Gonadectomy reduces the concentrations of putative receptors for arginine vasotocin in the brain of an amphibian

Bram Research, 541 (1991) 193-197

193

Elsevier BRES 16313

Gonadectomy reduces the concentrations of putative receptors for arginine vasotocin in the brain of an amphibian Sunny K. Boyd and Frank L Moore Department of Zoology, Oregon State University, Corvalhs, OR 97331 (U S A ) (Accepted 4 September 1990)

Key words Newt, Argmlne vasotocm receptor, Amygdala, Sexual behawor, Vasopressm

Putatwe receptors for argmme vasotocm (AVT) in the brain of the newt (Tancha granulosa) were measured using quantltatwe autoradmgraphy with tntmm-labelled vasopressm Speofic binding sites were observed m the olfactory bulb, medial (hlppocampal) palhum, dorsal palhum, amygdala pars laterahs and tegmental region of the medulla oblongata In both male and female newts, concentrations of binding sites m the amygdala, but not m the other four areas, were slgmhcantly lower m gonadectomtzed ammals than m sham-operated controls The eqmhbrlum dlssocmtlon constants (Kos) were not altered by gonadectomy Since long-term castratmn abohshes the effect of AVT mjectmn on sexual behawors, these results support the hypothesis that gonadal stermds maintain sexual behawors m this amphlbmn by maintaining AVT receptors m the amygdala INTRODUCTION G o n a d a l steroid hormones have been shown to activate reproductive behaviors in m a n y vertebrates 7'2°'21'24 For example, sexual behaviors of male rough-skinned

and are restored by treatment with gonadal steroids suggested to us that gonadal steroids are acting at one or more sites in the neurochemical pathway between the receptors for those neuropeptides and m o t o r out-

newts (Tartcha granulosa) are abolished by castration and are m a i n t a i n e d in castrates by the administration of androgen11 z5 Typically, however, sexual behaviors in

put Thus, as a starting point, we hypothesized that gonadal steroid hormones m a i n t a i n sexual behaviors by maintaining receptors for the behaviorally-active peptldes Spe-

m a n y vertebrates persist for weeks or months after the testes are removed, long after gonadal steroid hormones have decreased to undetectable levels2'5'19

cifically, we hypothesized that, in the behaviorallyimportant brain areas in T granulosa, gonadal steroid hormones maintain high concentrations of A V T recep-

G o n a d a l steroid hormones appear to maintain the behavioral actions of those neuropeptldes which have been shown to activate sexual behaviors in vertebrates For example, lutelnlzmg hormone-releasing h o r m o n e ( L H R H ) InJections can enhance lordosis behavior in estrogen-treated, but not in untreated, ovanectomized female rats 27 Likewise, L H R H injections can stimulate copulatory behaviors in testosterone-treated, but not in untreated, castrated male voles 3 In male rough-skinned newts, argmine vasotocin (AVT) injections can stimulate sexual behaviors m intact males and in androgen-treated castrates 26"34 In these newts, it takes several weeks

tors and, therefore, we predicted that gonadectomy would reduce the concentrations of A V T receptors in specific brain loci We used In vitro quantitatwe autoradlography to determine the concentrations of putative A V T receptors in specific brain areas of male and female newts that were sham-operated or gonadectomlzed during the breeding season

before untreated castrates fall to respond behaviorally to A V T rejections 34 The slow post-castration decreases in spontaneous and peptIde-lnduced sexual behaviors could be related phen o m e n a The finding that the behaworal responses to mjections of neuropeptides are abolished by castration

MATERIALS AND METHODS Adult male and female rough-skinned newts (T granulosa) were collected locally at the height of the breeding seasons (March) Under benzocalne anesthesia (Sigma Chemicals, immersed in 1% solution), newts were e~ther sham-operated or gonads were removed through a single abdominal mosion (n = 15 per sex for each treatment initially) Newts were treated with antibiotics for 2 h 3 times per week, were fed chopped beef heart once weekly, and were mamtamed at 10 °C in dechionnated water on a photopenod of 12L 12D (lights out at 22 00 h) After 30 days, newts were decapitated and their brains immedl-

Correspondence S K Boyd Present address Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556, U S A 0006-8993/91/$03 50 (~) 1991 Elsevier Soence Pubhshers B V (Biomedical Division)

