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Distribution of Progesterone Receptor Immunoreactivity in the Midbrain and Hindbrain of Postnatal Rats Princy S. Quadros,1 Lisa J. Schlueter,2 Christine K. Wagner2 1

Department of Biological Sciences, Delaware State University, Dover, Delaware 19901

2

Department of Psychology and Center for Neuroscience Research, University at Albany—SUNY, Albany, New York 12222

Received 10 April 2008; revised 11 June 2008; accepted 13 June 2008

ABSTRACT: Nuclear steroid hormone receptors are powerful transcription factors and therefore have the potential to influence and regulate fundamental processes of neural development. The expression of progesterone receptors (PR) has been described in the developing forebrain of rats and mice, and the mammalian brain may be exposed to significant amounts of progesterone, either from maternal sources and/or de novo synthesis of progesterone from cholesterol within the brain. The present study examined the distribution of PR immunoreactive (PRir) cells within the midbrain and hindbrain of postnatal rats. The results demonstrate that PR is transiently expressed within the first 2 weeks of life in specific motor, sensory and reticular core nuclei as well as within midbrain dopaminergic cell groups such as the substantia nigra and the ventral teg-

INTRODUCTION Steroid hormones play a pivotal role in the differentiation of the central nervous system acting via their cognate receptors, a class of proteins belonging to a larger \superfamily" of nuclear receptors that act as Correspondence to: P.S. Quadros ([email protected]). Contract grant sponsor: NIH; contract grant number: HD37244. Contract grant sponsor: NRSA; contract grant number: MH65874. ' 2008 Wiley Periodicals, Inc. Published online 19 August 2008 in Wiley InterScience (www. interscience.wiley.com). DOI 10.1002/dneu.20664

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mental area. Additionally, robust PRir was observed in cells of the lower rhombic lip, a transient structure giving rise to precerebellar nuclei. These results suggest that progestins and progesterone receptors may play a fundamental role in the postnatal development of numerous midbrain and hindbrain nuclei, including some areas implicated in human disorders. Additionally, these findings contribute to the increasing evidence that steroid hormones and their receptors influence neural development in a wide range of brain areas, including many not typically associated with reproduction or neuroendocrine function. ' 2008 Wiley Periodicals, Inc. Develop Neurobiol 68: 1378–1390, 2008

Keywords: PR; SNc; rhombic lip; ontogeny; steroid hormones; VTA; motor nuclei; sensory nuclei; development

powerful transcription factors. This class of nuclear steroid hormone receptors includes estrogen receptors (ER), progesterone/progestin receptors (PR), androgen receptors (AR), glucocorticoid receptors (GR), and mineralicorticoid receptors (MR). Accumulating evidence demonstrates that steroid hormones can affect fundamental processes of neural developmental such as embryonic stem cell proliferation (e.g., Brannvall et al., 2002, 2005; Suzuki et al., 2004; Sundberg et al., 2006), programmed cell death (e.g., Davis et al., 1996; Gould et al., 1997; Park et al., 1998; Chung et al., 2000; Nilsen et al., 2000; Nunez et al., 2000; for review see Forger, 2006), establishment of target-dependent connectivity (Ibanez et al.,

