- Email: [email protected]
Expression of c-fos, fos B, and egr-1 in the medial preoptic area and bed nucleus of the stria terminalis during maternal behavior in rats
Brain Research 792 Ž1998. 348–352
Short communication
Expression of c-fos, fos B, and egr-1 in the medial preoptic area and bed nucleus of the stria terminalis during maternal behavior in rats Michael Numan
) ,1
, Marilyn J. Numan, Sandra R. Marzella, Andrea Palumbo
Department of Psychology, Boston College, Chestnut Hill, MA 02167, USA Accepted 3 March 1998
Abstract The spatial and temporal pattern of expression of the protein products of immediate early genes ŽIEGs. c-fos, fos B, and egr-1 were mapped in medial preoptic area ŽMPOA. and ventral bed nucleus of stria terminalis ŽVBST. during maternal behavior in rats. Immunocytochemical analysis indicated significant increases in the number of cells expressing c-Fos after 2 h of pup exposure, while Fos B levels showed a delayed response, reaching maximal levels after 6 h. q 1998 Elsevier Science B.V. Keywords: Maternal behavior; Medial preoptic area; Immediate early gene; c-Fos; Fos B
Medial preoptic area ŽMPOA. and ventral bed nucleus of stria terminalis ŽVBST. are critical brain regions controlling maternal behavior in rats. Lesions to these regions disrupt maternal behavior w21,25x, and hormone implants Žestradiol, prolactin. into these regions facilitate the behavior w3,9,24x. Recent studies using c-Fos immunocytochemistry to determine the pattern of neural activation in maternal rodents found a strong increase in c-Fos expression within the MPOArVBST after 1–2 h of pup exposure w5,10,19,20,22,31x. Importantly, these studies indicated that c-Fos expression was closely tied to the performance of maternal behavior, suggesting that c-Fos is marking neurons within the preoptic region that are regulating maternal responsiveness. The protein product of the immediate early gene ŽIEG. c-fos is c-Fos. IEGs produce proteins which serve as transcription factors, and therefore are positioned to couple extracellular signals to long-term genomic responses. The c-fos gene is a member of the fos family which includes
)
Corresponding author. Departamento de Psicobiologia, Universidad Nacional de Educacion a Distancia, Ciudad Universitaria srn, P.O. Box 60.148, 28040 Madrid, Spain. Fax: q34-1-398-6287; E-mail: [email protected] 1 After July 29, 1998 and for reprints, correspondence address is: Department of Psychology, Boston College, McGuinn Hall, 140 Commonwealth Avenue, Chestnut Hill, MA 02167, USA. Fax: 1-617-5520523; E-mail: [email protected]. 0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 2 5 7 - 1
fos B, fra 1, and fra 2. Another IEG is egr-1, also referred to as krox-24, zif-268, or ngfi-a w17,18x. The purpose of the present study was to compare the expression of c-Fos with that of Fos B and Egr-1 within the MPOArVBST during maternal behavior in rats. Analysis of Fos B is important because of the finding that a mouse line with a knockout mutation of the fos B gene showed a lack of maternal responsiveness w4x. Since egr-1 mRNA in the MPOA does not increase in maternal sheep w6x, we predicted that Egr-1 in MPOArVBST would not increase during maternal behavior in the rat. Our underlying perspective is that Fos proteins alter genomic responses within MPOArVBST neurons of postpartum ŽPP. females, and that such effects are essential for various aspects of maternal responsiveness Žsee Ref. w22x.. The general design and methods of this experiment, and housing conditions of the subjects, were as previously described w22x. Primiparous female rats ŽCharles River CD strain. gave birth ŽDay 1 PP. in observation cages and their litters were reduced to six pups. On Days 2 and 4 PP each litter was weighed and each female was given a 20-min maternal behavior test. On the afternoon of Day 4, each female was separated from her litter. On Day 7 PP, four groups were formed: no exposure to pups Ž0 h group, n s 10., and either 2 h Ž n s 9., 4 h Ž n s 10., or 6 h Ž n s 10. exposure to pups. A maternal behavior test was given to all females exposed to pups during the first 2 h of the exposure period Žsee Ref. w22x.. Retrieval and nursing behavior were recorded. At the end of the exposure times,
M. Numan et al.r Brain Research 792 (1998) 348–352
