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Localization of prolactin binding sites in ring dove brain by quantitative autoradiography
Brain Research, 487 (1989) 245-254
245
Elsevier BRE 14484
Localization of prolactin binding sites in ring dove brain by quantitative autoradiography John H. Fechner Jr.* and John D. Buntin Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee 53201 (U.S.A.) (Accepted 25 October 1988)
Key words: Prolactin receptor; Ring dove; Streptopelia risoria; Hypothalamus; Quantitative autoradiography; Telencephalon; Choroid plexus; Sex difference
Specific binding sites for prolactin have been previously detected and characterized in ring dove brain membranes. In order to map the distribution of these sites, specific binding of ~25I-ovine prolactin was examined in slide-mounted sections of 6 male and 6 female ring dove brains by in vitro film autoradiography and densitometry. Analysis of 34 brain regions revealed a sex-specific pattern in specific binding activity. Although no significant sex differences were observed in any individual brain region, a trend in this direction was observed in the preoptic area. Specific binding levels in which the lower limit of the 99% confidence interval was greater than zero were detected in choroid plexus, medial habenula, lateral mesencephalic nucleus, hippocampus, parahippocampal area, preoptic area and 4 hypothalamic sites: paraventricular nucleus, ventromedial nucleus, suprachiasmatic nucleus, and tuberal region. Autoradiographic analysis of specific binding in cerebellum, ventromedial hypothalamus, and hippocampus/parahippocampus yielded relative differences that closely approximated those obtained in binding studies on tissue homogenates from these regions. These results suggest possible sites of prolactin action in altering behavioral state and neuroendocrine function in this species.
INTRODUCTION Prolactin ( P R L ) p r o m o t e s a variety of physiological changes in v e r t e b r a t e s which subserve osmoregulatory, reproductive, and d e v e l o p m e n t a l functions 9. A l t h o u g h m a n y of these effects are m e d i a t e d through h o r m o n a l actions in peripheral target tissues, recent evidence suggests that P R L may also influence the activity of the central nervous system (CNS). Neurons responsive to systemically injected P R L 1° and iontophoretically applied P R L 46"55 have been r e p o r t e d in some m a m m a l i a n species. In addition, P R L - i n d u c e d changes in d o p a m i n e turnover in the t u b e r o i n f u n d i b u l a r region have been well d o c u m e n t e d in the rat 41 and effects of P R L on several o t h e r n e u r o t r a n s m i t t e r systems have also b e e n o b s e r v e d 1s'35'53. Finally, changes in feeding behavior 5"34"4°, sexual b e h a v i o r 21'25, and parental
behavior a'3z'43 have b e e n r e p o r t e d following central or p e r i p h e r a l administration of P R L in various v e r t e b r a t e species. While the b l o o d - b r a i n b a r r i e r precludes direct access of b l o o d - b o r n e P R L to CNS tissue, a specialized u p t a k e mechanism m a y exist in choroid plexus to t r a n s p o r t P R L into the brain by way of the cerebrospinal fluid 52. F u r t h e r m o r e , pituitary-independent, prolactin-like immunoreactivity has been d e t e c t e d in h y p o t h a l a m i c and extrahypothalamic regions in several v e r t e b r a t e s 13'16'2°'23'48'54. This leaves open the possibility that brain function is also altered by P R L - l i k e proteins of CNS origin. Despite evidence for direct n e u r o m o d u l a t o r y effects of P R L , little is known a b o u t the sites or m o d e s of P R L action in the CNS. Specific binding sites for P R L have been described and partially characterized in h o m o g e n a t e s or m e m b r a n e fractions p r e p a r e d from rabbit h y p o t h a l a m u s and substantia nigra 14'15,
* Present address: Molecular Endocrinology Section, Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, Bethesda, MD 20892, U.S.A. Correspondence: J. D. Buntin, Department of Biological Sciences, P.O. Box 413, University of Wisconsin-Milwaukee, Milwaukee, WI 53201, U.S.A. 0006-8993/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
246 ring dove forebrain and midbrain 6, and toad cerebrum, midbrain, and olfactory bulb 37. PRL binding sites have also been detected in the choroid plexus of the rat, pig, sheep, rabbit, and ring dove 7"45"51 by conventional receptor binding techniques or by autoradiography. However, with the exception of the choroid plexus, the discrete localization of PRL binding sites within the CNS has yet to be determined for any vertebrate species. The purpose of this study, accordingly, was to map the distribution of CNS binding sites for PRL in the ring dove (Streptopelia risoria), a species that exhibits marked behavioral and physiological changes following systemic 2s'32"5°, or intracranial PRL administration 5' 8 and possesses saturable CNS binding sites with high affinity and specificity for PRL 6 which could conceivably mediate such effects. Specific binding of radioiodinated oPRL in vitro was examined in 34 brain regions using quantitative autoradiography. Competitive binding studies were also conducted on tissue homogenates from 3 of these regions in order to evaluate the validity of the mapping data obtained by autoradiography. Because the effect of centrally administered PRL on food intake has been previously reported to be sexually dimorphic in this species 5, sex differences in PRL binding were also examined in each region. MATERIALS AND METHODS
Animals Tissue was obtained from non-breeding adult ring doves which were housed individually in visual isolation cages (41 x 41 × 53 cm) under a 14L:10D photoperiod, with lights on at 07.00 h. Temperature was maintained at 19 °C. Food and water were continuously available. Birds were sacrificed between 08.00 and 10.00 h by decapitation and the brains were removed quickly and frozen in powdered dry ice. Sectioning commenced within 4 h of sacrifice.
Competitive binding studies on tissue homogenates Frozen brains were mounted on a chuck using embedding matrix (Lipshaw M-l). 300 p m coronal sections were taken using a clinical microtome, transferred to microscope slides, thawed briefly and refrozen to adhere the sections to the slide. Brain
regions were dissected over dry ice with the aid of a light box and a dissecting microscope using the pigeon brain atlas of Karten and Hodos 29 as a reference. The ventromedial hypothalamic nucleus (VMN) was removed bilaterally using a micropunch 44 made from stainless steel tubing (2 mm o.d., 0.2 mm wall, Small Parts Inc., Miami, FL). The hippocampal/parahippocampal region (Hp/APH) and the cerebellum (Cb) were microdissected using a scalpel blade. Corresponding regions from two or more doves were pooled and suspended in homogenizing buffer (25 mM Tris-HC1, 10 mM CaC12, 0.1% bacitracin, 0.1% sodium azide, pH 7.6) using polyurethane microhomogenizers (Kontes, Vineland, N J). Protein concentration was determined using a modification of the Lowry method 26 with bovine serum albumin (BSA) (Fraction V, Sigma Chemical Co., St. Louis, MO) as the protein standard. Homogenates were snap-frozen in liquid nitrogen and stored at -70 °C until assayed. Ovine PRL (NIADDK-oPRL-I-2) was labelled with carrier-free 1251 to an average specific activity of 56 pCi/pg using a lactoperoxidase procedure 6 and repurified on a 1 × 25 cm Sephadex G-100 column immediately prior to use. Specific binding was determined by methods described previously 6. Homogenates were thawed, rehomogenized and diluted to appropriate protein concentration with assay buffer (homogenizing buffer plus 0.1% BSA). Incubations were carried out in 12 × 75 polystyrene tubes containing 200 pl tissue homogenate, 100/tl 125I-ovine PRL (250 pM), and 200/al assay buffer or unlabelled ovine PRL competitor ( N I A D D K - o P R L 18; 83 nM). Assay tubes were incubated for 96 h at 19 °C unless indicated otherwise. At the end of the incubation period, 3 ml cold assay buffer without bacitracin and sodium azide was added to each tube to terminate the assay. After centrifugation for 1 h at 850 g (4 °C), the supernatants were decanted and the tubes were inverted over absorbent paper. Pellets were counted on a Packard PRIAS C G D Autogamma Spectrometer (65% counting efficiency). Three-5 total binding and non-specific binding tubes were used to determine mean _+ S.E.M. specific binding in each tissue sample. A total of 6 binding assays were conducted, each consisting of one or two pooled tissue samples from each of the 3 brain regions.