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2001; for review see Simerly, 2005), the expression of neurotrophic factors and their receptors (e.g., Viant et al., 2000; Carrer and Cambiasso, 2002; Solum and Handa, 2002; Roskoden et al., 2004; Franklin and Perrot-Sinal, 2006), as well as neuronal migration (e.g., Henderson et al., 1999; Wolfe et al., 2005). Although the cascade of cellular and molecular events initiated by steroid hormones are diverse and remain poorly understood, steroid hormones and their receptors clearly have the capacity to organize and direct critical aspects of neural development, which ultimately lead to changes in brain function and behavior. Much research has focused on the mechanisms by which testicular-derived hormones, (i.e., testosterone and its metabolite, estradiol) sexually differentiate the central nervous system and alter the development of brain regions with known reproductive and/or neuroendocrine function (for reviews see Cooke et al., 1998; De Vries and Simerly, 2002; Morris et al., 2004; Negri-Cesi et al., 2004; Simerly, 2005; OhtaniKaneko, 2006). However, in addition to testicular hormones, the developing mammalian brain is likely exposed to progesterone. Although the perinatal ovary is quiescent until the second week of life (Schlegel et al., 1967; Quattropani and Weisz, 1973; Greco and Payne, 1994), there appear to be at least two alternate sources of progesterone: the maternal ovary and/or de novo synthesis within the developing brain itself. Maternal progesterone levels are high, not only during gestation, but also during lactation (Pepe and Rothchild, 1974; Martin et al., 1977; Sanyal, 1978; Quadros and Wagner, 1999) and progesterone may pass to neonates through mother’s milk (Betrabet et al., 1987; Toddywalla et al., 1995). In addition, the perinatal rodent brain expresses all the enzymes necessary for the de novo synthesis of progesterone from cholesterol (Compagnone et al., 1995a; Kohchi et al., 1998; Ukena et al., 1998; Zwain and Yen, 1999), potentially producing locally high concentrations of progesterone within specific brain regions. In general, the sensitivity of the brain to a particular steroid hormone is conferred by the expression of the appropriate steroid receptor protein and indeed, various regions of the forebrain express PR during specific periods of development. These include hypothalamic and preoptic regions involved in reproductive and neuroendocrine functions (Quadros et al., 2007), as well as regions not classically associated with reproduction, such as the thalamus, hippocampus, (Quadros et al., 2007) and neocortex (Shughrue et al., 1991; Kato et al., 1993; Lopez and Wagner, 2000; Wagner et al., 2001). Indeed, progesterone and

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its receptor have been implicated in the development of many forebrain systems, including the expression of neurotransmitter enzymes (Franz et al., 1978; Snyder et al., 1979), neuronal morphology (Menzies, 1982; Quadros et al., 2002a) as well as aggressive (Hull et al., 1980; Schneider et al., 2003) and sexual behavior (Hull, 1981; Weinstein et al., 1992; Phelps et al., 1998; Lonstein et al., 2001; Schneider et al., 2005). In contrast to the forebrain, there is very little information on the sensitivity of midbrain and hindbrain structures to progesterone during development. Using the cellular-level resolution of immunocytochemistry, the present study, examined PR expression in the midbrain and hindbrain of postnatal rats and has identified numerous regions that express significant levels of PR immunoreactivity during periods of neural maturation. The results suggest that PR may play a significant role in the development of some motor, sensory, reticular, and dopaminergic cell groups within the midbrain and hindbrain, including brain regions implicated in human disorders.

METHODS Animals The subjects from the following experiments were obtained from 15 nulliparous and primiparous Sprague–Dawley female rats, aged 60–100 days that were mated with males of the same strain. The day copulatory plugs were found, was designated as day 1 of gestation (E1). Pregnant females were housed singly in plastic tubs with bedding and given food and water ad libitum on a 10 h light:14 h dark cycle in a room that was held at a constant temperature of (25 6 2)8C. Pregnant females were left undisturbed for the duration of their pregnancy and allowed to deliver normally. The day of birth was considered postnatal day 1 (P1). On P1, P7, and P14, male and female pups were sacrificed and tissue was collected and prepared as described below All animal procedures used in these experiments were approved by the Institutional Animal Care and Use Committee at the University at Albany–SUNY.

Tissue Preparation Approximately 2 males and 2 females were randomly chosen from separate litters such that the pups sacrificed on P1 were derived from a total of five different litters, while pups sacrificed on P7 or P14 were derived from a total of 11 different litters. All P1 animals were sacrificed by cryoanesthesia followed by decapitation. Brains were removed immediately and immersion-fixed in 5% acrolein in 0.1M phosphate buffer (PB, pH 7.6) for approximately 6 h, followed by cryDevelopmental Neurobiology

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oprotection in 30% sucrose in 0.1 M PB (pH 7.6). Animals sacrificed on P7 or P14 were anesthetized with a lethal dose of nembutal (Pentobarbital sodium, Henry Schein, NY) and intracardially perfused with 0.9% saline followed by 5% acrolein in 0.1 M PB (pH 7.6). All P7 and P14 brains were removed and postfixed in 5% acrolein in 0.1 M PB (pH 7.6) for 2 h followed by cryoprotection in 30% sucrose in 0.1 M PB (pH 7.6). All brainstems were frozen and cut in the coronal plane on a rotary microtome at 50-lm thickness and stored in cryoprotectant (30% sucrose, 0.1% polyvinyl–pyrrolidone-40 in ethylene glycol and 0.1 M PB) at 208C until immunocytochemical processing.