females were anesthetized with Nembutal Ž100 mgrkg, i.p.. and when they were unconscious, a vaginal smear was taken. Each female was then intracardially perfused. All testing and perfusions occurred during the dark phase of the light–dark cycle. Importantly, the 0-h exposure females were perfused at different times throughout the dark period to match the perfusion times of females in the different pup exposure groups. Frozen brain sections were cut at 40 m m on a microtome through the hypothalamus in three series for immunocytochemical detection of either c-Fos, Fos B, or Egr-1 labeled cells, using specific polyclonal antibodies purchased from Santa Cruz Biotechnology ŽSanta Cruz, CA.. Brain sections were incubated in rabbit anti-c-Fos Ž1:15 000, cat a SC-52. or anti-Fos B Ž1:1000, cat a SC-48; this antibody detects both the long and short forms of Fos B. for 48 h at 48C, or in rabbit anti-Egr-1 Ž1:500, cat a SC-110. overnight at room temperature. Antibody complexes were visualized by Elite ABC and DAB Substrate Kits ŽVector Labs, Burlingame, CA.. Control tests verified the specificity of our immunocytochemical procedures. Immunocytochemical labeling was eliminated when primary antibody was removed from the procedure or when primary antibody was preabsorbed with an excess of the peptide used to raise the particular antibody. Moreover, preabsorption of the c-Fos antibody with the Fos B peptide, and vice versa, had no effect on the detection of either c-Fos or Fos B immunoreactivity. Brain sections were microscopically examined at 100 = magnification. Labeled cells were counted in the MPOA and VBST on three rostral-to-caudal hemisections through the preoptic region, approximating Plates 19–21 in Swanson’s w30x atlas, and matched for all females. Counting was done blind to treatment and analyzed by ANOVA followed by Fisher’s LSD post-hoc test. P F 0.05 was considered significant. As a control site, labeled cells were similarly counted on three rostral-to-caudal hemisections through the ventromedial nucleus of the hypothalamus ŽVMN. approximating Plates 27–28 in Swanson’s atlas. Maternal behavior in the various groups did not differ on Days 2, 4, and 7 PP: all females retrieved, there were no differences in litter weights, in time spent nursing, nor in vaginal cytology at the time of perfusion. For the Day 7 PP test there was very little variability in maternal responsiveness, with females nursing their young for the major portion of the 2-h test. Correlational analyses indicated that for the 0-h group, the number of c-Fos, Fos B and Egr-1 labeled cells in the MPOA and VBST did not vary significantly with time of perfusion, indicating that baseline expression of these IEGs was constant throughout the dark cycle. Fig. 1 shows the number of MPOArVBST cells Žsummed across three hemisections. labeled with c-Fos, Fos B, and Egr-1 in the various treatment groups. For c-Fos expression, the number of cells increased significantly at 2 h, and remained high throughout the 6 h of pup exposure. In contrast, the
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Fig. 1. Mean number of cells summed across three hemisections ŽqS.E.M.. containing c-Fos, Fos B, or Egr-1 immunoreactivity in the MPOA and VBST in PP rats after various durations of pup exposure. For c-Fos and Egr-1: ) ssignificantly different from remaining groups; For Fos B: ) ssignificantly different from 0 h group; )) ssignificantly different from 0 h and 2 h groups.
number of Fos B-expressing cells increased with duration of pup exposure, and the highest cell counts for both areas occurred at 6 h. Finally, the number of cells in the MPOA and VBST that contained Egr-1 showed a small increase over baseline at 2 h, and this effect was no longer evident at later times. Fig. 2 shows the pattern of Fos expression in the preoptic region at various time points. Note the similarity in pattern for both c-Fos and Fos B: highly concentrated counts occur in the ventromedial and dorsolateral medial preoptic nucleus, and in ventral, magnocellular, and posterodorsal preoptic subnuclei of VBST Žsee Swanson w30x for subdivisions of VBST.. With respect to IEG products in the VMN, our results found only low numbers of c-Fos, Fos B, and Egr-1
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Fig. 2. Distribution of Fos B and c-Fos labeled cells in the MPOA and VBST of PP rats. The upper left hand plate shows a frontal section through the preoptic region, and the dashed area indicates the border of the region that was analyzed for the presence of labeled cells. For Fos B, the spatial distribution of labeled cells is shown for a representative female in the 0-h and 6-h groups. For c-Fos, the spatial distribution of labeled cells is shown for a representative female in the 0-h, 2-h, and 6-h groups. Note the similarity in the spatial distribution of labeled cells for all females exposed to pups. Each dot represents five labeled cells. Abbreviations: AC s anterior commissure; F s fornix; IC s internal capsule; MPNs medial preoptic nucleus; OC s optic chiasm.