247
Autoradiographic study Frozen 20 #m coronal sections were cut in a cryostat (Lipshaw Model 1500) at -15 °C and thawmounted on to gelatin-coated slides. Successive sections were mounted alternately on two sets of slides so that total binding and non-specific binding values could be obtained from adjacent sections. Slides were then air-dried at 4 °C for 24 h and stored at -20 °C for no more than 5 days. Following a 30 min ,preincubation period at 19 °C in Tris-HCl assay buffer (see above), total binding sections were incubated for an additional 24 h at the same temperature in fresh buffer containing 75 pM 125I-ovine PRL (NIADDK-oPRL-I-2). Non-specific binding sections were preincubated in assay buffer containing 83 nM ovine PRL (NIADDK-oPRL-18) and then incubated with 75 pM labelled ovine PRL plus 83 nM unlabelled ovine PRL competitor. Radiolabelled ovine PRL was prepared as described above. Incubation was terminated by washing the slides for 20 min with fresh assay buffer without bacitracin and sodium azide at 4 °C. After two more 20-min washes, the slides were rinsed with cold distilled water, placed on absorbent paper and allowed to dry overnight at room temperature.
Tissue sections and slide-mounted 125I plastic polymer standards (Autoradiographic [I-125] Microscales, Amersham, Arlington Heights, IL) were placed in X-ray cassettes and exposed to Kodak XAR-5 film for 7-10 days at room temperature. Autoradiograms were then developed and sections were stained with thionin for histological examination. The autoradiograms were analyzed using the E Y E C O M Model 850 computer-enhanced densitometry system, version 2.0 (Spatial Data Systems, Melbourne, FL). The 1251 standards, corrected for decay, were used to generate a calibration equation of the general form concentration = a (optical density) + b, where concentration units are dpm/mg polymer. The correlation coefficient of the equation averaged 0.96. Using this equation, the system quantified dpm/mg polymer in regions defined by user-generated templates. Templates were constructed by outlining anatomically distinct brain areas from video images of stained brain sections using the capabilities of the system software. Two brain atlases were used as references in this regard 29' 3~. A total of 34 regions were examined in each brain (Table I). This represented all brain regions that
TABLE I
Dove brain regions analyzed by autoradiography1 Nomenclature taken from Karten and nodos29(extrahypothalamicareas) and Kuenzel and van Tienhoven3a (hypothalamus).
Abbreviation
Region
Abbreviation Region
mc
Nucleus accumbens Nucleus rostralis (anterior) hypothalami Area parahippocampalis Bulbus olfactorius Cerebellum Chiasma opticum Choroid plexus Fasciculus diagonalis Brocae Hyperstriatum accessorium Hyperstriatum dorsale Nucleus habenularis medialis Hippocampus Hyperstriatum ventrale Hyperstriatum ventrale dorso-ventrale Hyperstriatum ventrale ventro-ventrale Nucleus intercollicularis Regio lateralis hypothalami Nucleus mesencephalicuslateralis, pars dorsalis
MLD N NC Nil Ov PMI POA* PT PVN Rt SGP SL SCN SRt TSM TU* * VMN
AM APH BO Cb CO CP FDB HA HD HM Hp HV HVdv HVvv ICo LHy MLd
Nucleus mesencephalicus lateralis, pars dorsalis Neostriatum Neostriatum caudale Nervus oculomotorius Nucleus ovoidalis Nucleus paramedianus internus thalami Preoptic area Nucleus pretectalis Nucleus paraventricularis Nucleus rotundus Substantia grisea et fibrosa periventricularis Nucleus septalis lateralis Nucleus suprachiasmaticus Nucleus subrotundus Tractus septomesencephalicus Tuberal region Nucleus ventromedialis hypothalami
* This area comprisesthe nucleus preopticus medialis (POM) and the nucleus magnocellularispreopticus (PPM). ** This region includes the nucleus inferior hypothalami (IH) and the nucleus infundibuli (IN).
248 appeared upon preliminary visual inspection to contain detectable levels of specific binding as well as several other regions that appeared to contain little or no specific binding. Areas of high specific binding were consistently associated with anatomically distinct regions that were readily detectable in stained sections. Accordingly, the template procedure yielded a reasonably accurate representation of the distribution of specific binding activity. For each region, the dpm/mg polymer values from sections incubated in the non-specific binding condition were subtracted from values obtained from adjacent sections incubated in the total binding condition to yield specific binding estimates. For each region in each brain 4-12 specific binding values were averaged. A total of 6 assays were conducted, each consisting of data from one male and one female. Statistics Preliminary analyses of the autoradiography data revealed significant heterogeneity of variance 3 which necessitated the use of transformed specific binding scores in the analysis of variance (Levene's test F33.374 = 2.23, P < 0.001). Specific binding data from the autoradiographic study and the competitive binding study on tissue homogenates were analyzed separately using three factor mixed design analyses of variance (assay, sex, region). However, sample sizes were not sufficient to statistically evaluate assay by sex interaction effects. Pairwise comparisons of specific binding values were performed using t-tests. A two-tailed significance level of P < 0.05 was used in all analyses. However, significance levels were adjusted by the Dunn-Bonferroni method 38 when multiple pairwise comparisons were performed. Specific binding in a particular brain region was considered significant if the lower limit of the 99% confidence interval was greater than zero.
i
,4.50-
4003501
,--. VMN
, - - A Hp/APH I1--11 Cb
L
T
/"'~ ~ /
1
/
o
200-
0
24
48
72
98
120
144-
INCUBATIONTIME (HOURS) Fig. 1. Effect of incubation time on specific binding of ~251-ovine PRL to homogenates prepared from the nucleus ventromedialis hypothalami (VMN), the hippocampus and area parahippocampalis (Hp/APH), and the cerebellum (Cb). Tissues from 10 female ring doves were pooled for assay. Specific binding estimates (mean + S.E.M.) for each region were based on 4-5 total binding and 4-5 non-specific binding tubes. Homogenate concentrations used for assay were 30 pg/tube for VMN and 100 #g/tube for HP/APH and Cb.
exhibited asymptotic levels by 96 h (Fig. 1). Accordingly, this incubation period was used in all further studies. As shown in Fig. 2, specific binding in each region also increased in linear fashion with increasing protein concentration. Using tissue homogenate protein concentrations in the linear range for each region, 3 assays were conducted to compare levels of specific binding in the 3 brain areas (Fig. 3). No significant effect of sex was observed in the analysis nor did a sex by region interaction effect
~ 500 t
,°°1
e--
•
VMN
,--A
Hp/APH
---,
c~
l
.
i
300-
¢3
z°
200-
~
100-
E3 Z
RESULTS 0
Competitive binding studies on tissue homogenates Specific binding to tissue homogenates represented 10-20% of total binding in all assays. The incubation time required for specific binding of 125I-ovine PRL to reach maximum levels varied across the 3 brain regions; however, all 3 regions
50 1O0 150 200 PROTEINCONCENTRATION~u~ protein/tube)
Fig. 2. Effect of tissue homogenate protein concentration on specific binding of ]25I-ovine PRL to homogenates of 3 microdissected brain regions. See Fig. 1 for abbreviations. Tissues from 8 female ring doves were pooled for assay. Specific binding estimates (mean __ S.E.M.) for each region were based on 3-5 total binding and 3-5 non-specific binding tubes.
249 greater than zero) when data from males and females were pooled for analysis.