Immunocytochemistry for PR Free-floating brains sections were processed for immunocytochemistry using a rabbit polyclonal antibody (DAKO, Glostrup, Denmark) directed against a region adjacent to the DNA-binding domain of the human PR. This antibody detects both the A and B isoforms of PR (Traish and Wotiz, 1990). All incubations were performed at room temperature unless otherwise indicated. Sections were rinsed in tris-buffered saline (TBS, pH 7.6) three times for 5 min to remove any residual cryoprotectant solution. Sections were then incubated in 1% sodium borohydride in TBS for 10 min, rinsed in TBS four times for 5 min each, and then incubated in TBS containing 20% normal goat serum (NGS), 1% H2O2 and 1% bovine serum albumin for approximately 30 min. PR antiserum was diluted to 1:1000 in TBS containing 2% NGS, 0.3% Triton X-100, and 0.02% sodium azide for 72 h at 48C. Following three rinses (5 min each) in TBS containing 2% NGS, 0.3% Triton X-100, and 0.02% sodium azide, the sections were incubated for 60 min in biotinylated goat antirabbit IgG (Vector Laboratories, Burlingame, CA) at a concentration of 3 lg/mL in TBS containing 2% NGS, 0.3% Triton X-100, and 0.02% sodium azide. After two rinses (5 min each) in TBS containing 2% NGS, 0.3% Triton X-100, and 0.02% sodium azide and two rinses (5 min each) in TBS, the sections were incubated in the Avidin-Biotin complex reagent (Vectastain Elite Kit, Vector laboratories, Burlingame, CA) for 60 min. Following three rinses (5 min each) in TBS, the sections were incubated in TBS containing 0.05% diaminobenzidine, 0.75 mM nickel ammonium sulfate, 0.15% -A-D-glucose, 0.04% ammonium chloride, and 0.001% glucose oxidase for *20 min. The sections were then rinsed three times (5 min each) in TBS, mounted onto gelatin-coated slides and allowed to air-dry. Sections were then dehydrated, delipidated and coverslipped with Permount (Fisher Scientific).

Immunocytochemical Controls Immunocytochemical controls for this PR antiserum have been previously reported (Traish and Wotiz, 1990; Haywood et al., 1999; Quadros et al., 2007). In the present study additional brain sections were collected and prepared as described earlier and were used to test for the specificity of immunoreactivity. Brain sections were incubated in either Developmental Neurobiology

1:1000 or 1:500 DAKO PR antisera that had been preabsorbed overnight at 408C with either 200 or 300 lg/mL of the antigen peptide, respectively (Traish and Wotiz, 1990; amino acids 533–547; Genosys Biotechnologies, The Woodlands, TX). Additionally other brain sections were incubated with untreated PR antisera as a positive control or were incubated in buffer alone, with the primary antisera omitted. PR immunoreactivity was abolished in all brain sections exposed to antisera preabsorbtion or antisera omission.

Drawings and Semi-Quantitative Analysis Because PRir is sexually dimorphic in certain regions of the developing forebrain (Wagner et al., 1998; Quadros et al., 2002b), the distribution of PRir in the brainstem of both postnatal males and females was investigated. However, upon systematic observations of the distribution and ontogeny of PR expression, no obvious sex differences in PRir were observed in any region, at any of the ages examined. Therefore, a representative female from each age group P1, P7, and P14 was selected for drawings. Drawings depicting the distribution of individual PRir nuclei throughout the midbrain and hindbrain were made as follows. Digital images of all sections were captured with a Model 1.3.0 SPOT digital camera (Diagnostic Instruments) attached to a Nikon E600 microscope using a 103 objective. Using Adobe Photoshop on a Macintosh G3 computer, line drawings were superimposed over photomicrographs of select midbrain and hindbrain sections that contained PRir. Black dots were used to indicate the presence of an individual cell nucleus that contained PRir defined by consistent criteria that were applied by an investigator blind to experimental group. Criteria for selecting a positively stained PRir nucleus were as follows: (1) distinct nucleus, visible above background; (2) circular or ovoid in shape; and (3) light gray through black staining intensity. To determine the identity of nuclear cell groups in the perinatal rat forebrain that were positive for PRir, two prenatal (Paxinos et al., 1994; Altman and Bayer, 1995) and one adult (Paxinos and Watson, 1998) rat brain atlases were used as points of reference. The abbreviations for various structures can be found in Table 1. For quantification, a single representative section was selected for each brain region, in each animal at each age. Semiquantification was conducted as follows. The relative total amount of PRir, which takes into account number of PRir cells as well as intensity of stain, was assessed in each brain region on a microscope with a 103 objective by an experimenter blind to the treatment group. PR expression was scored throughout the brain ranging from the lowest expression (þ/) to low expression (þ) to moderate expression (þþ) to high expression (þþþ) to the very highest level of expression (þþþþ). Brain regions that contained relatively low levels of PRir, such as the P14 ventral tegmental area (VTA), were assigned a plus-or-minus sign. On the other hand, brain regions that contained substantially high levels of PRir, such as the P1 lower rhombic lip (LRL), were assigned four plus signs. All other brain regions were visually compared