labeled cells, and this level of expression did not increase above baseline Ž0-h group. during maternal behavior. A qualitative analysis of other brain regions indicated that our results conformed to other studies w12x. In particular, constitutive expression Žas measured in the 0-h group. of Fos B and Egr-1 was high in the striatum, while the number of striatal cells constitutively expressing c-Fos was low. Finally, we correlated the amount of time spent nursing during the 2-h test on Day 7 PP with the number of MPOA
or VBST cells that were labeled with each of the IEG products in the 2-h, 4-h, and 6-h exposure groups. None of these correlations reached statistical significance. These results are most likely due to the homogeneity in maternal responsiveness during the 2-h test. Variability in number of labeled cells within groups, particularly for the 2-h exposure groups, was most likely the result of variations in the immunocytochemical processing procedure. The most important findings of this study are that the expression of both c-Fos and Fos B is a dominant response
M. Numan et al.r Brain Research 792 (1998) 348–352
of MPOArVBST cells during maternal behavior in rats, while the observed increase in Egr-1 expression is very mild and transient. In addition, we have described the spatial pattern of Fos expression and have quantitatively analyzed the temporal pattern of IEG expression in MPOArVBST during maternal behavior. The facts that the MPOArVBST shows a robust c-Fos and Fos B response during maternal behavior, and that a mouse line with a fos B knockout mutation does not show maternal behavior w4x indicate that future work should explore the effects on maternal behavior of infusing antisense oligodeoxynucleotides to c-fos and fos B mRNA into MPOArVBST. That IEG products were not induced in the VMN during maternal behavior is consistent with our previous work with c-Fos w19x and with findings which indicate that the VMN plays an inhibitory role in maternal behavior w2,29x. It should be noted that other forms of stimulation are capable of inducing c-Fos and Egr-1 in the VMN w27x. Polston and Erskine w27x have reported that mating stimulation in female rats is associated with a dramatic activation of both c-Fos and Egr-1 in MPOA, which contrasts with the strong c-Fos and weak Egr-1 response during maternal behavior. These results suggest that extracellular signals and intracellular responses that regulate MPOA neurons during maternal behavior differ from those which are operative during mating. We found that the temporal pattern of maternal behavior-associated c-Fos and Fos B expression within the MPOArVBST differed. The number of cells expressing c-Fos increased dramatically after 2 h of pup exposure, while the expression of Fos B rose more slowly. One can speculate that with increasing durations of pup exposure, c-Fos expression might have declined, while Fos B expression might have continued to increase. Perhaps during maternal behavior c-Fos mediates the transcription effects of acute stimulation while Fos B mediates the effects of chronic stimulation, as has been suggested for other neural systems w8,11,13x. An alternative interpretation of our temporal data is that mother–pup interactions may have changed over time, and this change may have induced Fos B. Since our behavioral observations were restricted to the first 2 h of pup exposure, we cannot provide data relevant to this point. In contrast to the temporal pattern, the spatial pattern of expression of c-Fos and Fos B within MPOArVBST during maternal behavior was very similar, suggesting that they may be expressed in the same cells. Future doublelabeling studies will be needed to prove this point. The c-Fos and Fos B proteins are more than just markers of neural activation. They have the potential to provide detailed information on the molecular biology of MPOArVBST neurons relevant to maternal behavior. The c-fos and fos B genes contain SRE and CRE sites in their promoter regions w17,18x. Future work should concentrate on uncovering the extracellular signals within the
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MPOArVBST which are capable of affecting these elements. Also, MPOArVBST neurons contain prolactin and oxytocin receptors w1,16,26x, and neurotensin w15x. Since the genes for each of these proteins contain AP-1 sites within their regulatory regions w7,14,28x, and since each of these peptides may also be involved in the regulation of maternal behavior w23x, these genes represent candidates that may be regulated by c-FosrFos B during maternal behavior. Interestingly, the cellular pattern of Fos expression in the MPOArVBST matches the spatial distribution of prolactin receptor mRNA Žcf. Ref. w1x..