DO" mm9
Autoradiographic study As in the competitive binding study on tissue homogenates, specific binding in the autoradiographic study accounted for less than 30% of total binding. Although some specific binding was detected in the majority of regions studied, a significant overall difference was observed across the 34 brain a r e a s ( F 3 3 , 1 6 5 = 18.74, P < 0.001). Levels of specific binding generally tended to be higher in males than in females but this trend was not statistically significant (F1, 5 = 5.19, P = 0.07). However, a significant sex-by-region interaction was found (F33,165 = 1.70, P < 0.02). W h e n the Bonferroni inequality was used to adjust for the number of statistical comparisons performed, no significant differences in specific binding were found between males and females in any of the 34 brain regions. Nevertheless, some evidence for a sexually dimorphic pattern was obtained in the preoptic area, where average specific binding in males was twice that observed in females (Fig. 4). This area was also the only region in which unadjusted t-tests yielded a significant sex difference (tlo = 2.85, P < 0.02). Significant specific binding, as defined by the 99% confidence limit criterion, was mainly concentrated in the preoptic-hypothalamic continuum (Figs. 4 and 5). Within this region, significant specific binding
'o IO0
g m
t/
5°
VMN
Hp/APH
Cb
REGION
Fig. 3. Specific binding of 125I-ovinePRL to homogenates of 3 microdissected brain regions from male and female ring doves. See Fig. 1 for abbreviations. Data shown are mean + S.E.M. specific binding from 6 assays. In each assay, homogenates from two doves were pooled and assayed using 5 total binding and 5 non-specific binding tubes to generate specific binding estimates in each region. Asterisk signifies regions in which the lower limit of the 99% confidence interval of specific binding values is greater than zero.
emerge. However, specific binding did vary significantly across the three regions (F2,t2 = 35.27, P < 0.001), with the ventromedial hypothalamic nucleus exhibiting significantly higher levels of specific binding that the hippocampus/parahippocampus (Bonferr o n i /3.33 = 2.86, P < 0.05) or the cerebellum (Bonferroni/3,33 = 4.01, P < 0.01). Further analysis revealed that the cerebellum was the only region that failed to exhibit significant specific binding (i.e., specific binding with lower 99% confidence limit
•"L"
350. 300.
r---l o"
•
9
,
*
250. m
2001,50. 100. 50-
N o.
o
I REGION
Fig. 4. Specific binding of 125I-ovinePRL to 34 ring dove brain regions as determined by autoradiography and computer-assisted densitometry. Data represent mean _+ S.E.M. specific binding estimates from 6 males and 6 females. Asterisk signifies regions in which the lower limit of the 99% confidence interval of specific binding values is greater than zero. See Table I for abbreviations.
250
A4.75
A 6.50
V APH~-~
V-~~~~
NC HM TSM
A8.50
HA
~
A9.00
St
"
Fig. 5. Autoradiograms depicting areas of significant specific binding of 125I-ovinePRL at 4 different coronal levels of the dove brain. The pigeon atlas of Karten and Hodos 29 with modified nomenclature31 is shown for reference in each panel. Autoradiograms from sections incubated with 75 pM ~25I-ovinePRL alone (total binding) are shown in the top position in each panel while those from adjacent sections incubated with 75 pM ~25I-ovine PRL plus 83 nM unlabelled ovine PRL (non-specific binding) are presented in the bottom position. Principal areas of specific binding depicted in each panel are as follows: A4.75: TU and HM; A6.50: VMN, PVN and CP; A8.50: SCN and POA (POM and PPM); A9.00: POA. Abbreviations for structures not listed in Table I are as follows: AL: ansa lenticularis; PPM: nucleus magnocellularis preopticus; POM: nucleus preopticus medialis; V: cerebral ventricle.
was d e t e c t e d in the preoptic area, suprachiasmatic nucleus, and v e n t r o m e d i a l nucleus in both sexes and in the paraventricular nucleus in males. The tuberal region of the hypothalamus also exhibited significant specific binding in males and females, but binding in the m e d i a n eminence, which lies ventral to this region, could not be reliably assessed due to tissue d a m a g e or loss in many of the brain samples. A m o n g the extrahypothalamic sites surveyed, the medial
habenula and the choroid plexus displayed significant binding at levels c o m p a r a b l e to those in the preoptic area and h y p o t h a l a m u s in one or both sexes. A l t h o u g h specific binding in most other extrahypothalamic regions t e n d e d to be low (Figs. 4 and 5), significant levels were d e t e c t e d in the hippocampus in both sexes, the p a r a h i p p o c a m p a l area in females, and the lateral mesencephalic nucleus (pars dorsalis) in males.
251 DISCUSSION The results of this study provide evidence for PRL binding sites in the choroid plexus and in discretely localized areas within the ring dove forebrain. The validity of these mapping results is strengthened by the close correspondence between patterns of specific binding obtained by autoradiography in the ventromedial nucleus of the hypothalamus, the hippocampal/parahippocampal area, and the cerebellum and binding patterns obtained in these same areas in competitive binding studies using microdissected tissue homogenates. Using tissue homogenates, the ratio of specific binding estimates in VMN, Hp/APH, and Cb was 1.00:0.35:0.14, respectively, in females and 1.00:0.34:0.08 in males. By comparison, the corresponding ratio obtained by autoradiography/densitometry was 1.00:0.36:0.10 in females and 1.00:0.31:0.26 in males after specific binding values for the hippocampus and parahippocampal regions were averaged. The percentage of total binding represented by specific binding was low in both studies, although the values obtained were similar to those detected previously in crude membrane fractions prepared from dove brain homogenates 6. The factors responsible for the relatively high non-specific binding levels in dove brain remain to be determined. However, the response of brain tissue in this regard is not unique, since a similar pattern has been observed in binding studies on membrane fractions prepared from dove liver4 and pigeon crop sac 19 when 125I-ovine PRL was used as the radioligand. As was observed in a previous binding study on dove brain membrane fractions6, autoradiographic analysis revealed low levels of specific binding of lzSI-oPRL in most ring dove brain regions. However, areas of significant specific binding, as defined by the 99% confidence limit criterion, were restricted to the choroid plexus, hippocampus and parahippocampus, medial habenula, lateral mesencephalic nucleus, preoptic area, and 4 hypothalamic regions. In mammals, neurons in two of these regions, the preoptic area and ventromedial nucleus of the hypothalamus, have been previously reported to be responsive to PRL by electrophysiological criteria 1°'46'55. In addition, previous studies have identified cell bodies or nerve terminals containing