194 ately removed and stored at -80 °C Using a cryostat at -20 °C, serml coronal sections were made at 48 pm thickness, except in the caudal telencephalon where sections were taken at 16/tm These sections were used for quantitative in vitro autoradlography for [3H]arglnine vasopresstn ([3H]AVP) binding sites, using techniques we have previously described and vahdated for newt brains 31 Entire serially sectioned brains from each newt (48 ~m sections) were incubated with 10 × 1 0 -9 M [3H]AVP (New England Nuclear, 40 Ci/mmol spec act ) In 2 ml Tris-HCl buffer per slide for 30 mln at 23 °C Adjacent sections for non-specific binding determination were incubated under the same conditions except for the addlUon of 10 p M unlabeUed AVT (Sigma) The incubation buffer was 50 mM TrIs-HCl (pH 7 4) containing 5 mM MgCl2, 2 mg/ml bovine serum albumin, 0 5 mg/ml bacltracm, and 10/~g/ml aprotmin (all from Sigma) Slides were washed, following incubation, in two 30-s rinses of ice-cold buffer (10 mM Tris-HCl, 1 mM MgCl2, and 1 mg/ml bovine serum albumin) They were briefly dipped m ice-cold double distilled water and dried on a slide warmer at 40 °C Slides were stored desiccated overnight at room temperature then apposed to tntmm-sensltlVe Ultrofllm (LKB Instruments) m X-ray cassettes Films were exposed for 2 months at room temperature and developed in Kodak D-19 at 21 °C for 4 rain Tissue sections were lightly stained with Cresyl violet Since any differences in bindmg observed with the procedure above could result from changes in either binding site concentration or affinity, sections from caudal telencephalon of each animal were also included in a saturation binding experiment Specifically, 16 # m sections were distributed (2 sectlons/shde) across 10 pairs of shdes Each pair consisted of one slide for determination of total binding and another shde, containing anatomically adjacent sections, for determination of nonspeciflc bmdmg Pairs of slides from each animal were randomly assigned to receive one of 10 different concentrations of [3H]AVP Thus, duplicate determinations from each animal were included at each concentration of [3H]AVP used to generate saturation binding isotherms and for Scatchard analysis of data Binding procedures were the same as above except ten different concentrations of [3H]AVP (ranging from 0 07 to 17 4 nM) were used Differences m optical densIUes on autoradiograms were determined using an IBM PC-based image analysis system 31 This system quantified average optical density under a 0 1 mm z area which was centered within each neuroanatomlcal locus (for each side of the brain) Preliminary studies indicated that there was no slgmflcant variation in specific brain areas from rostral to caudal OpUcal density values were converted to femtomoles [3H]AVP bound/rag protein using tntium brain-mash standards prepared from newts using the procedure of Rainbow et al 29 Sections through five different standards were made at 16pm and mounted on slides One set of standards was included m each cassette Standards had optical densiUes that ranged from 0 073 to 0 865, values which were within detectable limits of the fdm and camera The coefficient of variation for the standards on different films was less than 2 5% Other sections of brain-mash standards were homogenized m doubledistilled water and used for liquid scmtdlatlon counting or protein determinations These values produced a standard curve relating optical density to nCi/mg protein which was linear (r = 0 97) The tntmm concentration of each brain structure was in turn converted into fmol [3H]AVP/mg protein by dividing by the specific radioactivity of the [3H]AVP Data were analyzed using LIGAND 2s

incubation medium abolished binding of [3H]AVP and r e s u l t e d in h o m o g e n e o u s b a c k g r o u n d b i n d i n g a c r o s s t h e sections D e n s e c o n c e n t r a t i o n s o f b i n d i n g sites w e r e o b s e r v e d in t h e o l f a c t o r y n e r v e as it e n t e r e d t h e o l f a c t o r y b u l b T h i s b i n d i n g d i s a p p e a r e d r o s t r a l to t h e a c c e s s o r y o l f a c t o r y bulb

