Vasotocinergic innervation of the septal region in the Japanese quail: sexual differences and the influence of testosterone. C. Viglietti-Panzica 1, G.C. Anselmetti ...
Cell Tissue Res (1992) 267:261 265
Cell&Tissue Research 9 Springer-Verlag 1992
Vasotocinergic innervation of the septal region in the Japanese quail: sexual differences and the influence of testosterone C. Viglietti-Panzica 1, G.C. Anselmetti 1, J. Balthazart 2, N. AstC'
2
and G.C. Panzica t
1 Department of Human Anatomy and Physiology, Section Neuroanatomy and Neuroembryology, University of Torino, c.so M. D'Azeglio 52, 1-10126 Torino, Italy 2 Laboratory of General and Comparative Biochemistry, University of Li6ge, Li6ge, Belgium Received August 26, 1991 / Accepted September 27, 1991
Summary. Vasotocin (VT)-immunoreactive fibres were observed in the nuclei of the quail (Coturnix coturnix japonica) septal region. Their distribution in the nucleus septalis lateralis (SL) and the nucleus striae terminalis (nST) was sexually dimorphic: a dense network of immunoreactive fibres was seen in adult sexually stimulated males but not in females. Experimental manipulation of the hormonal environment influenced this distribution only in males. VT immunoreactivity was absent in SL and nST when male quail were exposed to a shortday photoperiod or castrated. The immunoreactivity was restored to its original level in castrated males by silastic implants of testosterone.
Key words: Sexual dimorphism - Vasotocin - Testosterone Nucleus septalis lateralis - Nucleus striae terminalis - Quail
rat and the canary, this innervation is dependent on gonadal hormones (De Vries etal. 1985; Voorhuis etal. 1988); it is reduced in gonadectomized males and is restored by treatment with testosterone (T). The Japanese quail is a useful model for the study of sexual behaviour and the underlying neural plasticity (Panzica et al. 1990). The VT/VP telencephalic innervation is present in those vertebrate brain areas intimately involved in the control of male copulatory behaviour (e.g., the nucleus striae terminalis or the amygdala) and is regulated by steroid hormones in a manner similar to the regulation of behaviour in the male. Therefore, in the present study, we examined (1) the VT-innervation of the septal complex in quail and (2) the possible existence of sexual differences and steroid control.
Materials and methods Animals and treatments
The topographical distribution of vasotocin (VT)-immunoreactive neurons and fibres has been studied in the diencephalon of several avian species (for references, see Korf et al. 1988). However, the extradiencephalic distribution of the avian VT-ergic system has been described in only a few species studies. Immunopositive fibres are observed within the archistriatum, the lateral septum, the mesencephalon, the pons, and the spinal cord, whereas immunoreactive cell bodies have been found in some mesencephalic and telencephalic locations (for references, see Kiss et al. 1987; Panzica et al. 1988). The septal complex appears to be a phylogenetically stable target for the VT/vasopressin (VP) system in vertebrates (toad: Jokura and U r a n o 1985; lizard: Stoll and Voorn 1985; Thepen et al. 1987; canary: Voorhuis et al. 1988; rat: De Vries 1985; De Vries et al. 1985; mouse: Shaw et al. 1987). A peculiarity of this projection is represented by a sexual dimorphism in its innervation, which is always denser in males than in females. In the
Offprint requests to .' C. Viglietti-Panzica
This study was performed on 36 adult (18 males and 18 females) Japanese quail (Coturnix coturnix japonica) bought from a local breeder at the age of 3 weeks. Immediately after their arrival in the laboratory, the birds were randomly assigned to one of the following experimental groups. Short day: 5 males (MSD) and 4 females (FSD) were exposed to light for 6 h per day (6L:18D), a condition in which they do not reach full sexual maturation (Balthazart et al. 1979). Long day: 5 males (MLD) and 6 females (FLD) were exposed to a photoperiod of 16 h light each day (16L:8D); birds treated this way reach full sexual maturation within 6 weeks of age (Ottinger and Brinkley 1979). The other 8 males and 8 females were gonadectomized as previously described (Panzica et al. 1991) and maintained under a long-day photoperiod (16L:8D). Starting 9 days later, these gonadectomized birds were subdivided in two groups that were treated with T [60 mm silastic implants filled with crystalline T; 5 males (MT) and 4 females (FT)] or received empty silastic implants as controls [3 males (MCX) and 4 females (FOVX); see Panzica et al. ]991 for details of the procedures]. The masculine sexual behaviour of all birds was quantified during a 3-min test (see Panzica et al. 1991 for details). After the test, the area of the cloacal gland was measured with a caliper to the nearest millimeter (area=largest length x largest width). The birds were killed and the brains were removed for histology.