Acknowledgements This research was supported by NSF Grant IBN 9319315. Special thanks to Teige Sheehan for critically reviewing the manuscript.
References w1x J.C. Bakowska, J.I. Morrell, Atlas of the neurons that express mRNA for the long form of the prolactin receptor in the forebrain of the female rat, J. Comp. Neurol. 386 Ž1997. 161–177. w2x R.S. Bridges, P.E. Mann, Prolactin–brain interactions in the induction of maternal behavior in rats, Psychoneuroendocrinology 19 Ž1994. 611–622. w3x R.S. Bridges, M. Numan, P.M. Ronsheim, P.E. Mann, C.E. Lupini, Central prolactin infusions stimulate maternal behavior in steroidtreated nulliparous rats, Proc. Natl. Acad. Sci. U.S.A. 87 Ž1990. 8003–8007. w4x J.R. Brown, H. Ye, R.T. Bronson, P. Dikkes, M.E. Greenberg, A defect in nurturing in mice lacking the immediate early gene fos B, Cell 86 Ž1996. 297–309. w5x G. Calamandrei, E.B. Keverne, Differential expression of Fos protein in the brain of female mice dependent upon pup sensory cues and maternal experience, Behav. Neurosci. 108 Ž1994. 113–120. w6x A.P.C. Da Costa, K.D. Broad, K.M. Kendrick, Olfactory memory and maternal-behavior-induced changes in c-fos and zif r 268 expression in sheep brain, Mol. Brain Res. 46 Ž1997. 63–76. w7x P.R. Dobner, E. Kislaukis, B.P. Bullock, Cooperative regulation of neurotensinrneuromedin N gene expression in PC12 cells involves AP1 transcription factors, Ann. New York Acad. Sci. 668 Ž1992. 17–29. w8x J.P. Doucet, Y. Nakebeppu, P.J. Bedard, B.T. Hope, E.J. Nestler, B.J. Jasmin, J.S. Chen, M.J. Iadarola, M. St. Jean, N. Wigle, P. Blanchet, R. Grondin, G.S. Robertson, Chronic alterations in dopaminergic neurotransmission produce a persistent elevation of D Fos B-like proteinŽs. in both rodent and primate striatum, Eur. J. Neurosci. 8 Ž1996. 365–381. w9x S.E. Fahrbach, D.W. Pfaff, Effect of preoptic region implants of dilute estradiol on the maternal behavior of ovariectomized, nulliparous rats, Horm. Behav. 20 Ž1986. 354–363. w10x A.S. Fleming, E.J. Suh, M. Korsmit, B. Rusak, Activation of Fos-like immunoreactivity in the MPOA and limbic structures by maternal and social interactions in rats, Behav. Neurosci. 108 Ž1994. 724–734. w11x P. Gass, T. Herdegen, R. Bravo, M. Kiessling, Induction of immediate early gene encoded proteins in the rat hippocampus after bicuculline-induced seizures: differential expression of krox-24, Fos and Jun proteins, Neuroscience 48 Ž1992. 315–324.
