins [1,23,24,29,30], we have examined in more detail the localisation of 5-HT and NADPH-diaphorase in the rat dorsal raphe nucleus. For this analysis, N A D P H diaphorase histochemistry was combined with 5-HT immunofluorescence. Six male Wistar rats (250 300 g) were deeply anaesthetized with sodium pentobarbitone (Sagatal; 60 mg/kg) and perfused through the ascending aorta with 50 ml of vascular rinse solution (0.01 M phosphate buffer, pH 7.4 containing 0.006 M NaHCO3, 0.025% KC1 and 8% NaCI saturated with 95% OJ5% COe prior to perfusion) followed by 400 ml of 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4. The brains were removed and postfixed for 2 h at room temperature and then cryoprotected in 20% sucrose overnight at 4°C. 30/am sections were cut on a freezing microtome and collected in 0.1 M phosphate buffer, pH 7.4. Free-floating sections were pre-

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(;. Wother,spoon et al./ Neuro~ciencc Letters 173 ( 19947 31 36

incubated with 10% normal goat serum for 1 h then incubated overnight with 1:5000 rabbit anti 5-HT antibody at 4°C, washed in phosphate buffered saline (PBS) and incubated for 3 h with rhodamine-conjugated goat anti rabbit secondary antibody at room temperature. The staining characteristics of the 5-HT antibody have been described previously [8,15]. Sections were washed briefly in PBS and then either mounted for viewing or processed further for NADPH-diaphorase histochemistry. The NADPH-diaphorase reaction was demonstrated using the method of Vincent and Kimura [27]. 0.1 M phosphate buffer, pH 7.4, containing 0.3% Triton X-100, 0.1 mg/ml Nitroblue tetrazolium and 1 mg/ml flNADPH was applied to the sections, the slides coverslipped and incubated in a sealed humid chamber at 37°C for 30-60 min. Following the reaction the coverslips were removed and the slides washed in 0.1 M phosphate buffer before coverslipping in PBS/glycerol (1:3) containing DABCO (1,4-diazabicyclo-[2,2,2]octane). The sections were examined by epifluorescence and brightfield microscopy. The relationship between 5-HT and NOS containing profiles was observed by combining epifluorescence and transmitted bright-field illumination. Examination of sections immunostained for 5-HT revealed a characteristic distribution of immunostained cells in the dorsal raphe (B7 cell subgroup). At a level corresponding to A 0.7 (0.7 mm anterior to the interaural line) in the atlas of Paxinos and Watson [20], four distinct cell groups were visible (Fig. 1A). The two lateral subgroups contained large multipolar cells which were strongly immunofluorescent. The dorsomedial and ventromedial subgroups contained less strongly immunofluorescent cells and, at some levels, could be divided into a compact midline region and more dispersed lateral regions (Fig. 1A). Similar subdivisions have been reported by other workers [25,12]. NADPH-diaphorase staining showed a similar pattern to that of 5-HT in the dorsal raphe although the staining was much more variable in its intensity, being stronger in the lateral regions than the medial (Fig. 1B). Closer examination revealed coexistence of 5-HT and NADPH-diaphorase in a number of cells in the dorsomedial and ventromedial (Fig. 1C,D) subgroups. The proportion of coexistence varied slightly according to the anatomical level and subgroup (Table 1) but averaged about 31% of 5-HT cells and 67% of NADPH-diaphorase cells. No major difference was seen between the dorsomedial and ventromedial subgroups or between their midline and lateral regions. In contrast to the dorsomedial and ventromedial subgroups, no double labelling of cells was observed in the lateral subgroups (Fig. 1E,F). In this region, despite the strong intensity of NADPH-diaphorase staining, 5-HT and NADPH-diaphorase positive cells formed two distinct populations. In addition to labelled cell bodies, numerous 5-HT