262
Imrnunohistochernistry The birds were transcardially perfused with a saline solution followed by Bouin's fixative without acetic acid. Brains were postfixed for 48 h at 4~ C in the same fixative, embedded in Histosec (Merck, Darmstadt, FRG) and serially cut in the coronal plane at a thickness of 10 gm. One section every 100 gm was selected for processing for VT-immunohistochemistry(anti-VT at a dilution of 1 :4000 ; the antibody was a gift of D.A. Gray, Bad Nauheim, FRG) according to our previously described protocol (Viglietti-Panzica 1986). The specificity of the reaction was verified by omitting the anti-VT, or by using a preadsorbed antiserum (see Viglietti-Panzica 1986 for details). Brains that were not properly sectioned or had a high background staining after immunoreaction were discarded; thus, all numbers mentioned in this study (see Table 1) refer to the final number of animals that were subjected to the semiquantitative analysis. The sections corresponding to the septal region were coded for each animal, and the subsequent semiquantitative analysis was performed by one operator (CVP) with no knowledge of the sex or treatment of the bird. The density of immunoreactive fibres was evaluated on an ordinal scale, using the highest VTimmunoreactivity within the septum as a reference; + + + represents abundant fibres, + + represents medium density, + represents low density of fibres, and -- corresponds to a complete absence of immunoreactive material. In this way, we evaluated the VT-innervation of the nucleus striae terminalis (nST), nucleus septalis lateralis (SL), nucleus commissurae pallii (nCPa) in each animal. Two diencephalic regions that were previously shown to contain dense VT-innervation [e.g., the dorsal diencephalic region (DD) and the periventricular hypothalamic region (P); VigliettiPanzica 1986] were also quantified as control areas.
Table 1. Density of VT fibres in the septal and diencephalic regions of male and female quail submitted to different experimental treatments. MLD, long-day males; FLD, long-day females; MSD, short-day males; FSD, short-day females; MCX, castrated males; FOVX, ovariectomized females; MT, T-treated males; FT, T-treated females. Each entry represents one animal : - , complete absence of fibres; +, very few fibres; § +, moderate fibre density; + + +, high fibre density, nST, nucleus striae terminalis; SL, septum laterale; nCPa, nucleus commissurae pallii; DD, dorsal diencephalic region; P, hypothalamic periventricular region
MLD
FLD
MSD
FSD
nST
SL
nCPa
DD
P
++
--
+
_
_
_
+++ +++ ++
+++ +++ ++
+++
+++ ++ ++ ++
+++ ++ +++ +++ ++
-.