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w12x T. Herdegen, K. Kovary, A. Buhl, R. Bravo, M. Zimmermann, P. Gass, Basal expression of the inducible transcription factors c-Jun, JunB, c-Fos, FosB, and Krox-24 in the adult rat brain, J. Comp. Neurol. 354 Ž1995. 39–56. w13x B. Hope, B. Kosofsky, S.E. Hyman, E.J. Nestler, Regulation of immediate early gene expression and AP-1 binding in the rat nucleus accumbens by chronic cocaine, Proc. Natl. Acad. Sci. U.S.A. 89 Ž1992. 5764–5768. w14x Z. Hu, L. Zhuang, M. Dufau, Multiple and tissue-specific promoter control of gonadal and non-gonadal prolactin receptor gene expression, J. Biol. Chem. 271 Ž1996. 10242–10246. w15x G. Ju, L.W. Swanson, R.B. Simerly, Studies on the cellular architecture of the bed nuclei of the stria terminalis in the rat: II. Chemiarchitecture, J. Comp. Neurol. 280 Ž1989. 603–621. w16x P. Kremarik, J. Freund-Mercier, M.E. Stoeckel, Oxytocin and vasopressin binding sites in the hypothalamus of the rat: histoautoradiographic detection, Brain Res. Bull. 36 Ž1995. 195–203. w17x P.S. Lazo, K. Dorfman, T. Noguchi, M-G. Mattei, R. Bravo, Structure and mapping of the fos B gene: Fos B downregulates the activity of the fos B promoter, Nucleic Acids Res. 20 Ž1992. 343–350. w18x J.I. Morgan, T. Curran, Stimulus–transcription coupling in the nervous system: involvement of the inducible proto-oncogenes fos and jun, Annu. Rev. Neurosci. 14 Ž1991. 421–451. w19x M. Numan, M.J. Numan, Expression of Fos-like immunoreactivity in the preoptic area of maternally behaving virgin and postpartum rats, Behav. Neurosci. 108 Ž1994. 379–394. w20x M. Numan, M.J. Numan, Importance of pup-related sensory inputs and maternal performance for the expression of Fos-like immunoreactivity in the preoptic area and ventral bed nucleus of the stria terminalis of postpartum rats, Behav. Neurosci. 109 Ž1995. 135–149. w21x M. Numan, M.J. Numan, A lesion and neuroanatomical tract-tracing analysis of the role of the bed nucleus of the stria terminalis in retrieval behavior and other aspects of maternal responsiveness in rats, Dev. Psychobiol. 29 Ž1996. 23–51.
w22x M. Numan, M.J. Numan, Projection sites of medial preoptic area and ventral bed nucleus of the stria terminalis neurons that express Fos during maternal behavior in female rats, J. Neuroendocrinol. 9 Ž1997. 369–384. w23x M. Numan, T.P. Sheehan, The neuroanatomical circuitry for mammalian maternal behavior, Ann. New York Acad. Sci. 807 Ž1997. 101–125. w24x M. Numan, J.S. Rosenblatt, B.R. Komisaruk, Medial preoptic area and onset of maternal behavior in the rat, J. Comp. Physiol. Psychol. 91 Ž1977. 146–164. w25x M. Numan, K.P. Corodimas, M.J. Numan, E.M. Factor, W.D. Piers, Axon-sparing lesions of the preoptic region and substantia innominata disrupt maternal behavior in rats, Behav. Neurosci. 102 Ž1988. 381–396. w26x C.A. Pedersen, J.D. Caldwell, C. Walker, G. Ayers, G.A. Mason, Oxytocin activates the postpartum onset of rat maternal behavior in the ventral tegmental and medial preoptic areas, Behav. Neurosci. 108 Ž1994. 1163–1171. w27x E.K. Polston, M.S. Erskine, Patterns of induction of the immediateearly genes c-fos and egr-1 in the female rat brain following differential amounts of mating stimulation, Neuroendocrinology 62 Ž1995. 370–384. w28x F. Rozen, C. Russo, D. Banville, H.H. Zingg, Structure, characterization, and expression of the rat oxytocin receptor gene, Proc. Natl. Acad. Sci. U.S.A. 92 Ž1995. 200–204. w29x T.P. Sheehan, M. Numan, Microinjection of the tachykinin neuropeptide K into the ventromedial hypothalamus disrupts the hormonal onset of maternal behavior in female rats, J. Neuroendocrinol. 9 Ž1997. 677–687. w30x L.W. Swanson, Brain Maps: Structure of the Rat Brain, Elsevier, Amsterdam, 1992. w31x C.J. Walsh, A.S. Fleming, A. Lee, J.E. Magnusson, The effects of olfactory and somatosensory desensitization on Fos-like immunoreactivity in the brains of pup-exposed postpartum rats, Behav. Neurosci. 110 Ž1996. 134–153.