Within

campal)

the

pallium,

telencephalon, dorsal

pallium,

the

medial

and

amygdala

(hlppopars

l a t e r a h s c o n t a i n e d specific b i n d i n g sites f o r [ 3 H ] A V P B i n d i n g in t h e m e d i a l p a l l i u m e x t e n d e d f o r t h e e n t i r e length of the telencephalon,

whereas

dorsal pallium was best-developed levels a n d was n o t p r e s e n t

b i n d i n g in t h e

at m i d - h e m i s p h e r i c

at t h e r o s t r a l o r c a u d a l

extremities of the telencephalon

B i n d i n g in t h e a m y g -

a

RESULTS Measurements grams

revealed

of optical density on the autoradioseveral

nervous system of

discrete

T granulosa

areas

b

in t h e c e n t r a l

with high concentrations

o f [ 3 H ] A V P b i n d i n g sites (Figs 1 a n d 2) B i n d i n g in t h e s e a r e a s was specific, a d d i t i o n o f u n l a b e l l e d A V T t o t h e

Fig 1 Autoradlograms showing [3H]AVP binding in the olfactory' bulb (a) and medial palhum (b) Section used In (a) was taken at a level which corresponds to Fig 2A whale (b) corresponds to a level intermediate between Fig 2B and 2C Bar = 0 25 mm

195

Fig 2 Schematic diagram of the newt brain with regions containing putative A V T receptors indicated by closed triangles T h e top figure is an e x t e n o r side view Figures labelled A - E are typical frontal sections at the levels indicated on the side view amy, amygdala pars laterahs, b oi , olfactory bulb, cb, cerebellum, dp, dorsal pallium, hyth, hypothalamus, rap, interpeduncular nucleus, Ip, lateral p a l h u m , mot, medml olfactory tract, m p , medml p a l h u m , ha, nucleus accumbens, on, olfactory nerve terminal field, pit, anterior pltmtary, poa, preoptlc area, sep, septal nucleus, teg, t e g m e n t u m V and VII, I I - X , cranial nerves

TABLE I

Concentrations of [3H]AVP binding sttes m brains from shamoperated newts or newts gonadectomtzed for 30 days Data reported are m e a n s + S E M h e m i s p h e r e s for all a m m a l s in group

Bra:n area

for each area In both

dala was solely m the pars laterahs in the caudal telencephalon Finally, dense binding was o b s e r v e d in the lateral hlndbraln in the t e g m e n t a l region o f the medulla oblongata These binding sites were located in the tegmental r o o t region extending from roots of cranial nerves V - V I I I The location of receptors in all these areas was almost exclusively o v e r the neuroptl

Stte concentratton (fmol/mg proteIn) Sham-operated

Gonadectomtzed

Males Olfactory bulb Medial pallium Dorsal pallium Amygdala Tegmentum

(n = 60 7 76 2 72 7 76 0 61 4

14) _+ 5 + 4 + 3 _+ 2 + 3

1 0 4 1 2

(n = 13) 62 2 + 4 77 5 _+ 2 74 7 _+ 2 64 1 _+ 3 62 7 _+ 4

Females Olfactory bulb Medial p a l h u m Dorsal pallium Amygdala Tegmentum

(n = 13) 64 0 _+ 4 76 9 _+ 3 77 0 _+ 2 73 8 + 2 64 3 + 3

0 8 4 0 2

(n = 64 6 78 7 73 6 66 6 63 3

4 2 0 0* 0

8) +_ 2 6 _+ 4 9 + 2 1 _+ 1 9* + 2 9

* A N O V A comparison of binding site concentrations within the amygdala showed that g o n a d e c t o m y had a slgmficant effect, compared with sham-operation, m both male and female newts (F = 14 29, P < 0 0001) T h e r e were no significant differences in A N O V A comparisons of other brain areas

T A B L E II

A V T receptor dtssoczatzon constants and concentrattons wtthm the amygdala pars laterahs of sham-operated or gonadectomtzed male and female newts a Data presented are m e a n s + standard errors of the estimates The 95% confidence intervals for all four groups overlapped and therefore KdS were not significantly different b D a t a presented are m e a n s _+ standard errors The 95% confidence intervals for sham-operated newts overlapped However, s h a m - o p e r a t e d a m m a l s differed significantly from gonadectomlzed newts and gonadectomlzed males and females differed from each o t h e r

Group

n

g da (nM)