PR in Developing Brainstem Table 1 Abbreviations of Structures Within the Brain

Table 1

3 6 3V 4V 7n A5 Aq APT Bp cp CIC CB cc CPx DC DLL DMTeg DpMe DRN EW Gi hbc IC icp IF IO OC IP LGN LPB LRL mcp MGN Mo5 MM MPB NTS PAG PAGd PAGdl PAGv PC pn Pn PPTg Pr5 PrC PRH R RL RPn RRF RTA SC SNc SNr s5

Sp5 su3 Teg tz URL VE VTA

Oculomotor nucleus Abducens nucleus Third ventricle Fourth ventricle Facial nerve Adrenergic cell group Aqueduct Anterior pretectal nucleus Brachium pontis Cerebral peduncle Inferior colliculus, central nucleus Cerebellum Central canal Choroid plexus Dorsal cochlear nucleus Dorsal nucleus, lateral lemniscus Dorsomedial tegmental nucleus Deep mesencephalic nucleus Dorsal raphe nucleus Edinger-Westphal nucleus Gigantocellular reticular nucleus Habenular commissure Inferior colliculus Inferior cerebellar peduncle Interfascicular nucleus Inferior olive Inferior olive, central nucleus Interpeduncular nucleus Lateral geniculate nucleus Parabrachial nucleus, lateral division Lower rhombic lip Middle cerebellar peduncle Medial geniculate nucleus Motor trigeminal nucleus Mammillary nuclei Parabrachial nucleus, medial division Nucleus of the solitary tract Periaqueductal gray Periaqueductal gray, dorsal nucleus Periaqueductal gray, dorsolateral nucleus Periaqueductal gray, ventral nucleus Posterior commissure Paranigral region Pontine nucleus Pedunculopontine tegmental nucleus Principal sensory trigeminal root Precommissural nucleus Prepositus hypoglossal nucleus Red nucleus Rhombic lip Raphe pontis nucleus Retrorubral field Reticular formation Superior colliculus Substantia nigra, compact Substantia nigra, reticular Sensory root trigeminal nerve

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(Continued) Spinal trigeminal tract Supraoculomotor central gray Tegmental area Trapezoid body Upper rhombic lip Vestibular nucleus Ventral tegemental area

with these regions, using consistent light levels, and assigned a relative value which was recorded (see Table 2). Brain areas that did not demonstrate any PRir were not assigned any symbol. This process was replicated for all brain regions, at all ages, examined.

Table 2 The Relative Amount of Progesterone Receptor Immunoreactivity (PRir) in the Postnatal Rat Midbrain and Hindbrain P1 Motor Nuclei Substantia nigra, compact Substantia nigra, reticular Ventral tegmental Area Edinger Westphal Nucleus Retrorubral field Abducens nucleus Interfascicular nucleus Paranigral region Sensory Nuclei Inferior colliculus Nucleus of the solitary tract Vestibular nucleus Parabrachial nucleus, lateral Parabrachial nucleus, medial Anterior pretectal nucleus Medial geniculate nucleus Reticular core nuclei Periaqueductal gray, dorsal Periaqueductal gray, ventral Periaqueductal gray, dorsolateral Dorsal raphe nucleus Reticular formation Gigantocellular reticular region Raphe pontis nucleus Pedunculopontine tegmental nucleus Miscellaneous Choroid plexus Cerebellum Lower rhombic lip

P7

P14

þþþ þþþ þ/ þ/ þ  þþþþ þþþþ þþ(þ) þ/ þ/  þ(þ) þ þ/ þ/ þ  þþ þþ þ/ þ þ þ/ þ þ/ þ(þ)

þ(þ) þ þ/ þþ þ

þ/

þ/

þ/ þ/

þþ þþ(þ) þ/  þþþ þþ(þ)

þ/ þ/ þ þ þþ þ/ þþþþ

þ/ þ þ/ þ(þ) þ/ þ 

þ/  þ/ þ/ þ/ þ/ þ þ(þ) þþ(þ) þþþ þþ þþ þþ

  

Scores are scaled as follows: (þ/), lowest expression; (þ), low expression; (þþ), moderate expression; (þþþ), high expression; (þþþþ), highest expression; P, postnatal day.