Short communication
Expression of c-fos, fos B, and egr-1 in the medial preoptic area and bed nucleus of the stria terminalis during maternal behavior in rats Michael Numan
) ,1
, Marilyn J. Numan, Sandra R. Marzella, Andrea Palumbo
Department of Psychology, Boston College, Chestnut Hill, MA 02167, USA Accepted 3 March 1998
Abstract The spatial and temporal pattern of expression of the protein products of immediate early genes ŽIEGs. c-fos, fos B, and egr-1 were mapped in medial preoptic area ŽMPOA. and ventral bed nucleus of stria terminalis ŽVBST. during maternal behavior in rats. Immunocytochemical analysis indicated significant increases in the number of cells expressing c-Fos after 2 h of pup exposure, while Fos B levels showed a delayed response, reaching maximal levels after 6 h. q 1998 Elsevier Science B.V. Keywords: Maternal behavior; Medial preoptic area; Immediate early gene; c-Fos; Fos B
Medial preoptic area ŽMPOA. and ventral bed nucleus of stria terminalis ŽVBST. are critical brain regions controlling maternal behavior in rats. Lesions to these regions disrupt maternal behavior w21,25x, and hormone implants Žestradiol, prolactin. into these regions facilitate the behavior w3,9,24x. Recent studies using c-Fos immunocytochemistry to determine the pattern of neural activation in maternal rodents found a strong increase in c-Fos expression within the MPOArVBST after 1–2 h of pup exposure w5,10,19,20,22,31x. Importantly, these studies indicated that c-Fos expression was closely tied to the performance of maternal behavior, suggesting that c-Fos is marking neurons within the preoptic region that are regulating maternal responsiveness. The protein product of the immediate early gene ŽIEG. c-fos is c-Fos. IEGs produce proteins which serve as transcription factors, and therefore are positioned to couple extracellular signals to long-term genomic responses. The c-fos gene is a member of the fos family which includes
)
Corresponding author. Departamento de Psicobiologia, Universidad Nacional de Educacion a Distancia, Ciudad Universitaria srn, P.O. Box 60.148, 28040 Madrid, Spain. Fax: q34-1-398-6287; E-mail: [email protected] 1 After July 29, 1998 and for reprints, correspondence address is: Department of Psychology, Boston College, McGuinn Hall, 140 Commonwealth Avenue, Chestnut Hill, MA 02167, USA. Fax: 1-617-5520523; E-mail: [email protected]. 0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved. PII S 0 0 0 6 - 8 9 9 3 Ž 9 8 . 0 0 2 5 7 - 1
fos B, fra 1, and fra 2. Another IEG is egr-1, also referred to as krox-24, zif-268, or ngfi-a w17,18x. The purpose of the present study was to compare the expression of c-Fos with that of Fos B and Egr-1 within the MPOArVBST during maternal behavior in rats. Analysis of Fos B is important because of the finding that a mouse line with a knockout mutation of the fos B gene showed a lack of maternal responsiveness w4x. Since egr-1 mRNA in the MPOA does not increase in maternal sheep w6x, we predicted that Egr-1 in MPOArVBST would not increase during maternal behavior in the rat. Our underlying perspective is that Fos proteins alter genomic responses within MPOArVBST neurons of postpartum ŽPP. females, and that such effects are essential for various aspects of maternal responsiveness Žsee Ref. w22x.. The general design and methods of this experiment, and housing conditions of the subjects, were as previously described w22x. Primiparous female rats ŽCharles River CD strain. gave birth ŽDay 1 PP. in observation cages and their litters were reduced to six pups. On Days 2 and 4 PP each litter was weighed and each female was given a 20-min maternal behavior test. On the afternoon of Day 4, each female was separated from her litter. On Day 7 PP, four groups were formed: no exposure to pups Ž0 h group, n s 10., and either 2 h Ž n s 9., 4 h Ž n s 10., or 6 h Ž n s 10. exposure to pups. A maternal behavior test was given to all females exposed to pups during the first 2 h of the exposure period Žsee Ref. w22x.. Retrieval and nursing behavior were recorded. At the end of the exposure times,
M. Numan et al.r Brain Research 792 (1998) 348–352