PRL-like immunoreactivity in rat brain regions that correspond to areas of significant PRL binding activity in dove brain. These include the preoptic area, the ventromedial and paraventricular nuclei of the hypothalamus, and tuberal hypothalamic regions 2°'24'25'48. These results, together with similar reports of PRL-like immunoreactivity in the preoptic area and periventricular hypothalamus of a teleost 23 and in the lamprey 54, suggest that preoptic and hypothalamic sites of PRL action may be common to a wide range of vertebrate species. Nevertheless, immunocytochemical, biochemical, and neurophysiological studies on various species have suggested relationships between PRL and extrahypothalamic regions that were not identified as areas of significant PRL binding in the present s t u d y 16'25'48. Thus, the lower levels of specific binding observed in other dove brain regions should not be dismissed as unimportant given the relatively conservative definition of significant specific binding used in the present study and the relatively low sensitivity of the film employed for autoradiographic mapping. Although no single brain region exhibited a significant, sexually dimorphic pattern of PRL binding activity when adjustments were made for the total number of individual male-female comparisons performed, the fact that sex differences in specific binding in the preoptic area would have achieved statistical significance in an unadjusted t-test comparison suggests a trend towards a sex-specific pattern of binding in this region. Moreover, recent reports of sex differences in nuclear morphology22" 27.49 and neurochemistry 1'11'17 in the avian and mammalian preoptic area provides some support for this interpretation. However, the physiological basis of a putative sex difference in PRL binding activity in this region is difficult to evaluate, since any binding sites that were already occupied by endogenous dove PRL most likely went undetected in this study. As a result, a sex difference in specific binding in this region could reflect differences in native PRL concentrations in this region instead of differences in receptor binding affinity or capacity. Our results also corroborate previous in vivo autoradiographic evidence for specific lzSI-oPRL binding sites in the dove choroid plexus 7. In contrast to the present results, however, no specific binding was detected in other brain areas following intrave-
252 nous injection of ~25I-ovine PRL in this earlier study. This discrepancy may relate to the short (5 min) interval between injection of the labeled oPRL and sacrifice which may not have provided sufficient time for uptake and binding of 125I-ovine PRL to PRLsensitive sites in the CNS. PRL binding sites have been reported in the choroid plexus in several mammalian species 45'51 and, in the rat, there is evidence that these sites may be part of a receptormediated transport mechanism by which PRL gains access to the cerebrospinal fluid (CSF) from the peripheral circulation 36'52. Many of the major sites of PRL binding observed in the dove brain are situated adjacent to the ventricles and may therefore be accessible to CSF-borne substances. Based on these results, it is conceivable that the choroid plexus plays an important role in the CSF-mediated uptake and delivery of blood-borne PRL to PRL-sensitive target cells in the dove CNS. While our results suggest that PRL binding sites are concentrated in specific loci within the preoptichypothalamic continuum, the role of these regions in mediating PRL-induced behavioral and physiological changes remains to be characterized. Gonadotropin-releasing hormone (GnRH) immunoreactivity has been reported in the avian preoptic area and tuberal hypothalamic region 39'47 and, as the present results indicate, these areas are also sites of significant PRL binding activity in dove brain. There is currently no direct evidence to suggest that PRL acts at these sites to alter GnRH secretion; however, this possibility is plausible in view of the marked dosedependent suppression of plasma LH levels and gonadal weight observed in male doves given i.c.v. injections of ovine PRL 5. Food intake is also dramatically increased in ring doves given i.c.v. injections of ovine PRL 5'~ and it is therefore noteREFERENCES 1 Balthazart, J. and Schumacher, M., Testosterone metabolism and sexual differentiation in quail. In J. Balthazart, E. Prove and R. Gilles (Eds.), Hormones and Behaviour in Higher Vertebrates, Springer, Berlin, 1983, pp. 237-260. 2 Bridges, R.S., DiBiase, R., Loundes, D.D. and Doherty, P.C., Prolactin stimulation of maternal behavior in female rats, Science, 227 (1985) 782-784. 3 Brown, M.B. and Forsythe, A.B., Robust tests for the equality of variances, J. Am. Statist. Assoc., 69 (1974) 364-367. 4 Buntin, J.D., Keskey, T. and Janik, D., Properties of
worthy that the paraventricular nucleus and the ventromedial nucleus of the hypothalamus, which are implicated in the regulation of food intake ~2' 30.33,42, are also areas of significant prolactin binding activity in doves of one or both sexes. Indirect evidence from systemic injection studies also implicates PRL in the expression of 3 reproductive behaviors in the ring dove: maintenance of incubation 28, nest defense 5°, and parental feeding activity32. However, with the exception of the incubation response, which may involve a peripheral site of PRL action 5, the degree to which PRL acts directly on the dove CNS to promote these activities remains to be assessed. The mapping results reported here, together with data already obtained on the binding affinity, capacity, saturability, and specificity of PRL binding sites in dove membrane fractions 6 should prove helpful in subsequent investigations of the mechanisms by which PRL promotes these and other alterations in brain function.
ACKNOWLEDGEMENTS We would like to thank the National Hormone and Pituitary Program, NIDDK, for the allocation of purified ovine prolactin for these experiments and the Biology Department of Marquette University for use of the computerized densitometry system. We also wish to acknowledge Drs. N. Horseman and E. Stein for assistance with the densitometry system, Elaine Ruzycki for expert technical assistance, and Dr. M.D. Davis for his helpful comments on the manuscript. This work was supported by NSF Grant DCB 8303026, NIMH Grant MH41447, and discretionary research funds provided by the Shaw Foundation. hepatic binding sites for prolactin in the ring dove, Gen. Comp. Endocrinol., 55 (1984) 418-428. 5 Buntin, J.D. and Tesch, D., Effects of intracranial prolactin administration on maintenance of incubation readiness, ingestive behavior and gonadal condition in the ring dove, Horm. Behav., 19 (1985) 188-203. 6 Buntin, J.D. and Ruzycki, E., Characterization of prolactin binding sites in the brain of the ring dove (Streptopelia risoria), Gen. Comp. Endocrinol., 65 (1987) 243-253. 7 Buntin, J.D. and Walsh, R.J., In vivo autoradiographic analysis of prolactin binding sites in the brain and choroid plexus of the domestic ring dove, Cell Tissue Res., 251 (1988) 105-109.
253 8 Buntin, J.D., Lea, R.W. and Figge, G., Reductions in plasma LH concentrations and testes weight in ring doves following intracranial injection of prolactin or growth hormone, J. Endocrinol., 118 (1988) 33-40. 9 Clarke, W.C. and Bern, H.A., Comparative endocrinology of prolactin. In C.H. Li (Ed.), Hormonal Proteins and Peptides, Vol. 8, Academic, New York, 1980, pp. 105-197. 10 Clemens, J.A., Gallo, R.V., Whitmoyer, D.I. and Sawyer, C.H., Prolactin responsive neurons in the rabbit hypothalamus, Brain Research, 25 (1971) 371-379. 11 Commins, D. and Yahr, P., Acetylcholinesterase activity in the sexually dimorphic area of the Mongolian gerbil brain, J. Comp. Neurol., 224 (1984) 123-131. 12 Denbow, D.M., Food intake control in birds, Neurosci. Biobehav. Rev., 9 (1985) 223-232. 13 DeVito, W.J., Distribution of immunoreactive prolactin in the male and female rat brain: effects of hypophysectomy and intraventricular administration of colchicine, Neuroendocrinology, 47 (1988) 284-289. 14 DiCarlo, R. and Muccioli, G., Presence of specific prolactin binding sites in the rabbit hypothalamus, Life Sci., 28 (1981) 2299-2307. 15 DiCarlo, R., Muccioli, G., Lando, D. and Bellussi, G., Further evidence for the presenc6 of specific binding sites for prolactin in the rabbit brain. Preferential distribution in the hypothalamus and substantia nigra, Life Sci., 36 (1985) 375-382. 16 Emanuele, N.V., Metcalfe, L., Wallock, L., Tendler, J., Hagen, T.C., Beer, C.T., Martinson, D., Gout, P.W., Kirsteins, L. and Lawrence, A.M., Extrahypothalamic brain prolactin: characterization and evidence for independence from pituitary prolactin, Brain Research, 421 (1987) 255-262. 17 Fischette, C.T., Biegon, A. and McEwen, B.S., Sex differences in serotonin 1 receptor binding in rat brain, Science, 222 (1983) 333-335. 18 Foreman, M. and Porter, J., Prolactin augmentation of dopamine release and norepinephrine release from superfused medial basal hypothalamic fragments, Endocrinology, 108 (1981) 800-804. 19 Forsyth, I.A., Buntin, J.D. and Nicoll, C.S., A pigeon crop sac radioreceptor assay for prolactin, J. Endocrinol., 79 (1978) 349-356. 20 Fuxe, H., HOkfelt, T., Eneroth, P., Gustafson, J.-A. and Skett, P., Prolactin-like immunoreactivity: localization in nerve terminals of rat hypothalamus, Science, 196 (1977) 899-900. 21 Giorgio, M., Giacoma, C., Vellano, C. and Mazzi, V., Prolactin and sexual behavior in the crested newt (Triturus cristatus carnifex Laur.), Gen. Comp. Endocrinol., 47 (1982) 139-147. 22 Gorski, R.A., Gordon, J.H., Shryne, J.E. and Southam, A.M., Evidence for a morphological sex difference within the medial preoptic area of the rat brain, Brain Research, 148 (1978) 333-346. 23 Hansen, B.L. and Hansen, G.N., Immunocytochemical demonstration of somatotropin-like and prolactin-like activity in the brain of Calamoichthys calabaricus (Actinopterygii), Cell Tissue Res., 222 (1982) 615-627. 24 Hansen, B.L., Hansen, G.N. and Hagen, C., Immunoreactive material resembling ovine prolactin in perikarya and nerve terminals of the rat hypothalamus, Cell Tissue Res., 226 (1982) 121-131. 25 Harlan, R.E., Shivers, B.D. and Pfaff, D.W., Midbrain