immunostained or NADPH-diaphorase labelled varicose fibres were present. Coexistence in fibres was not observed, but contacts between fibres and labelled cells were common. In a few cases single NADPH-diaphorase labelled fibres were seen wrapped around NADPH-diaphorase labelled cell bodies (Fig. 2B), but more commonly labelled fibres were seen to make just one or two en passant (Fig. 2C) or terminal (Fig. 2D,E) contacts on labelled cells. Contacts between NADPH-diaphorase labelled fibres and NADPH-diaphorase/5-HT double labelled cells (Fig. 2A E) were equally common in both the dorsomedial and ventromedial subgroups. Contacts between NADPH-diaphorase labelled fibres and 5-HT singly labelled cells were difficult to identify but a few examples were observed in the lateral subgroups (Fig. 2F, G). Our results on the distribution of NADPH-diaphorase labelled cells and 5-HT immunoreactive cells confirm and extend the findings of other workers [27,13]. NADPH-diaphorase and 5-HT coexist in neurones of the dorsal raphe nucleus, but coexistence is largely restricted to the midline subgroups. Our results are similar to those of Johnson and Ma [13] who used a different double labelling procedure, namely NADPH-diaphorase histochemistry combined with ABC (avidin/biotin/peroxidase) immunocytochemistry for 5-HT. They report that approximately 70% of 5-HT cells in the medial dorsal raphe nucleus exhibit NADPH-diaphorase activity while less than 10% of cells in the lateral subgroups show coexistence. All their quantitation was carried out in young rats (postnatal P2-P7). Our results show that a similar difference in coexistence between the midline and lateral subgroups applies in the adult rat brain and also give some more information regarding the distribution of double labelled cells. Double labelled cells appear to be evenly distributed throughout the dorsomedial and ventromedial subgroups. In contrast the large, intensely fluorescent, cells in the lateral subgroups never show coexistence. The difference of detail between the percentage coexistence that we observed and that of Johnson and Ma [13] is probably mainly due to the different methods employed and to a difference in definition of dorsal raphe subgroups. However, there may also be a developmental change in the degree of coexistence. A quantitative developmental study employing dual cotour immunofluorescence to localise NOS and 5-HT would clarify this issue, as well as confirming that NADPHdiaphorase activity in the dorsal raphe is equivalent to NOS. No previous studies examined NADPH-diaphorase labelled axons and terminals in the dorsal raphe nucleus. Our results show that many NADPH-diaphorase containing varicose fibres are present and that they contact the population of cells containing both 5-HT and NADPH-diaphorase. Contacts on the 5-HT single labelled population are less evident. Although we have not carried out electron microscopic studies to establish

G. Wotherspoon et al. / Neuroseience Letters 173 (1994) 31-36

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Fig. 1. Low l A,B) and high (C,D) magnification micrographs of 5-HT/NADPH-diaphorase double labelled sections in the region of the dorsal raphe nucleus (B7 5-HT cell group). 5-HT immunofluorescence (A,C.E) and NADPH-diaphorase histochemistry (B,D,F). A,B: 5-HT immunoreactive cells can be seen in four distinct subgroups of the dorsal raphe nucleus (A) namely a dorsomedial subgroup (asterisk), ventromedial subgroup (star) and lateral groups (arrows). The boundaries of the dorsomedial and ventromedial subgroups are marked. At this level of the nucleus, the dorsoventral subgroup consists of a compact midline region and more dispersed lateral regions. Cells that express NADPH-diaphorase are present in all subgroups and regions (B). C+D: midline ventromedial subgroup of the dorsal raphe. In this region many of the NADPH-diaphorase labelled cells also contain 5-HT immunoreactivity. Arrows indicate double labelled cells with intense NADPH-diaphorase reaction. Open arrows indicate double labelled cells with light NADPH-diaphorase reaction. E,F: lateral subgroup of the dorsal raphe nucleus. Although many 5-HT immunofluorescent cells can be seen in the vicinity of strongly labelled NADPH-diaphorase positive cells, there is no coexistence. Arrows indicate the position of NADPH-diaphorase singly labelled cells. Bar = 200 ¢tm (A,B); 50 ~m (C F)+

34

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114'~Hlerg~ooHcg ,t/ /~euro.wiemu Letlcr,s' 1 73 1994; 3l 36