females were anesthetized with Nembutal Ž100 mgrkg, i.p.. and when they were unconscious, a vaginal smear was taken. Each female was then intracardially perfused. All testing and perfusions occurred during the dark phase of the light–dark cycle. Importantly, the 0-h exposure females were perfused at different times throughout the dark period to match the perfusion times of females in the different pup exposure groups. Frozen brain sections were cut at 40 m m on a microtome through the hypothalamus in three series for immunocytochemical detection of either c-Fos, Fos B, or Egr-1 labeled cells, using specific polyclonal antibodies purchased from Santa Cruz Biotechnology ŽSanta Cruz, CA.. Brain sections were incubated in rabbit anti-c-Fos Ž1:15 000, cat a SC-52. or anti-Fos B Ž1:1000, cat a SC-48; this antibody detects both the long and short forms of Fos B. for 48 h at 48C, or in rabbit anti-Egr-1 Ž1:500, cat a SC-110. overnight at room temperature. Antibody complexes were visualized by Elite ABC and DAB Substrate Kits ŽVector Labs, Burlingame, CA.. Control tests verified the specificity of our immunocytochemical procedures. Immunocytochemical labeling was eliminated when primary antibody was removed from the procedure or when primary antibody was preabsorbed with an excess of the peptide used to raise the particular antibody. Moreover, preabsorption of the c-Fos antibody with the Fos B peptide, and vice versa, had no effect on the detection of either c-Fos or Fos B immunoreactivity. Brain sections were microscopically examined at 100 = magnification. Labeled cells were counted in the MPOA and VBST on three rostral-to-caudal hemisections through the preoptic region, approximating Plates 19–21 in Swanson’s w30x atlas, and matched for all females. Counting was done blind to treatment and analyzed by ANOVA followed by Fisher’s LSD post-hoc test. P F 0.05 was considered significant. As a control site, labeled cells were similarly counted on three rostral-to-caudal hemisections through the ventromedial nucleus of the hypothalamus ŽVMN. approximating Plates 27–28 in Swanson’s atlas. Maternal behavior in the various groups did not differ on Days 2, 4, and 7 PP: all females retrieved, there were no differences in litter weights, in time spent nursing, nor in vaginal cytology at the time of perfusion. For the Day 7 PP test there was very little variability in maternal responsiveness, with females nursing their young for the major portion of the 2-h test. Correlational analyses indicated that for the 0-h group, the number of c-Fos, Fos B and Egr-1 labeled cells in the MPOA and VBST did not vary significantly with time of perfusion, indicating that baseline expression of these IEGs was constant throughout the dark cycle. Fig. 1 shows the number of MPOArVBST cells Žsummed across three hemisections. labeled with c-Fos, Fos B, and Egr-1 in the various treatment groups. For c-Fos expression, the number of cells increased significantly at 2 h, and remained high throughout the 6 h of pup exposure. In contrast, the
349
Fig. 1. Mean number of cells summed across three hemisections ŽqS.E.M.. containing c-Fos, Fos B, or Egr-1 immunoreactivity in the MPOA and VBST in PP rats after various durations of pup exposure. For c-Fos and Egr-1: ) ssignificantly different from remaining groups; For Fos B: ) ssignificantly different from 0 h group; )) ssignificantly different from 0 h and 2 h groups.
number of Fos B-expressing cells increased with duration of pup exposure, and the highest cell counts for both areas occurred at 6 h. Finally, the number of cells in the MPOA and VBST that contained Egr-1 showed a small increase over baseline at 2 h, and this effect was no longer evident at later times. Fig. 2 shows the pattern of Fos expression in the preoptic region at various time points. Note the similarity in pattern for both c-Fos and Fos B: highly concentrated counts occur in the ventromedial and dorsolateral medial preoptic nucleus, and in ventral, magnocellular, and posterodorsal preoptic subnuclei of VBST Žsee Swanson w30x for subdivisions of VBST.. With respect to IEG products in the VMN, our results found only low numbers of c-Fos, Fos B, and Egr-1
350
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Fig. 2. Distribution of Fos B and c-Fos labeled cells in the MPOA and VBST of PP rats. The upper left hand plate shows a frontal section through the preoptic region, and the dashed area indicates the border of the region that was analyzed for the presence of labeled cells. For Fos B, the spatial distribution of labeled cells is shown for a representative female in the 0-h and 6-h groups. For c-Fos, the spatial distribution of labeled cells is shown for a representative female in the 0-h, 2-h, and 6-h groups. Note the similarity in the spatial distribution of labeled cells for all females exposed to pups. Each dot represents five labeled cells. Abbreviations: AC s anterior commissure; F s fornix; IC s internal capsule; MPNs medial preoptic nucleus; OC s optic chiasm.