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Figure 1 The distribution of progesterone receptor immunoreactive (PRir) nuclei in representative coronal sections from a postnatal day 1 female rat brainstem P1, day of birth. Sections are arranged from rostral (A) to caudal (H). Black dots represent individual PRir nuclei. All ventricles are shaded gray. Refer Table 1 for abbreviations.

RESULTS We observed PRir in several distinct nuclear groups within the postnatal midbrain and brainstem. Drawings for representative females, one from each age, can be found in Figures 1–3. Many brainstem nuclei demonstrated dynamic changes in the expression of PRir over the three postnatal ages that were investigated. A semiquantitative analysis of these relative changes is reported in Table 2. Detailed descriptions of the distribution of PRir within specific brainstem nuclear groups over the first 2 weeks of life are reported below.

Motor Nuclei The motor nuclei demonstrating PRir during postnatal life were found primarily in the dopaminergic cell groups, including the substantia nigra, compact (SNc), substantia nigra, reticular (SNr), VTA, retrorubral field (RRF), paranigral region (pn), and interfascicular nucleus (IF). Other motor nuclei that demonstrated PRir were the Edinger-Westphal nucleus (EW) and abducens nucleus. Of these eight, the most abundant PRir was observed in the SNc and VTA (see Fig. 4). PRir was present in the SNc and VTA at Developmental Neurobiology

all ages examined with the greatest amount of staining present at P1 [Fig. 1(A,B)]. Like the SN and VTA, the RRF is part of the mesostriatal and the mesocortical dopaminergic system. A moderate level of PRir was observed in this region of the brainstem at all postnatal ages [Figs. 1(C), 2(C), and 3(C)]. Although the relative levels of PRir were lower in the RRF compared to the SNc and VTA, PR expression in the RRF was highest at P1 and lowest at P14 (see Fig. 5). Two other dopaminergic cell groups that demonstrated PRir during postnatal development were the IF and pn. Although the pn had comparatively fewer cells than the IF, both regions demonstrated PRir at all three postnatal ages examined. Both P1 and P7 females demonstrated PRir in the pn [Figs. 1(B) and 2(B)]. However, by P14, negligible PRir was present in this nucleus. In the IF, a reduction was observed in the level of PRir by P14 [Figs. 1(B), 2(B), and 3(B)]. PRir was also present in the SNr [Figs. 1(B) and 2(B)] and EW [Figs. 1(C) and 2(C)] at P1 and P7 but absent at P14. The cells that did express PRir at both ages were fewer in number relative to the other motor nuclei in the brainstem. The abducens nucleus (6/7n) demonstrated unusual PR expression [Figs. 1(D) and 2(E)]. Like the SNr and EW, PR expression was pres-

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Figure 2 The distribution of progesterone receptor immunoreactive (PRir) nuclei in representative coronal sections from a postnatal day 7 female rat brainstem. Sections are arranged from rostral (A) to caudal (J). Black dots represent individual PRir nuclei. All ventricles are shaded gray. Refer Table 1 for abbreviations.

ent in this nucleus at P1 and P7 but not at P14. What was unusual about the expression was that nuclei expressing PR were much smaller in size and appeared punctate in comparison to the nuclei in other cellular groups (see Fig. 6), suggesting a different cell population, possibly nonneuronal.

Sensory Nuclei Many sensory nuclei were positive for PR during postnatal development. From rostral to caudal, they included anterior pretectal nucleus (APT), medial geniculate nucleus (MGN), parabrachial nucleus (PB), inferior colliculus (IC), superior colliculus (SC), nucleus of the solitary tract (NTS), and vestibular nucleus (VE). Of these, the PB displayed the highest levels of PRir. However, in comparison to the SNc and VTA, the number of PRir cells was much lower in the PB. Two divisions of the PB expressed PRir during postnatal life, the lateral division (LPB) and the medial division (MPB). Although the LPB displayed PRir at all ages examined [Figs. 1(D), 2(E), and 3(D)], the onset of PR expression in the postnatal MPB was not evident until P7 [Figs. 2(E) and 3(F)]. The LPB and MPB expressed PR at relatively the same levels.