26 27
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31
32 33 34 35
36 37
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microinfusions of prolactin increase the estrogen dependent behavior, lordosis, Science, 219 (1983) 1451-1453. Hartree, E.F., Determination of protein: a modification of the Lowry method that gives a linear photometric response, Anal. Biochem., 48 (1972) 422-427. Hines, M., Davis, EC., Coquelin, A., Goy, R.W. and Gorski, R.A., Sexually dimorphic regions in the medial preoptic area and bed nucleus of the stria terminalis of the guinea pig brain: a description and an investigation of their relationship to gonadal steroids in adulthood, J. Neurosci., 5 (1985) 40-47. Janik, D.S. and Buntin, J.D., Behavioural and physiological effects of prolactin in incubating ring doves, J. Endocrinol., 105 (1985) 201-209. Karten, H.J. and Hodos, W., A Stereotaxic Atlas of the Brain of the Pigeon (Columba livia), Johns Hopkins, Baltimore, 1967. Kuenzel, W.J., Central neural structures affecting food intake in birds: the lateral and ventral hypothalamic areas. In C.G. Scanes, M.A. Ottinger, A.D. Kenny, J. Balthazart, J. Cronshaw and I. Chester-Jones (Eds.), Aspects of Avian Endocrinology: Practical and Theoretical Implications, Texas Tech University, Lubbock, 1982, pp. 211-216. Kuenzel, W.J. and Van Tienhoven, A., Nomenclature and location of avian hypothalamic nuclei and associated circumventricular organs, J. Comp. Neurol., 200 (1982) 293-313. Lehrman, D.S., The physiological basis of parental feeding behavior in the ring dove (Streptopelia risoria), Behaviour, 7 (1955) 241-286. Liebowitz, S.F., Paraventricular nucleus: a primary site mediating adrenergic stimulation of feeding and drinking, Pharm. Biochem. Behav., 8 (1978) 163-175. Licht, P., Interaction of prolactin and gonadotrophins on appetite, growth and tail regeneration in the lizard, Anolis carolinensis. Gen. Comp. Endocrinol., 9 (1967) 49-53. Locatelli, V., Apud, J.A., Gudelsky, G.A., Cocchi, D., Masotto, C., Casanueva, E, Racagni, G. and Muller, E.E., Prolactin in the cerebrospinal fluid increases the synthesis and release of hypothalamic gamma-aminobutyric acid, J. Endocrinol., 106 (1985) 323-328. Login, I.S. and MacLeod, R.M., Prolactin in human and rat serum and cerebrospinal fluid, Brain Research, 132 (1977) 477-483. Lfithy, I.A., Segura, E.T., L(ithy, V.I., Charreau, E.H. and Calandra, R.S., Prolactin binding sites in the brain and kidneys of the toad, Bufo arenarum Hensel, J. Comp. Physiol. B., 155 (1985) 611-614. Miller, M.G., Jr., Simultaneous Statistical Inference, 2rid edn., Springer, New York, 1966. Mikami, S.-I., Hasegawa, Y. and Miyamoto, K., Localization of avian LHRH-immunoreactive neurons in the hypothalamus of the domestic fowl, Gallus domesticus, and the Japanese quail, Coturnix coturnix, Cell Tissue Res., 251 (1988) 51-58. Moore, B.J., Gerado-Gattens, T., Horowitz, B.A. and Stern, J.S., Hyperprolactinemia stimulates food intake in the female rat, Brain Res. Bull., 17 (1986) 563-569. Moore, K.E., Interactions between prolactin and dopaminergic neurons, Biol. Reprod., 36 (1987) 47-58. Morley, J.E., Levine, A.S., Gosnell, B.A., Billington, C.J. and Krahn, D.D., Control of food intake. In E.E. Muller, R.M. MacLeod and L.A. Frohman (Eds.), Neuroendocrine Perspectives, Vol. 4, Elsevier, Amsterdam,
254 1985, pp. 145-190. 43 Opel, H., Induction of incubation behavior in the hen by brain implants of prolactin, Poult. Sci., 50 (1971) 1613. 44 Palkovits, M., Isolated removal of hypothalamic or other brain nuclei of the rat, Brain Research, 59 (1973) 449-450. 45 Posner, B.I., van Houton, M., Patel, B. and Walsh, R.J., Characterization of lactogen binding sites in the choroid plexus, Exp. Brain Res., 49 (1983) 300-306. 46 Poulain, P. and Carette, B., Actions of iontophoretically applied prolactin on septal and preoptic neurons in the guinea pig, Brain Research, 116 (1976) 172-176. 47 Sterling, R.J. and Sharp, P.J., The localisation of LH-RH neurones in the diencephalon of the domestic hen, Cell Tissue Res., 222 (1982) 283-298. 48 Toubeau, G., Desclin, J., Parmentier, M. and Pasteels, J., Cellular localization of a prolactin-like antigen in the rat brain, J. Endocrinol., 83 (1979) 261-266. 49 Viglietti-Panzica, C., Panzica, G.C., Fiori, M.G., Calcagni, M., Anselmetti, G.C. and Balthazart, J., A sexually dimorphic nucleus in the quail preoptic area, Neurosci. Lett., 64 (1986) 129-134. 50 Vowles, D.M. and Harwood, P., The effect of exogenous
51
52
53
54
55
hormones on aggressive and defensive behaviour in the ring dove (Streptopelia risoria), J. Endocrinol., 36 (1966) 35-51. Walsh, R.J., Posner, B.I., Kopriwa, B.M. and Brauer, J.R., Prolactin binding sites in the rat brain, Science, 201 (1978) 1041-1043. Walsh, R.J., Slaby, F.J. and Posner, B.I., A receptormediated mechanism for transport of prolactin from blood to cerebrospinal fluid, Endocrinology, 120 (1987) 18461850. Wood, P.L., Cheney, D.L. and Costa, E., A prolactin action on acetylcholine metabolism in striatum, hippocampus and thalamus, J. Neurochem., 34 (1980) 10531057. Wright, G.M., Immunocytochemical demonstration of growth hormone, prolactin and somatostatin-like immunoreactivities in the brain of larval, young adult and upstream migrant adult sea lamprey, Petromyzon rnarinus, Cell Tissue Res., 246 (1986) 23-31. Yamada, Y., Effects of iontophoreticaUy-applied prolactin on unit activity of the rat brain, Neuroendocrinology, 18 (1975) 263-271.
245
Elsevier BRE 14484
Localization of prolactin binding sites in ring dove brain by quantitative autoradiography John H. Fechner Jr.* and John D. Buntin Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee 53201 (U.S.A.) (Accepted 25 October 1988)
Key words: Prolactin receptor; Ring dove; Streptopelia risoria; Hypothalamus; Quantitative autoradiography; Telencephalon; Choroid plexus; Sex difference
Specific binding sites for prolactin have been previously detected and characterized in ring dove brain membranes. In order to map the distribution of these sites, specific binding of ~25I-ovine prolactin was examined in slide-mounted sections of 6 male and 6 female ring dove brains by in vitro film autoradiography and densitometry. Analysis of 34 brain regions revealed a sex-specific pattern in specific binding activity. Although no significant sex differences were observed in any individual brain region, a trend in this direction was observed in the preoptic area. Specific binding levels in which the lower limit of the 99% confidence interval was greater than zero were detected in choroid plexus, medial habenula, lateral mesencephalic nucleus, hippocampus, parahippocampal area, preoptic area and 4 hypothalamic sites: paraventricular nucleus, ventromedial nucleus, suprachiasmatic nucleus, and tuberal region. Autoradiographic analysis of specific binding in cerebellum, ventromedial hypothalamus, and hippocampus/parahippocampus yielded relative differences that closely approximated those obtained in binding studies on tissue homogenates from these regions. These results suggest possible sites of prolactin action in altering behavioral state and neuroendocrine function in this species.