Sham males Sham females Castrated males Ovariectomlzed females

14 13 13 8

0 0 0 0

25 25 20 23

_+ 0 + 0 + 0 _+ 0

B maxb (fmol/mg protein) 02 02 03 01

75 87 46 69

3+ 6 9 -+ 7 1 __. 5 4+ 6

1 3 8 6

196 For both males and females, when serial sections were incubated with a single saturatmg concentration of [3H]AVP, the concentration of binding sites in the amygdala pars laterahs was significantly lower in gonadectomized animals than sham-operated controls (Table I) Similar results were obtained when concentrations in the amygdala were determined from the B m a x (Table II) Analysis of saturation binding isotherms showed that the dissociation constant (Kd) of sites m the amygdala was not altered by gonadectomy (Table II) Castrated male newts had a 40% reduction in putative receptor concentration in the amygdala, compared with sham-operated control males Ovarlectomized female newts had a 20% decrease m concentration in this area, compared with sham-operated females This effect of gonadectomy on [3H]AVP binding site concentrations was specific to the amygdala, the concentrations of sites in the olfactory bulb, medial and dorsal palhum, and tegmentum were not affected by gonadectomy (Table I) There was no observable sexual dlmorphlsm in blndmg site concentrations (Tables I and II) DISCUSSION Concentrations of putatwe AVT receptors in the amygdala pars laterahs of gonadectomlzed male and female newts were significantly reduced compared with sham-operated controls These results support the hypothesis that gonadal steroid hormones may maintain sexual behawors in this amphibian by maintaining AVT receptors in the amygdala Therefore, one action of gonadal steroid hormones on neural substrates, which could be added to the growing list of proposed actions in vertebrates, is to maintain receptors for behaviorally active neuropeptldes Our earher study 31 provided evidence that the [3H]AVP bmdIng sites In the brains of newts are authentic AVT receptors These specihc and discretelydistributed [3H]AVP binding sites may represent the preose sites of action of AVT in controlling reproductive behawors in th~s species Alternatively, these sites may have other, yet to be identtfied, functions All regions with large amounts of binding contain detectable concentrations of lmmunoreactive AVT m T granulosa, except the olfactory nerve 35 In general, then, there is a correlation between the neuroanatomical distribution of AVT and putative AVT receptors in the brain of newts However, areas with high concentrations of AVT lmmunoreactlvlty35 do not necessarily correspond to areas wtth high concentrations of [3H]AVP binding sites The neuroanatomlcal distribution of putative AVT receptors in newts is similar to that described for central

AVP receptors in rats TM 6 8 i6,22 32 AVP receptors have been reported In the olfactory nucleus, hippocampus, amygdala and dorsal hmdbraln, among other areas These areas may correspond in this amphibian to the olfactory bulb, hippocampal palhum, amygdala pars laterahs, and dorsal tegmental region of the medulla Consistencies in location of [3H]AVP binding sites in amphibian and mammalian brains suggest similarities in the function of these areas across vertebrate groups Of the 5 neuroanatomlcal areas examined, the amygdala was the only one which exhibited changes m AVT receptor concentrations following castration or ovanectomy Interestingly, the amygdala has been reported to be sexually dlmorphic in amphibians 3° and the volume of this nucleus in males decreases following castration 17 Furthermore, the amygdala contains cells which concentrate radiolabelled estradiol in f r o g s TM Our finding that gonad removal changes AVT receptor concentration in this area, therefore, may represent an effect of estradlol on these receptors Gonadal steroids may modulate density of putative AVT receptors through direct or indirect mechanisms Vasopressmerglc lnnervation in some areas of the rat brain IS sexually dlmorphlc with the number of AVP neurons as well as their biosynthetic capacity being greater in males than in f e m a l e s 13'23'33 Gonad removal in etther sex reduces concentrations of immunoreactlve AVP and steroid replacement therapy restores levels to normall2 14 15 Thus, effects of gonadectomy on vasopressin or vasotocin receptors may be indirectly due to changes in peptlde levels As well, steroids may directly alter receptor densities as proposed for the abdtty of estradlol to increase concentrations of putative oxytocln receptors in female rat bram 9 i0 Thirty days following gonadectomy, the behavioral responses of newts to AVT injections disappear in ovarlectomized females (Moore, Boyd, and Wood, unpublished data) and castrated males 34 However, in the present study, gonadectomy of male and female newts resulted in only a 20-40% decrease in the concentration of putative AVT receptors in the amygdala, not a complete disappearance This decrease may reflect subpopulations of neurons in the amygdala that are specifically involved m sexual behavior As well, it is likely that any effect of gonadal hormones on AVT receptors is only one part of the mechanisms regulating the display of reproductwe behaviors

Acknowledgements This research was supported by National InstJtutes of Health research Grant RO1 HD13508 and a Medical Research Foundation of Oregon eqmpment grant to F L M

197

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