The IC demonstrated the second highest level of PRir in this category of brainstem nuclei and almost all the PR-positive cells in this division expressed the receptor with high intensity. Unlike the PB, the onset of PR expression in this region did not occur until P7 [Fig. 2(E)]. PRir was reduced in the IC by P14. The noradrenergic nucleus, NTS, and the VE demonstrated PRir at all postnatal ages [Figs. 1(F,G), 2(G–I), and 3(G)]. There appeared to be fewer PRir cells in the VE compared with the NTS. However, both nuclei demonstrated a slight reduction in the number of PRir cells on P14. In the MGN, PRir was observed at all postnatal ages [Figs. 1(A,B) and 2(A)]. As a point of comparison, the levels of PRir in the MGN were similar to what was observed in the NTS and VE. In addition, similar to the NTS and VE, a slight reduction in PRir was observed at P14. In the APT, PRir was absent until P14, at which point few but strongly labeled nuclei were observed in this region [Fig. 3(A)].

Reticular Core Nuclei Several reticular core nuclei demonstrated PRir during postnatal development. They included, in the Developmental Neurobiology

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Figure 3 The distribution of progesterone receptor immunoreactive (PRir) nuclei in representative coronal sections from a postnatal day 14 female rat brainstem. Sections are arranged from rostral (A) to caudal (G). Black dots represent individual PRir nuclei. All ventricles are shaded gray. Refer Table 1 for abbreviations.

order from highest to lowest levels of expression, periaqueductal gray, dorsolateral nucleus (PAGdl), pedunculopontine tegmental nucleus (PPTg), periaqueductal gray, dorsal nucleus (PAGd), periaqueductal gray, ventral (PAGv), raphe pontis nucleus (RPn), reticular formation (RTA), dorsal raphe nucleus (DRN), and gigantocellular reticular nucleus (Gi). The PAGd and PAGv demonstrated PRir at all three postnatal days [Figs. 1(A,B), 2(A–D), and 3(B–D)]. By P14, the PAGv was devoid of PR expression but still present in the PAGd. Unlike the PAGd and PAGv, PR expression was not evident in the PAGdl until P7 but continued on into P14 (see Fig. 7). The PPTg is a player in one of the major ascending arousal systems in the brainstem and is also thought to be involved in locomotion (for review see Winn, 2006). This particular nucleus of the brainstem expressed PRir at all postnatal ages [Fig. 2(D)]. On P1, very few PRir cells were observed. However, a higher level of PRir was observed on both P7 and P14. The RTA also displayed PRir during postnatal life [Figs. 1(E–G), 2(F–I), and 3(E,G)]. PRir was present in this region on the day of birth and remained conDevelopmental Neurobiology

stant for the rest of the postnatal ages examined. The Gi region of the RTA demonstrated moderately dark PRir cells but only at P7 [Fig. 2(E)]. No PRir was observed in this region at either P1 or at P14. PRir was present in the RPn at all ages [Fig. 3(F)]. In the DRN, very few PRir cells were present throughout the DRN [Figs. 1(C) and 2(D)]. However, unlike the RPn that demonstrated an increase in PRir at later postnatal ages, a reduction in PRir was observed in the DRN at P14.

Miscellaneous Structures The rhombic lip is a structure in the brainstem that is present only during a period of neural development (Wingate, 2001). A region of the rhombic lip, the LRL, displayed the highest levels of PRir in the entire postnatal brainstem [Figs. 1(D,E), 2(E), and 8]. Preliminary data indicate that PRir is present in this structure during fetal life. However, the present ontogeny began on P1. PRir was observed in the LRL at P1 and P7 but not at P14. The absence of PRir at P14 is most likely the result of the dissolution of the

PR in Developing Brainstem

Figure 4 SN and VTA. (A) Progesterone receptor immunoreactive (PRir) nuclei in a representative coronal section from a rostral section of the SNc in a postnatal day 7 female rat. PRir nuclei in (A0 ) the rostral SNc [delineated by the box in (A)] at a higher magnification. (B) PRir nuclei a representative coronal section containing medial SNc and rostral VTA. (B0 ) PRir in the SNc and SNr, delineated by the box in (B) at a higher magnification. (B00 ) PRir in the VTA [delineated by the box in (B)] at a higher magnification. Bars: A and B ¼ 200 lm; A0 , B0 , and B00 ¼ 100 lm. Refer Table 1 for abbreviations.