Fig. 2. High magnification micrographs of 5-HT/NADPH-diaphorase double labelled sections showing contacts between NADPH-diaphorase labelled fibres and cell bodies in the dorsal raphe nucleus. 5-HT immunofluorescence (A,F) and NADPH-diaphorase histochemistry (B-E,G). A,B: two 5-HT/NADPH-diaphorase double labelled cells (1, 2) and two 5-HT single labelled cells (3, 4) are shown viewed using either epifluorescent (A) or transmitted bright field (B) illumination. The cells are in the dorsomedial subgroup. The cell body and proximal dendrite of the 5-HT/NADPHdiaphorase labelled cell #l can be seen in (B) to be wrapped around by a NADPH-diaphorase labelled varicose fibre. Arrows indicate possible contacts between the fibre and the cell. C E: NADPH-diaphorase contacts on 5-HT/NADPH-diaphorase double labelled cells. The cells are shown viewed using transmitted bright field illumination. Epifluorescence illumination confirmed that the cells express 5-HT in addition to NADPH-diaphorase. C: a cell body (star) in the ventromedial subgroup is shown labelled (or NADPH-diaphorase. A NADPH-diaphorase labelled fibre with two varicosities (arrows) contacts the cell body. D,E: two focal depths through a cell body (star) in the dorsomedial subgroup labelled for NADPHdiaphorase. A NADPH-diaphorase labelled fibre makes two contacts (arrows) with the cell body. F,G: a group of 5-HT single labelled cells are shown viewed using either epifluorescent (F) or transmitted bright-tield (G) illumination. The cells are m one of the lateral subgroups. Three sample cells (1 3) are labelled in F. The cell body of cell 1 can be seen m (G) to be contacted by a fine NADPH-diaphorase labelled varicose fibre lsmall arrowsL A second, large, NADPH-diaphorase labelled fibre Iopen arrows in F, Gt crosses the group of cells but does nol contact them. Bars = 50/Ira (A,B,f=,G): 25/nn (C E).

whether NADPH-diaphorase varicosities synapse with t a r g e t cells, t h e d i f f u s a b l e n a t u r e o f n i t r i c o x i d e m a k e s a f u n c t i o n a l i n t e r a c t i o n h i g h l y likely. T h e o r i g i n o f the N A D P H - d i a p h o r a s e l a b e l l e d fibres is u n k n o w n , b u t the fact t h a t t h e y d o n o t c o n t a i n 5 - H T m a k e s it u n l i k e l y that t h e y are s i m p l y local a x o n c o l l a t e r a l s . O n e p o s s i b i l i t y is t h a t t h e y are d e r i v e d f r o m the N O S / c h o l i n e r g i c cell

g r o u p s e i t h e r in the a d j o i n i n g d o s o l a t e r a l a n d p e d u n c u l o p o n t i n e t e g m e n t a l nuclei o r in the b a s a l f o r e b r a i n . T h e s e r e g i o n s are k n o w n to h a v e p r o j e c t i o n s to t h e d o r sal r a p h e [25,12]. R e l a t i v e l y few s u b s t a n c e s h a v e b e e n r e p o r t e d to c o e x ist w i t h 5 - H T in d o r s a l r a p h e n e u r o n e s [5,9,19,21] a n d N A D P H - d i a p h o r a s e is a d d i t i o n a l l y u n u s u a l in b e i n g re-

G. Wotherspoon el aL / Neuroscience Letters 173 (1994) 31 36

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Table 1 N u m b e r s of cells expressing 5-HT and/or N A D P H - d i a p h o r a s e ( N A D P H - D ) at various levels of the dorsal raphe nucleus % 5-HT cells containing NADPH-D

% N A D P H - D cells containing 5-HT

26 57 I)

34 29 0

77 76 0

48 88 26

28 51 0

32 38 0

60 65 I)