INTRODUCTION Prolactin ( P R L ) p r o m o t e s a variety of physiological changes in v e r t e b r a t e s which subserve osmoregulatory, reproductive, and d e v e l o p m e n t a l functions 9. A l t h o u g h m a n y of these effects are m e d i a t e d through h o r m o n a l actions in peripheral target tissues, recent evidence suggests that P R L may also influence the activity of the central nervous system (CNS). Neurons responsive to systemically injected P R L 1° and iontophoretically applied P R L 46"55 have been r e p o r t e d in some m a m m a l i a n species. In addition, P R L - i n d u c e d changes in d o p a m i n e turnover in the t u b e r o i n f u n d i b u l a r region have been well d o c u m e n t e d in the rat 41 and effects of P R L on several o t h e r n e u r o t r a n s m i t t e r systems have also b e e n o b s e r v e d 1s'35'53. Finally, changes in feeding behavior 5"34"4°, sexual b e h a v i o r 21'25, and parental
behavior a'3z'43 have b e e n r e p o r t e d following central or p e r i p h e r a l administration of P R L in various v e r t e b r a t e species. While the b l o o d - b r a i n b a r r i e r precludes direct access of b l o o d - b o r n e P R L to CNS tissue, a specialized u p t a k e mechanism m a y exist in choroid plexus to t r a n s p o r t P R L into the brain by way of the cerebrospinal fluid 52. F u r t h e r m o r e , pituitary-independent, prolactin-like immunoreactivity has been d e t e c t e d in h y p o t h a l a m i c and extrahypothalamic regions in several v e r t e b r a t e s 13'16'2°'23'48'54. This leaves open the possibility that brain function is also altered by P R L - l i k e proteins of CNS origin. Despite evidence for direct n e u r o m o d u l a t o r y effects of P R L , little is known a b o u t the sites or m o d e s of P R L action in the CNS. Specific binding sites for P R L have been described and partially characterized in h o m o g e n a t e s or m e m b r a n e fractions p r e p a r e d from rabbit h y p o t h a l a m u s and substantia nigra 14'15,
* Present address: Molecular Endocrinology Section, Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, Bethesda, MD 20892, U.S.A. Correspondence: J. D. Buntin, Department of Biological Sciences, P.O. Box 413, University of Wisconsin-Milwaukee, Milwaukee, WI 53201, U.S.A. 0006-8993/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
246 ring dove forebrain and midbrain 6, and toad cerebrum, midbrain, and olfactory bulb 37. PRL binding sites have also been detected in the choroid plexus of the rat, pig, sheep, rabbit, and ring dove 7"45"51 by conventional receptor binding techniques or by autoradiography. However, with the exception of the choroid plexus, the discrete localization of PRL binding sites within the CNS has yet to be determined for any vertebrate species. The purpose of this study, accordingly, was to map the distribution of CNS binding sites for PRL in the ring dove (Streptopelia risoria), a species that exhibits marked behavioral and physiological changes following systemic 2s'32"5°, or intracranial PRL administration 5' 8 and possesses saturable CNS binding sites with high affinity and specificity for PRL 6 which could conceivably mediate such effects. Specific binding of radioiodinated oPRL in vitro was examined in 34 brain regions using quantitative autoradiography. Competitive binding studies were also conducted on tissue homogenates from 3 of these regions in order to evaluate the validity of the mapping data obtained by autoradiography. Because the effect of centrally administered PRL on food intake has been previously reported to be sexually dimorphic in this species 5, sex differences in PRL binding were also examined in each region. MATERIALS AND METHODS
Animals Tissue was obtained from non-breeding adult ring doves which were housed individually in visual isolation cages (41 x 41 × 53 cm) under a 14L:10D photoperiod, with lights on at 07.00 h. Temperature was maintained at 19 °C. Food and water were continuously available. Birds were sacrificed between 08.00 and 10.00 h by decapitation and the brains were removed quickly and frozen in powdered dry ice. Sectioning commenced within 4 h of sacrifice.
Competitive binding studies on tissue homogenates Frozen brains were mounted on a chuck using embedding matrix (Lipshaw M-l). 300 p m coronal sections were taken using a clinical microtome, transferred to microscope slides, thawed briefly and refrozen to adhere the sections to the slide. Brain
regions were dissected over dry ice with the aid of a light box and a dissecting microscope using the pigeon brain atlas of Karten and Hodos 29 as a reference. The ventromedial hypothalamic nucleus (VMN) was removed bilaterally using a micropunch 44 made from stainless steel tubing (2 mm o.d., 0.2 mm wall, Small Parts Inc., Miami, FL). The hippocampal/parahippocampal region (Hp/APH) and the cerebellum (Cb) were microdissected using a scalpel blade. Corresponding regions from two or more doves were pooled and suspended in homogenizing buffer (25 mM Tris-HC1, 10 mM CaC12, 0.1% bacitracin, 0.1% sodium azide, pH 7.6) using polyurethane microhomogenizers (Kontes, Vineland, N J). Protein concentration was determined using a modification of the Lowry method 26 with bovine serum albumin (BSA) (Fraction V, Sigma Chemical Co., St. Louis, MO) as the protein standard. Homogenates were snap-frozen in liquid nitrogen and stored at -70 °C until assayed. Ovine PRL (NIADDK-oPRL-I-2) was labelled with carrier-free 1251 to an average specific activity of 56 pCi/pg using a lactoperoxidase procedure 6 and repurified on a 1 × 25 cm Sephadex G-100 column immediately prior to use. Specific binding was determined by methods described previously 6. Homogenates were thawed, rehomogenized and diluted to appropriate protein concentration with assay buffer (homogenizing buffer plus 0.1% BSA). Incubations were carried out in 12 × 75 polystyrene tubes containing 200 pl tissue homogenate, 100/tl 125I-ovine PRL (250 pM), and 200/al assay buffer or unlabelled ovine PRL competitor ( N I A D D K - o P R L 18; 83 nM). Assay tubes were incubated for 96 h at 19 °C unless indicated otherwise. At the end of the incubation period, 3 ml cold assay buffer without bacitracin and sodium azide was added to each tube to terminate the assay. After centrifugation for 1 h at 850 g (4 °C), the supernatants were decanted and the tubes were inverted over absorbent paper. Pellets were counted on a Packard PRIAS C G D Autogamma Spectrometer (65% counting efficiency). Three-5 total binding and non-specific binding tubes were used to determine mean _+ S.E.M. specific binding in each tissue sample. A total of 6 binding assays were conducted, each consisting of one or two pooled tissue samples from each of the 3 brain regions.
247
Autoradiographic study Frozen 20 #m coronal sections were cut in a cryostat (Lipshaw Model 1500) at -15 °C and thawmounted on to gelatin-coated slides. Successive sections were mounted alternately on two sets of slides so that total binding and non-specific binding values could be obtained from adjacent sections. Slides were then air-dried at 4 °C for 24 h and stored at -20 °C for no more than 5 days. Following a 30 min ,preincubation period at 19 °C in Tris-HCl assay buffer (see above), total binding sections were incubated for an additional 24 h at the same temperature in fresh buffer containing 75 pM 125I-ovine PRL (NIADDK-oPRL-I-2). Non-specific binding sections were preincubated in assay buffer containing 83 nM ovine PRL (NIADDK-oPRL-18) and then incubated with 75 pM labelled ovine PRL plus 83 nM unlabelled ovine PRL competitor. Radiolabelled ovine PRL was prepared as described above. Incubation was terminated by washing the slides for 20 min with fresh assay buffer without bacitracin and sodium azide at 4 °C. After two more 20-min washes, the slides were rinsed with cold distilled water, placed on absorbent paper and allowed to dry overnight at room temperature.
Tissue sections and slide-mounted 125I plastic polymer standards (Autoradiographic [I-125] Microscales, Amersham, Arlington Heights, IL) were placed in X-ray cassettes and exposed to Kodak XAR-5 film for 7-10 days at room temperature. Autoradiograms were then developed and sections were stained with thionin for histological examination. The autoradiograms were analyzed using the E Y E C O M Model 850 computer-enhanced densitometry system, version 2.0 (Spatial Data Systems, Melbourne, FL). The 1251 standards, corrected for decay, were used to generate a calibration equation of the general form concentration = a (optical density) + b, where concentration units are dpm/mg polymer. The correlation coefficient of the equation averaged 0.96. Using this equation, the system quantified dpm/mg polymer in regions defined by user-generated templates. Templates were constructed by outlining anatomically distinct brain areas from video images of stained brain sections using the capabilities of the system software. Two brain atlases were used as references in this regard 29' 3~. A total of 34 regions were examined in each brain (Table I). This represented all brain regions that
TABLE I
Dove brain regions analyzed by autoradiography1 Nomenclature taken from Karten and nodos29(extrahypothalamicareas) and Kuenzel and van Tienhoven3a (hypothalamus).