e
s
u
l
t
s
Body weight a n d cloacal g l a n d size were a n a l y s e d by a t w o - w a y analysis o f v a r i a n c e ( A N O V A ) with the sex of the birds a n d their t r e a t m e n t as i n d e p e n d e n t factors. This c o n f i r m e d p r e v i o u s l y established (Panzica et al. 1991) e x p e r i m e n t a l effects a n d sex differences [larger b o d y weight o f females, larger cloacal glands i n males, increase in g l a n d size following exposure to T ( M L D , M T , FT)]. I n t a c t birds exposed to long days were sexually m a t u r e as evidenced by the large testis weight (5.14__ 1.27 grams, m e a n _ _ S D ) in males a n d the presence o f large ( > 15 m m ) o v a r i a n follicles in females. I n contrast, s h o r t - d a y males h a d small testes (0.091 + 0 . 1 3 4 grams) a n d females small follicles ( < 2 mm). D u r i n g the first b e h a v i o u r a l test t h a t was p e r f o r m e d , 4 o u t of 5 M L D birds a n d 2 o u t o f 5 M T birds showed c o p u l a t o r y b e h a v iour (at least one m o u n t attempt). All other birds were sexually inactive. Inactive birds in the M L D a n d M T groups were s u b s e q u e n t l y retested a n d all finally b e c a m e b e h a v i o u r a l l y active, b u t in some cases, this was a p p a r ent only d u r i n g the f o u r t h test (see below). I n o u r Nisslstained sections, we identified several n u c l e a r masses that c o u l d readily be c o m p a r e d with similar nuclei described in the chicken (Kuenzel a n d M a s s o n 1988): the nucleus a c c u m b e n s (Ac), the n u c l e u s septalis lateralis (SL) a n d medialis (SM), the n u c l e u s c o m m i s s u r a e pallii (nCPa), a n d the n u c l e u s striae t e r m i n a l i s (nST). I n M L D birds, we observed V T - i m m u n o r e a c t i v e fibres in the SL a n d nST, whereas i m m u n o r e a c t i v e fibres were irregularly f o u n d i n the n C P a (see Fig. 1). I n these nuclei, the i m m u n o r e a c t i v e m a t e r i a l often h a d the a p p e a r a n c e o f
FOVX
MT
FT
.
.
+
--
+++ ++ +++ +++
+++ ++ ++ +++ +++
+
+++ +++ ++ +++
+++ +++ +++ +++
-
++
+++
++ ++ ++ +++ +++
++ ++
++ ++
_
_
+
+
-
_
_
+
--
--
-
_
_
_
_
_
_
+++
+++
.
.
_
MCX
+
- -
_
. R
.
- -
-
.
.
.
.
_
.
_
++
_
_
_
-
-
-
_
_
_
_
_
_
+
+
+++ +++
+++ +++
+
+
++
++
+ + +++ ++ ++
+++ ++ ++ +++ +++
++ ++
++ ++ +
+
+
+
+
+
-
-
++
_
_
_
_
_
_
_
_
_
+ + +++ ++ ++
+ ++ +++ ++ ++
+ ++ +
-
-
-
_
_
+
_
_
_
+
-
+
-
+
-
+
p u n c t a t e structures, sometimes in close association with i m m u n o p o s i t i v e cell bodies (Fig. I a, c, d). T h e distribution o f VT-ergic n e u r o n s a n d fibres in the d i e n c e p h a l o n was c o n s i s t e n t with o u r p r e v i o u s l y p u b l i s h e d d a t a (Viglietti-Panzica 1986). A careful e x a m i n a t i o n revealed n o m a j o r c h a n g e in the cell n u m b e r or d i s t r i b u t i o n as a f u n c t i o n o f the sex o f the b i r d or its e x p e r i m e n t a l treatment. T h e results o f the s e m i q u a n t i t a t i v e e v a l u a t i o n o f the density of V T fibres are r e p o r t e d in Table 1 for each subject. As c a n be seen, the s i t u a t i o n observed in M L D birds was r e p r o d u c e d o n l y in the T - t r e a t e d males (MT). T h e r e was a c o m p l e t e or n e a r l y complete d i s a p p e a r a n c e of i m m u n o r e a c t i v e fibres in the n S T a n d SL o f castrated
263
Fig. 1. a, b VT-immunoreactivity in the septal complex of the male (a) and female (b) Japanese quail, x 100. CA Commissura anterior; n S T nucleus striae terminalis; S L nucleus septalis lateralis; * lateral ventricle, c-I VT-immunoreactivity in the SL and n S T of the maIe
Japanese quail exposed to different experimental treatments; c-d tong-day; e, h short-day; f, i castrated; g, I T-treated castrated, x 300
males (MCX), and of males exposed to short days (MSD). With few exceptions, immunoreactive fibres were never seen in the nST and SL of females, irrespective of their hormonal conditions. This situation was highly specific to the septal nuclei: in the diencephalic regions that were examined ( D D and P), no m a j o r