57 76 85

18 39 12

12 25 0

23 33 0

67 65 II

633 2 l4

299 50

199 0

31 0

67 II

N u m b e r of labelled cells

Anatomical level

Subgroup

A 1.2

dorsomedial ventromedial lateral

75 196 76

33 73 12

A 0.7

dorsomedial vent romedial lateral

99 130 53

A 0.2

dorsomedial ventromedial lateral

Total

medial lateral

5-HT

NADPH-D

5-HT + N A D P H - D

stricted to specific subgroups. The function of NOS in these 5-HT cells is not known but there are several possibilities. NO is a potent vasodilator and 5-HT containing dorsal raphe neurones are thought by some authors to innervate the cerebral vasculature [6]. Recently this view has been challenged [17] but it remains an open question and a role for 5-HT/NO in control of cerebral blood flow must be considered. Another possibility is that NO acts as an intercellular messenger [7,26], released either locally from cell bodies or in the target regions of ascending serotonergic projections. The dorsal raphe shows a topographical organization such that different subgroups project to different forebrain areas. This is most evident in the rostrocaudal organization of the nucleus [11] but there are also distinctions between the medial and lateral subgroups [4,22]. It will be important to determine whether the coexisting 5-HT/NOS cell population contributes only to certain ascending pathways. NOS has been implicated in both mechanisms of, and protection against, neurotoxicity [14,26]. The mechanism of action of monoamine neurotoxins is not clear [16], but recent studies indicate that NOS may play a role. It has been shown that inhibitors of NOS can block the neurotoxic effects of pCA and fenfluramine on 5-HT neurones [6] and of methamphetamine on dopaminergic neurones [3]. The location of the NOS responsible is not known but our results indicate that several different sites need to be considered. These include NOS in both serotonergic neurones and neighbouring non serotonergic neurones within the raphe nuclei, and NOS in terminals which innervate 5-HT/NADPH-diaphorase containing dorsal raphe cells. One interesting and unexplained feature of 5-HT neurotoxins is that different raphe nuclei [16] and even different subgroups within a single nucleus [2,23,24,29,30] show different degrees of vulnerability. Studies on several 5-HT neurotoxins have reported a

larger decrease in tryptophan hydroxylase (TrpOH) protein [29] and in serotonin transporter m R N A [23,30] in the ventral subgroup than in the dorsal or lateral subgroups. In contrast 5,7-dihydroxytryptamine causes a complete loss of both TrpOH m R N A [1] and 5-HT immunoreactivity [24] in the dorsal subgroup, yet no change [24] or even an increase [1] in the ventral subgroup. However, we observed no difference between the dorsomedial and ventromedial subgroups in either 5HT/NADPH-diaphorase coexistence or in contacts made by NADPH-diaphorase fibres. Thus for the dorsal raphe subgroups, as for the raphe nuclei generally [14], there seems to be no simple correlation between NADPH-diaphorase distribution and vulnerability to neurotoxins. Our study has identified several possible sites of interaction between 5-HT and NOS in dorsal raphe neurones. Further work is required to understand the role of NO in serotonergic function or disfunction and to determine the significance of the fact that it is present in only a subpopulation of 5-HT neurones. The financial support of the Medical Research Council (UK), the Wellcome Trust and St. Thomas's Hospital Research Endowments ( L o n d o n ) i s gratefully acknowledged. We acknowledge also generous provision of 5-HT antiserum by Dr. T. G6rcs (Budapest). [1] Bendotli, C., Servadio, A.. Forloni, G., Angereni, N. and Samanin, R., Increased tryptophan hydroxylasc m R N A in raphe serotonergic neurons spared by' 5,7-dihydroxylryptanmle, Mol. Brain Res.. 8 (1990) 343 348. [2] Benmansour, S. and Brunswick. D.J., Protection of amphetaminederivative-induced neuroloxicily to serotonm neurons by nitric oxide (NO) synthesis inhibition or N-methyl-[>aspartate ( N M D A ) antagonism, Soc. Ncurosci. Abstr.. 18 (1992l 913. [3] Clikeman, J.A.. Wei, S., Turkanis, S.A. and Finnegan, K T . , lnhibitors of nitric oxide synthesis reduce the ncurotoxic effects of m e t h a m p h e t a m i n e in mice, Soc. Neurosci. Abstr., IS (1992) 91~.

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G. ~ b t h e r . q ) o o n el a]. / N e u r o s c i e m ( ~ l , e t t e r s 173 ~ 1004 j 3 t

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