Abbreviation
Region
Abbreviation Region
mc
Nucleus accumbens Nucleus rostralis (anterior) hypothalami Area parahippocampalis Bulbus olfactorius Cerebellum Chiasma opticum Choroid plexus Fasciculus diagonalis Brocae Hyperstriatum accessorium Hyperstriatum dorsale Nucleus habenularis medialis Hippocampus Hyperstriatum ventrale Hyperstriatum ventrale dorso-ventrale Hyperstriatum ventrale ventro-ventrale Nucleus intercollicularis Regio lateralis hypothalami Nucleus mesencephalicuslateralis, pars dorsalis
MLD N NC Nil Ov PMI POA* PT PVN Rt SGP SL SCN SRt TSM TU* * VMN
AM APH BO Cb CO CP FDB HA HD HM Hp HV HVdv HVvv ICo LHy MLd
Nucleus mesencephalicus lateralis, pars dorsalis Neostriatum Neostriatum caudale Nervus oculomotorius Nucleus ovoidalis Nucleus paramedianus internus thalami Preoptic area Nucleus pretectalis Nucleus paraventricularis Nucleus rotundus Substantia grisea et fibrosa periventricularis Nucleus septalis lateralis Nucleus suprachiasmaticus Nucleus subrotundus Tractus septomesencephalicus Tuberal region Nucleus ventromedialis hypothalami
* This area comprisesthe nucleus preopticus medialis (POM) and the nucleus magnocellularispreopticus (PPM). ** This region includes the nucleus inferior hypothalami (IH) and the nucleus infundibuli (IN).
248 appeared upon preliminary visual inspection to contain detectable levels of specific binding as well as several other regions that appeared to contain little or no specific binding. Areas of high specific binding were consistently associated with anatomically distinct regions that were readily detectable in stained sections. Accordingly, the template procedure yielded a reasonably accurate representation of the distribution of specific binding activity. For each region, the dpm/mg polymer values from sections incubated in the non-specific binding condition were subtracted from values obtained from adjacent sections incubated in the total binding condition to yield specific binding estimates. For each region in each brain 4-12 specific binding values were averaged. A total of 6 assays were conducted, each consisting of data from one male and one female. Statistics Preliminary analyses of the autoradiography data revealed significant heterogeneity of variance 3 which necessitated the use of transformed specific binding scores in the analysis of variance (Levene's test F33.374 = 2.23, P < 0.001). Specific binding data from the autoradiographic study and the competitive binding study on tissue homogenates were analyzed separately using three factor mixed design analyses of variance (assay, sex, region). However, sample sizes were not sufficient to statistically evaluate assay by sex interaction effects. Pairwise comparisons of specific binding values were performed using t-tests. A two-tailed significance level of P < 0.05 was used in all analyses. However, significance levels were adjusted by the Dunn-Bonferroni method 38 when multiple pairwise comparisons were performed. Specific binding in a particular brain region was considered significant if the lower limit of the 99% confidence interval was greater than zero.
i
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INCUBATIONTIME (HOURS) Fig. 1. Effect of incubation time on specific binding of ~251-ovine PRL to homogenates prepared from the nucleus ventromedialis hypothalami (VMN), the hippocampus and area parahippocampalis (Hp/APH), and the cerebellum (Cb). Tissues from 10 female ring doves were pooled for assay. Specific binding estimates (mean + S.E.M.) for each region were based on 4-5 total binding and 4-5 non-specific binding tubes. Homogenate concentrations used for assay were 30 pg/tube for VMN and 100 #g/tube for HP/APH and Cb.
exhibited asymptotic levels by 96 h (Fig. 1). Accordingly, this incubation period was used in all further studies. As shown in Fig. 2, specific binding in each region also increased in linear fashion with increasing protein concentration. Using tissue homogenate protein concentrations in the linear range for each region, 3 assays were conducted to compare levels of specific binding in the 3 brain areas (Fig. 3). No significant effect of sex was observed in the analysis nor did a sex by region interaction effect
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Competitive binding studies on tissue homogenates Specific binding to tissue homogenates represented 10-20% of total binding in all assays. The incubation time required for specific binding of 125I-ovine PRL to reach maximum levels varied across the 3 brain regions; however, all 3 regions
50 1O0 150 200 PROTEINCONCENTRATION~u~ protein/tube)
Fig. 2. Effect of tissue homogenate protein concentration on specific binding of ]25I-ovine PRL to homogenates of 3 microdissected brain regions. See Fig. 1 for abbreviations. Tissues from 8 female ring doves were pooled for assay. Specific binding estimates (mean __ S.E.M.) for each region were based on 3-5 total binding and 3-5 non-specific binding tubes.
249 greater than zero) when data from males and females were pooled for analysis.
DO" mm9
Autoradiographic study As in the competitive binding study on tissue homogenates, specific binding in the autoradiographic study accounted for less than 30% of total binding. Although some specific binding was detected in the majority of regions studied, a significant overall difference was observed across the 34 brain a r e a s ( F 3 3 , 1 6 5 = 18.74, P < 0.001). Levels of specific binding generally tended to be higher in males than in females but this trend was not statistically significant (F1, 5 = 5.19, P = 0.07). However, a significant sex-by-region interaction was found (F33,165 = 1.70, P < 0.02). W h e n the Bonferroni inequality was used to adjust for the number of statistical comparisons performed, no significant differences in specific binding were found between males and females in any of the 34 brain regions. Nevertheless, some evidence for a sexually dimorphic pattern was obtained in the preoptic area, where average specific binding in males was twice that observed in females (Fig. 4). This area was also the only region in which unadjusted t-tests yielded a significant sex difference (tlo = 2.85, P < 0.02). Significant specific binding, as defined by the 99% confidence limit criterion, was mainly concentrated in the preoptic-hypothalamic continuum (Figs. 4 and 5). Within this region, significant specific binding
'o IO0
g m
t/
5°
VMN
Hp/APH
Cb
REGION
Fig. 3. Specific binding of 125I-ovinePRL to homogenates of 3 microdissected brain regions from male and female ring doves. See Fig. 1 for abbreviations. Data shown are mean + S.E.M. specific binding from 6 assays. In each assay, homogenates from two doves were pooled and assayed using 5 total binding and 5 non-specific binding tubes to generate specific binding estimates in each region. Asterisk signifies regions in which the lower limit of the 99% confidence interval of specific binding values is greater than zero.
emerge. However, specific binding did vary significantly across the three regions (F2,t2 = 35.27, P < 0.001), with the ventromedial hypothalamic nucleus exhibiting significantly higher levels of specific binding that the hippocampus/parahippocampus (Bonferr o n i /3.33 = 2.86, P < 0.05) or the cerebellum (Bonferroni/3,33 = 4.01, P < 0.01). Further analysis revealed that the cerebellum was the only region that failed to exhibit significant specific binding (i.e., specific binding with lower 99% confidence limit
•"L"
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Fig. 4. Specific binding of 125I-ovinePRL to 34 ring dove brain regions as determined by autoradiography and computer-assisted densitometry. Data represent mean _+ S.E.M. specific binding estimates from 6 males and 6 females. Asterisk signifies regions in which the lower limit of the 99% confidence interval of specific binding values is greater than zero. See Table I for abbreviations.
250
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St
"
Fig. 5. Autoradiograms depicting areas of significant specific binding of 125I-ovinePRL at 4 different coronal levels of the dove brain. The pigeon atlas of Karten and Hodos 29 with modified nomenclature31 is shown for reference in each panel. Autoradiograms from sections incubated with 75 pM ~25I-ovinePRL alone (total binding) are shown in the top position in each panel while those from adjacent sections incubated with 75 pM ~25I-ovine PRL plus 83 nM unlabelled ovine PRL (non-specific binding) are presented in the bottom position. Principal areas of specific binding depicted in each panel are as follows: A4.75: TU and HM; A6.50: VMN, PVN and CP; A8.50: SCN and POA (POM and PPM); A9.00: POA. Abbreviations for structures not listed in Table I are as follows: AL: ansa lenticularis; PPM: nucleus magnocellularis preopticus; POM: nucleus preopticus medialis; V: cerebral ventricle.
was d e t e c t e d in the preoptic area, suprachiasmatic nucleus, and v e n t r o m e d i a l nucleus in both sexes and in the paraventricular nucleus in males. The tuberal region of the hypothalamus also exhibited significant specific binding in males and females, but binding in the m e d i a n eminence, which lies ventral to this region, could not be reliably assessed due to tissue d a m a g e or loss in many of the brain samples. A m o n g the extrahypothalamic sites surveyed, the medial
habenula and the choroid plexus displayed significant binding at levels c o m p a r a b l e to those in the preoptic area and h y p o t h a l a m u s in one or both sexes. A l t h o u g h specific binding in most other extrahypothalamic regions t e n d e d to be low (Figs. 4 and 5), significant levels were d e t e c t e d in the hippocampus in both sexes, the p a r a h i p p o c a m p a l area in females, and the lateral mesencephalic nucleus (pars dorsalis) in males.