change in the VT-innervation could be detected by the present method. The innervation of the septum in two male birds was not in agreement with the treatment of the group to which they belonged. In one M L D bird, no VT fibres were seen in the nST and SL; this individual had a much
264 smaller combined testis weight than the other birds in the group (3.23 grams versus 5.61 +0.81 grams in the other four birds; mean+SD). In addition, during the first behavioural test, this bird did not show copulatory behaviour whereas the other four did. It had to be retested four times before even weak behaviour was observed. On the other hand, one MSD bird displayed a dense innervation of the examined septal nuclei, although this innervation was absent in the other subjects from the same group. The combined testis weight of this bird was very much increased (329.4 mg versus 31.4_+ 15.2 mg). Interestingly, there was also a relationship between the density of innervation in the septum and the copulatory behaviour of the MT birds. The majority of these birds did not show sexual behaviour during the first test (probably in relation to the relatively short duration of the treatment with T) and had therefore to be retested several times before they were active. We calculated the correlation coefficient (Spearman Rank correlation coefficient) between the number of tests before the first behaviour was observed and the density of the innervation (on a 0 to 3 scale); this coefficient was equal to -0.917, which is significant for P-0.05 provided that the direction of the correlation is predicted based on previous data (one-tailed test).
Discussion
Using an immunocytochemical procedure, we have demonstrated, in the present study, a sexual dimorphism of the VT-ergic innervation of two septal areas of the quail: the nST and SL. A dense innervation of VT-immunoreactive fibres was observed in males, but it was not detectable in females. In the male, the presence of such fibres was apparently controlled by T; it was strongly reduced or completely absent in birds that were either castrated or exposed to a short photoperiod, a condition suppressing full sexual maturation (Balthazart et al. 1979). Two weeks of T-treatment in castrated males restored the innervation to a density that was typically seen in sexually mature males. Interestingly, T-treatment of ovariectomized females produced no detectable changes in septal innervation. These results are substantially different from those previously demonstrated in the canary brain (Voorhuis et al. 1988), the only avian species investigated to date. In this species, a sexual dimorphism in the VT-ergic innervation of the septal region was also observed in sexually mature birds: innervation is dense in male and almost non-existent in female. However, a 4-week Ttreatment of adult ovariectomized females enhances the immunoreactivity in the region to a typical male level. It therefore appears that the sexual dimorphism observed in the adult canary only reflects a differential activation by T in the two sexes. The present data from quail suggest, on the contrary, that the sexual dimorphism in the septal VT-ergic innervation is organizational in nature. Indeed, even when ovariectomized females are treated with doses of T that are sufficient to induce full restoration of the innervation in castrated males, they
show no immunoreactive fibres. The final validation of this conclusion would however require the demonstration that longer treatments with T still fail to induce VT-immunoreactive fibres in the septum of female quail. The quail model is, in some sense, qualitatively similar to the situation observed in adult rats. In this species, there is also a sexual dimorphism in the induction by T of the VP-ergic innervation of the septum: a denser innervation is always observed in castrated males than in ovariectomized females after a treatment with identical doses of T (De Vries and A1-Shamma 1990). A clear anatomical specificity was observed in the sex differences and control by T of the VT-innervation: these factors affected the immunoreactivity in the two septal nuclei, but had no effect on the innervation of two hypothalamic regions that were considered as controls (cf. Table 1). As in the rat (De Vries et al. 1985), the VT-ergic