labeled cells, and this level of expression did not increase above baseline Ž0-h group. during maternal behavior. A qualitative analysis of other brain regions indicated that our results conformed to other studies w12x. In particular, constitutive expression Žas measured in the 0-h group. of Fos B and Egr-1 was high in the striatum, while the number of striatal cells constitutively expressing c-Fos was low. Finally, we correlated the amount of time spent nursing during the 2-h test on Day 7 PP with the number of MPOA
or VBST cells that were labeled with each of the IEG products in the 2-h, 4-h, and 6-h exposure groups. None of these correlations reached statistical significance. These results are most likely due to the homogeneity in maternal responsiveness during the 2-h test. Variability in number of labeled cells within groups, particularly for the 2-h exposure groups, was most likely the result of variations in the immunocytochemical processing procedure. The most important findings of this study are that the expression of both c-Fos and Fos B is a dominant response
M. Numan et al.r Brain Research 792 (1998) 348–352
of MPOArVBST cells during maternal behavior in rats, while the observed increase in Egr-1 expression is very mild and transient. In addition, we have described the spatial pattern of Fos expression and have quantitatively analyzed the temporal pattern of IEG expression in MPOArVBST during maternal behavior. The facts that the MPOArVBST shows a robust c-Fos and Fos B response during maternal behavior, and that a mouse line with a fos B knockout mutation does not show maternal behavior w4x indicate that future work should explore the effects on maternal behavior of infusing antisense oligodeoxynucleotides to c-fos and fos B mRNA into MPOArVBST. That IEG products were not induced in the VMN during maternal behavior is consistent with our previous work with c-Fos w19x and with findings which indicate that the VMN plays an inhibitory role in maternal behavior w2,29x. It should be noted that other forms of stimulation are capable of inducing c-Fos and Egr-1 in the VMN w27x. Polston and Erskine w27x have reported that mating stimulation in female rats is associated with a dramatic activation of both c-Fos and Egr-1 in MPOA, which contrasts with the strong c-Fos and weak Egr-1 response during maternal behavior. These results suggest that extracellular signals and intracellular responses that regulate MPOA neurons during maternal behavior differ from those which are operative during mating. We found that the temporal pattern of maternal behavior-associated c-Fos and Fos B expression within the MPOArVBST differed. The number of cells expressing c-Fos increased dramatically after 2 h of pup exposure, while the expression of Fos B rose more slowly. One can speculate that with increasing durations of pup exposure, c-Fos expression might have declined, while Fos B expression might have continued to increase. Perhaps during maternal behavior c-Fos mediates the transcription effects of acute stimulation while Fos B mediates the effects of chronic stimulation, as has been suggested for other neural systems w8,11,13x. An alternative interpretation of our temporal data is that mother–pup interactions may have changed over time, and this change may have induced Fos B. Since our behavioral observations were restricted to the first 2 h of pup exposure, we cannot provide data relevant to this point. In contrast to the temporal pattern, the spatial pattern of expression of c-Fos and Fos B within MPOArVBST during maternal behavior was very similar, suggesting that they may be expressed in the same cells. Future doublelabeling studies will be needed to prove this point. The c-Fos and Fos B proteins are more than just markers of neural activation. They have the potential to provide detailed information on the molecular biology of MPOArVBST neurons relevant to maternal behavior. The c-fos and fos B genes contain SRE and CRE sites in their promoter regions w17,18x. Future work should concentrate on uncovering the extracellular signals within the
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MPOArVBST which are capable of affecting these elements. Also, MPOArVBST neurons contain prolactin and oxytocin receptors w1,16,26x, and neurotensin w15x. Since the genes for each of these proteins contain AP-1 sites within their regulatory regions w7,14,28x, and since each of these peptides may also be involved in the regulation of maternal behavior w23x, these genes represent candidates that may be regulated by c-FosrFos B during maternal behavior. Interestingly, the cellular pattern of Fos expression in the MPOArVBST matches the spatial distribution of prolactin receptor mRNA Žcf. Ref. w1x..
Acknowledgements This research was supported by NSF Grant IBN 9319315. Special thanks to Teige Sheehan for critically reviewing the manuscript.
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