251 DISCUSSION The results of this study provide evidence for PRL binding sites in the choroid plexus and in discretely localized areas within the ring dove forebrain. The validity of these mapping results is strengthened by the close correspondence between patterns of specific binding obtained by autoradiography in the ventromedial nucleus of the hypothalamus, the hippocampal/parahippocampal area, and the cerebellum and binding patterns obtained in these same areas in competitive binding studies using microdissected tissue homogenates. Using tissue homogenates, the ratio of specific binding estimates in VMN, Hp/APH, and Cb was 1.00:0.35:0.14, respectively, in females and 1.00:0.34:0.08 in males. By comparison, the corresponding ratio obtained by autoradiography/densitometry was 1.00:0.36:0.10 in females and 1.00:0.31:0.26 in males after specific binding values for the hippocampus and parahippocampal regions were averaged. The percentage of total binding represented by specific binding was low in both studies, although the values obtained were similar to those detected previously in crude membrane fractions prepared from dove brain homogenates 6. The factors responsible for the relatively high non-specific binding levels in dove brain remain to be determined. However, the response of brain tissue in this regard is not unique, since a similar pattern has been observed in binding studies on membrane fractions prepared from dove liver4 and pigeon crop sac 19 when 125I-ovine PRL was used as the radioligand. As was observed in a previous binding study on dove brain membrane fractions6, autoradiographic analysis revealed low levels of specific binding of lzSI-oPRL in most ring dove brain regions. However, areas of significant specific binding, as defined by the 99% confidence limit criterion, were restricted to the choroid plexus, hippocampus and parahippocampus, medial habenula, lateral mesencephalic nucleus, preoptic area, and 4 hypothalamic regions. In mammals, neurons in two of these regions, the preoptic area and ventromedial nucleus of the hypothalamus, have been previously reported to be responsive to PRL by electrophysiological criteria 1°'46'55. In addition, previous studies have identified cell bodies or nerve terminals containing
PRL-like immunoreactivity in rat brain regions that correspond to areas of significant PRL binding activity in dove brain. These include the preoptic area, the ventromedial and paraventricular nuclei of the hypothalamus, and tuberal hypothalamic regions 2°'24'25'48. These results, together with similar reports of PRL-like immunoreactivity in the preoptic area and periventricular hypothalamus of a teleost 23 and in the lamprey 54, suggest that preoptic and hypothalamic sites of PRL action may be common to a wide range of vertebrate species. Nevertheless, immunocytochemical, biochemical, and neurophysiological studies on various species have suggested relationships between PRL and extrahypothalamic regions that were not identified as areas of significant PRL binding in the present s t u d y 16'25'48. Thus, the lower levels of specific binding observed in other dove brain regions should not be dismissed as unimportant given the relatively conservative definition of significant specific binding used in the present study and the relatively low sensitivity of the film employed for autoradiographic mapping. Although no single brain region exhibited a significant, sexually dimorphic pattern of PRL binding activity when adjustments were made for the total number of individual male-female comparisons performed, the fact that sex differences in specific binding in the preoptic area would have achieved statistical significance in an unadjusted t-test comparison suggests a trend towards a sex-specific pattern of binding in this region. Moreover, recent reports of sex differences in nuclear morphology22" 27.49 and neurochemistry 1'11'17 in the avian and mammalian preoptic area provides some support for this interpretation. However, the physiological basis of a putative sex difference in PRL binding activity in this region is difficult to evaluate, since any binding sites that were already occupied by endogenous dove PRL most likely went undetected in this study. As a result, a sex difference in specific binding in this region could reflect differences in native PRL concentrations in this region instead of differences in receptor binding affinity or capacity. Our results also corroborate previous in vivo autoradiographic evidence for specific lzSI-oPRL binding sites in the dove choroid plexus 7. In contrast to the present results, however, no specific binding was detected in other brain areas following intrave-
252 nous injection of ~25I-ovine PRL in this earlier study. This discrepancy may relate to the short (5 min) interval between injection of the labeled oPRL and sacrifice which may not have provided sufficient time for uptake and binding of 125I-ovine PRL to PRLsensitive sites in the CNS. PRL binding sites have been reported in the choroid plexus in several mammalian species 45'51 and, in the rat, there is evidence that these sites may be part of a receptormediated transport mechanism by which PRL gains access to the cerebrospinal fluid (CSF) from the peripheral circulation 36'52. Many of the major sites of PRL binding observed in the dove brain are situated adjacent to the ventricles and may therefore be accessible to CSF-borne substances. Based on these results, it is conceivable that the choroid plexus plays an important role in the CSF-mediated uptake and delivery of blood-borne PRL to PRL-sensitive target cells in the dove CNS. While our results suggest that PRL binding sites are concentrated in specific loci within the preoptichypothalamic continuum, the role of these regions in mediating PRL-induced behavioral and physiological changes remains to be characterized. Gonadotropin-releasing hormone (GnRH) immunoreactivity has been reported in the avian preoptic area and tuberal hypothalamic region 39'47 and, as the present results indicate, these areas are also sites of significant PRL binding activity in dove brain. There is currently no direct evidence to suggest that PRL acts at these sites to alter GnRH secretion; however, this possibility is plausible in view of the marked dosedependent suppression of plasma LH levels and gonadal weight observed in male doves given i.c.v. injections of ovine PRL 5. Food intake is also dramatically increased in ring doves given i.c.v. injections of ovine PRL 5'~ and it is therefore noteREFERENCES 1 Balthazart, J. and Schumacher, M., Testosterone metabolism and sexual differentiation in quail. In J. Balthazart, E. Prove and R. Gilles (Eds.), Hormones and Behaviour in Higher Vertebrates, Springer, Berlin, 1983, pp. 237-260. 2 Bridges, R.S., DiBiase, R., Loundes, D.D. and Doherty, P.C., Prolactin stimulation of maternal behavior in female rats, Science, 227 (1985) 782-784. 3 Brown, M.B. and Forsythe, A.B., Robust tests for the equality of variances, J. Am. Statist. Assoc., 69 (1974) 364-367. 4 Buntin, J.D., Keskey, T. and Janik, D., Properties of
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ACKNOWLEDGEMENTS We would like to thank the National Hormone and Pituitary Program, NIDDK, for the allocation of purified ovine prolactin for these experiments and the Biology Department of Marquette University for use of the computerized densitometry system. We also wish to acknowledge Drs. N. Horseman and E. Stein for assistance with the densitometry system, Elaine Ruzycki for expert technical assistance, and Dr. M.D. Davis for his helpful comments on the manuscript. This work was supported by NSF Grant DCB 8303026, NIMH Grant MH41447, and discretionary research funds provided by the Shaw Foundation. hepatic binding sites for prolactin in the ring dove, Gen. Comp. Endocrinol., 55 (1984) 418-428. 5 Buntin, J.D. and Tesch, D., Effects of intracranial prolactin administration on maintenance of incubation readiness, ingestive behavior and gonadal condition in the ring dove, Horm. Behav., 19 (1985) 188-203. 6 Buntin, J.D. and Ruzycki, E., Characterization of prolactin binding sites in the brain of the ring dove (Streptopelia risoria), Gen. Comp. Endocrinol., 65 (1987) 243-253. 7 Buntin, J.D. and Walsh, R.J., In vivo autoradiographic analysis of prolactin binding sites in the brain and choroid plexus of the domestic ring dove, Cell Tissue Res., 251 (1988) 105-109.
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