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midbrain dopaminergic cell groups (SNc, SNr, VTA, RRF, pn, IF). In addition, PRir was observed at extremely high levels in the CPx and the developmentally transient structure, the LRL, which gives rise to precerebellar structures. In the majority of these regions, PRir was evident on the day of birth suggesting that the onset of PR expression may be prenatal. Exceptions were the MPB and IC in which PRir was not observable until P7. In many regions, PR expression was transient during early postnatal life, with the relative amount of PRir being dramatically reduced or virtually absent in most areas by P14. PR expression was observed within many midbrain dopaminergic cell groups (e.g., SNc, SNr, VTA, RRF) on P1 and P7 but decreased or was absent by P14. Other studies have reported an absence of PRir in these nuclei in adulthood (Lonstein and Blaustein, 2004). Therefore, it can be assumed that PR expression is developmentally transient in these regions or that conversion of PR mRNA to protein is developmentally transient, as PR mRNA has been found in these structures in adulthood (CurranRauhut and Petersen, 2002). Interestingly, preliminary data from our lab indicate that PRir in SNc and VTA is localized in both tyrosine hydroxylase (TH)

rhombic lip at some point between the ages of P7 and P14. Nearly every cell at the medial edge of the P1 LRL demonstrated intense PRir, and a few scattered but intensely stained cells were present more laterally. By P7, most of the PRir cells were found in the lateral edge of the LRL. However, the number of PRir cells at P7 was dramatically reduced in comparison to P1. The choroids plexus (CPx), although not a neuronal structure, demonstrated moderately high levels of PRir at P1. PRir was also present in this structure at P7 but completely absent by P14. PRir was also observed in a distinct layer of the cerebellum at P1 and P7. By P14, PRir was no longer observable.

DISCUSSION The present study demonstrates the transient expression of PRir in numerous midbrain and hindbrain structures of the postnatal rat. PRir was found in motor-associated nuclei (EW, abducens), sensory nuclei (APT, PB, IC, SC, NTS, VE), reticular core structures (PAG, PPT, RPn, RTA, DRN, Gi), and

Figure 5 RRF. (A) Progesterone receptor immunoreactive (PRir) nuclei in a representative coronal section from the RRF of a postnatal day 7 female rat. PRir nuclei in the (B) RRF [delineated by the box in (A)] at a higher magnification. Bars: A ¼ 200 lm; B ¼ 100 lm. Refer Table 1 for abbreviations. Developmental Neurobiology

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Figure 6 Abducens. Progesterone receptor immunoreactive (PRir) nuclei in a representative coronal section from the abducens nucleus of (A) a postnatal day 1 female and (B) a postnatal day 7 female. Arrowheads point to the small punctate PRir nuclei found in the medial edge of the nucleus. Bar: 50 lm.

positive cells, as well as nondopaminergic cells (Quadros and Wagner, unpublished observations). Although the exact function of PR within these cell groups is unknown, it is possible that progesterone may be involved in developmental apoptosis. Dopaminergic cells of the SN undergo cell death during the first two postnatal weeks as they form connections with their striatal targets (Burke, 2003). Given the role of steroid receptors in apoptosis within the brain (Yao et al., 2005; Forger, 2006), it is possible that the expression of PR in TH-positive cells within the first two postnatal weeks influences the cell death process either directly, or by altering the sensitivity of dopaminergic cells to target-derived factors which, in turn, regulate cell fate. Indeed, blocking PR function during postnatal life altered the

Figure 7 PAG and PPTg. (A) Progesterone receptor immunoreactive (PRir) nuclei in a representative coronal section from the PAGdll and PPTg. PRir nuclei in the (B) PAG and (C) PPTg [delineated by the boxes in (A)] at a higher magnification. Arrowhead points to the cluster of PRir nuclei within the LPB. Bars: A ¼ 200 lm; C ¼ 100 lm. Refer Table 1 for abbreviations. Developmental Neurobiology

Figure 8 LRL. Progesterone receptor immunoreactive (PRir) nuclei in a representative coronal section from the LRL of (A) a postnatal day 1 female and (C) a postnatal day 7 female. (B and D) Higher magnifications of (A) and (C), respectively. Bars: C ¼ 200 lm; D ¼ 100 lm. Refer Table 1 for abbreviations.