system of quail therefore consists of elements that are sensitive and elements that are not sensitive to steroids. We do not have any information about the origin of the VT steroid-sensitive fibres in quail. Tracing studies in duck (Korf 1984) have demonstrated that the paraventricular nucleus projects to the septum but there is, to our knowledge, no evidence that VT-ergic elements are involved in these projections. In the canary, a weakly stained cell group has been identified in the dorsal diencephalon and septal complex of males and T-treated females (Voorhuis et al. 1988). The projections of these cells are, however, unknown so far. In the present material, we have found no such population of VTergic cell bodies, and no obvious change after T administration in males could be detected in the number of VTergic perikarya located in the periventricular and lateral hypothalamic regions. The origin of the septal VP-innervation in mammals is still a matter of discussion: in the guinea pig, the cells of origin were identified, by means of immunocytochemistry and retrograde labelling, in the paraventricular nucleus; contrasting data are available for the rat, in which the bed nucleus of the stria terminalis and the medial amygdaloid nucleus are considered to be the major source of this innervation (for discussions, see Staiger and Niirnberger 1989; De Vries and A1-Shamma 1990). In quail, cells concentrating T and/or its metabolites oestradiol and dihydrotestosterone have been identified by autoradiography (Watson and Adkins-Regan 1989) or by immunocytochemistry (Balthazart et al. 1989) in the lateral septum. The avian septal region, and in particular the nST, also contain aromatase-immunoreactive cells (Balthazart et al. 1990). This enzyme catalyses the conversion of T into oestradiol, one of the critical steps in the activation of sexual activity (for references, see Balthazart and Schumacher 1985). The present study demonstrates interesting correlations between the T-induced changes in the septal VTergic innervation and in masculine copulatory behaviour. Both are present in the male and not in the female, both are increased by T in males but not in females, and finally there is a positive correlation between the induction of behaviour and the density of the septal VT fibres in T-treated males. This suggests causal relation-
265 ships b e t w e e n these t w o T - d e p e n d e n t effects. A few studies in birds have i n d e e d p o i n t e d to a p u t a t i v e role o f V T in the c o n t r o l o f the r e p r o d u c t i v e b e h a v i o u r . K i h l s t r 6 m a n d D a n n i g e (1972) h a v e d e m o n s t r a t e d a s h o r t - t e r m increase o f m a t i n g b e h a v i o u r in m a l e chicken a n d p i g e o n after V T a n d o x y t o c i n systemic a d m i n i s t r a tion. V o o r h u i s (1990) suggests t h a t V T m i g h t be i n v o l v e d in the p r o d u c t i o n o f song in the c a n a r y . A n u m b e r o f studies also p r o v i d e direct e x p e r i m e n t a l evidence for a role o f the septal r e g i o n in the c o n t r o l o f s t e r o i d - d e p e n d e n t b e h a v i o u r in birds. Electrical s t i m u l a t i o n in o r n e a r the s e p t u m in the fowl induces a g o n i s t i c r e s p o n s e s (Phillips a n d Y o u n g r e n 1971). It is t h e r e f o r e p o s s i b l e t h a t the septal r e g i o n is i n v o l v e d in the a n d r o g e n i c r e g u l a t i o n of agonistic behaviour. T h e c o r r e l a t i o n s o b s e r v e d between sexual b e h a v i o u r a n d the septal V T - e r g i c i n n e r v a t i o n m i g h t t h e r e f o r e be t a k e n as evidence to suggest t h a t V T p l a y s a direct role in the c o n t r o l o f b e h a v i o u r . A l t e r n a t i v e l y , it is p o s s i b l e t h a t T i n d e p e n d e n t l y affects m a l e c o p u l a t o r y b e h a v i o u r a n d the V T - e r g i c i n n e r v a t i o n .
Acknowledgements. This study was supported by grants from the Italian Ministry for University and Scientific Researches (MURST 60%), CNR (89.03043.CT04, 90.02456.CT04), FRFC (9.4601.90 and 2.9003.91), and EEC (SC1-0230-C).
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