volume of a nucleus, the MPNc, whose size is dictated by developmental apoptosis (Davis et al., 1996; Quadros et al., 2002a). PR expression was also observed in several nuclei that form important ascending and descending circuits within the brain. For example, one of the main ascending projections of the PPTg to the cortex is via the dopaminergic cell groups of the SNc and VTA (Winn, 2006), while a descending projection of the PPTg is to the pontine and medullary reticular formation, where it may influence functions such as attention (Winn, 2006). Interestingly, all of these structures that form a circuit with the PPTg contained PRir during postnatal development. Similarly, the PAG, LPB and NTS all expressed PR at relatively constant levels during postnatal life. These nuclei form the major ascending pain pathway which begins in the spinal cord and continues onto the NTS and the LPB. The LPB, which also receives efferent input from the NTS, projects to the PAGvl, the ventromedial nucleus of the hypothalamus, the central nucleus of the amygdala and the bed nucleus of the stria terminalis (Gauriau and Bernard, 2002). Interestingly, these hypothalamic and extrahypothalamic structures also express intense levels of PRir during postnatal life (Quadros et al., 2007). Although speculative, perhaps synchronized PR expression, in nuclei that form functional circuits, may serve to coordinate the development of these structures and ensure proper connectivity within neural circuits. The robust expression of PRir specifically within the rhombic lip implicates this receptor in the devel-

PR in Developing Brainstem

opmental processes that occur in this transient structure. While cells of the upper rhombic lip (URL) give rise to the cerebellar granule cells (Gao and Hatten, 1994), the LRL gives rise to pontine nuclei and the inferior olive which project mossy and climbing fibers, respectively, to the cerebellum (for review see Wingate, 2001). In the present study, PRir was found exclusively within the LRL. Interestingly, nuclei derived from the LRL have been implicated in sudden infant death syndrome (SIDS). For example, developmental abnormalities have been found in the LRL itself (Panigraphy et al., 2000) and the inferior olive (IO; Kinney et al., 2002) in SIDS cases compared to control infants. It is possible that developmental processes of the LRL may go awry making some infants more vulnerable to SIDS. The fact that PR is highly expressed in the LRL implicates progesterone and its receptor in the normal development of precerebellar nuclei that regulate respiratory and blood pressure responses. The present results suggest that many midbrain and hindbrain regions may be transiently sensitive to progesterone and/or its metabolites during important periods of development and maturation. The perinatal female ovary begins to secrete progesterone after the first week of life (Schlegel et al., 1967; Quattropani and Weisz, 1973; Greco and Payne, 1994). However, the developing brain of both males and females may be exposed to significant levels of progesterone deriving from maternal sources (Pepe and Rothchild, 1974; Martin et al., 1977; Sanyal, 1978; Betrabet et al., 1987; Toddywalla et al., 1995; Quadros and Wagner, 1999) or through the de novo synthesis of progesterone from cholesterol within the developing brain itself (Compagnone et al., 1995a,b; Kohchi et al., 1998; Ukena et al., 1998, 1999). Although very few reports exist on the expression of PR in developing midbrain and hindbrain, the present results are consistent with previous findings demonstrating PR mRNA in homogenized ventral midbrain (Beyer et al., 2002) and with evidence suggesting the expression of PRir within Purkinje cells of the postnatal cerebellum (Sakamoto et al., 2001; Sakamoto et al., 2003). While the function of PR expression in these midbrain and hindbrain structures is, of course, unknown at this time, nuclear steroid receptors are powerful transcription factors, presumably capable of altering the expression of developmentally critical genes. In this way, PR expression within specific structures may be important for ensuring normal patterns of development in these regions. The present findings add to the growing evidence that steroid hormones, and their receptors contribute to the development of more than just forebrain nuclei,

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and include brain regions not directly associated with reproductive or neuroendocrine function. PR expression in midbrain and brainstem nuclei during fetal and neonatal development implicates progesterone in the normal development of these structures and in several neurodevelopmental disorders. Elucidating the steroid sensitivity of the developing brain also becomes clinically important as the use of progestins in the treatment of premature infants (Trotter et al., 1999, 2001; Trotter and Pohlandt, 2000) and of pregnant women for the prevention of premature delivery (Meis et al., 2003; Meis and Aleman, 2004; Dodd et al., 2005; Spong et al., 2005) increases.

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