Neurons expressing estrogen receptor α differentially innervate the periaqueductal gray matter of female rats

Journal of Chemical Neuroanatomy 97 (2019) 33–42

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Neurons expressing estrogen receptor α differentially innervate the periaqueductal gray matter of female rats

T

Silvana da Silva Pachecoa,b, Tatiane Araujo Rondinic, Jackson Cioni Bittencourta, ⁎ Carol Fuzeti Eliasd, a

Laboratory of Chemical Neuroanatomy, Department of Anatomy, Institute of Biomedical Sciences, University of São Paulo, São Paulo, SP 05508-900, Brazil University Hospital, University of São Paulo, São Paulo, SP 05508-900, Brazil c Universidade Nove de Julho, São Paulo, SP 01504-001, Brazil d Departments of Molecular and Integrative Physiology and of Obstetrics and Gynecology, University of Michigan, Ann Arbor, MI 48109-5622, United States b

ARTICLE INFO

ABSTRACT

Keywords: Lordosis Sexual behavior Hypothalamus Sex steroids

The periaqueductal gray matter (PAG) is a brainstem site involved in distinct autonomic and behavioral responses. Among them, the motor control of female sexual behavior, including lordosis, is well described. Lordosis reflex is highly dependent on increasing levels of estradiol that occur in the afternoon of the proestrus day in normally cycling females. This effect is thought to be mediated primarily via actions in the ventromedial nucleus of the hypothalamus (VMH). By binding to estrogen receptor α (ERα), estradiol changes the activity of VMH neurons that project to the PAG. Evidence also exists for the coordination of PAG outputs by estradiolresponsive neurons outside the VMH. However, a comprehensive analysis of these circuitries is not available. Using stereotaxic injection of the retrograde tracer Fluorogold in distinct columns of the PAG we performed a systematic mapping of neurons innervating the PAG and those coexpressing ERα immunoreactivity. We found that the forebrain projections to PAG columns are largely segregated and that most of the ERα expressing neurons preferentially target the lateral and the ventrolateral columns. Dual labeled neurons were mostly found in the intermediate subdivision of the lateral septal nucleus, the posterior aspect of the medial bed nucleus of the stria terminalis, the medial preoptic nucleus, the striohypothalamic nucleus and the ventrolateral VMH. Few dual labeled neurons were also observed in the arcuate nucleus, in the posterodorsal subdivision of the medial nucleus of the amygdala and in the ventral premammillary nucleus. Our findings indicate that ERα modulates sexual behavior in female rats via an integrated neural network that differentially innervate the columns of the PAG.

1. Introduction The periaqueductal gray matter (PAG) is a brainstem area involved in a variety of physiological responses associated with autonomic function and behavioral control (Behbehani, 1995; Loyd and Murphy, 2009; Comoli et al., 2003; Miranda-Paiva et al., 2003; Bandler and Shipley, 1994; Lonstein and Stern, 1997). Among them, the role of PAG in female sexual behavior is well defined (Behbehani, 1995; Sakuma and Pfaff, 1979a; Arendash and Gorski, 1983a; Daniels et al., 1999; Veening et al., 2014). In rats, estrous females exhibit a series of complex behaviors when in the presence of a sexually active male. Initially, increased locomotor activity, anogenital sniffing and proceptive behaviors amplify the interest and interactive responses of the male counterpart (Veening et al., 2014; Pfaff, 1999; Erskine, 1989). The receptive

⁎

phase follows with females exhibiting lordotic posture that facilitates male intromission. Lordosis is a well-characterized component of the female sexual behavior. It is achieved in response to sensory/cutaneous stimulation of the back and flanks in conditions of high estrogen milieu that, in cycling females, occur in the afternoon of the proestrus day prior to behavioral estrus (Pfaff and Sakuma, 1979a; Harlan et al., 1984). Estradiol acting in estrogen receptor α (ERα) expressed in hypothalamic sites are crucial for the initiation, modulation and achievement of lordotic posture (Ogawa et al., 1998). In particular, ERα neurons in the ventrolateral subdivision of the ventromedial nucleus of the hypothalamus (VMHvl) are necessary for successful lordosis (Pfaff and Sakuma, 1979b, c; Musatov et al., 2006). Administration of estradiol into the VMHvl induces lordosis in females and in castrated males

Corresponding author at: 1137 E Catherine St, 7732B Medical Science Building II, Ann Arbor, MI 48109-5622, United States. E-mail address: [email protected] (C. Fuzeti Elias).

https://doi.org/10.1016/j.jchemneu.2019.01.004 Received 29 November 2018; Received in revised form 7 January 2019; Accepted 9 January 2019 Available online 28 January 2019 0891-0618/ © 2019 Elsevier B.V. All rights reserved.

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(Barfield and Chen, 1977; Davis and Barfield, 1979; Malsbury et al., 1977). Lesions of the VMHvl, RNA silencing of VMH ERα or sectioning of descending pathways lateral to the VMHvl disrupt the ability of females to achieve complete lordotic posture (Pfaff and Sakuma, 1979b; Musatov et al., 2006; Malsbury et al., 1977; Mathews et al., 1983). However, stimulation of VMHvl neurons when estradiol is low is not sufficient to induce lordosis. Whether estradiol-induced changes in VMH neuronal activity and/or morphology, or alternative estradiolresponsive brain sites are required for successful lordosis is not completely known (Chung et al., 1988; Flanagan-Cato et al., 2001). The ERα expressing neurons in the VMHvl do not directly innervate the motor neurons of skeletal muscles responsible for lordotic posture. Rather, they project to a relay station in the PAG (Daniels et al., 1999; Akesson et al., 1994; Hennessey et al., 1990). The PAG is a mantle of neurons surrounding the mesencephalic aqueduct with anatomical and functional subdivisions organized in heterogeneous rostral-to-caudal columns (Bandler and Shipley, 1994; Beitz, 1985). Lesion of the PAG dramatically decreases lordosis and disrupt the ability of VMH stimulation to facilitate lordotic reflex (Hennessey et al., 1990; Lonstein and Stern, 1998; Sakuma and Pfaff, 1979b). However, the role of distinct subdivision(s) or column(s) is still debated. The dorsolateral column receives direct inputs from lumbar spinal cord, indicating this site is a receptive field of sensory/cutaneous stimulation (Behbehani, 1995; Sakuma and Pfaff, 1980; Van der Horst and Holstege, 1998). On the other hand, the ventrolateral column appears to play a role in motor responses as it projects directly, or via interneurons in the hindbrain, to lumbar motor neurons (Daniels et al., 1999; Veening et al., 2014; Van der Horst and Holstege, 1998). To gain insights into the neural network controlling lordosis in female rats, we performed a systematic analysis of the forebrain inputs to the dorsolateral, the lateral and the ventrolateral columns of the PAG. We further defined the topographic distribution of PAG afferents directly targeted by circulating estradiol, specifically those coexpressing ERα.

(pH 9.5 at 4 °C). The brains were removed, postfixed in the same fixative for 4 h at 4 °C, and cryoprotected in 0.1 M phosphate buffer (PBS, pH 7.4) containing 20% sucrose. The brains were cut in freezing microtome (30 μm-thickness, 5 series in frontal plan) and sections were stored in cryoprotectant at −20 °C until they were processed for single or dual label immunohistochemistry. Four adult female rats (180–220 g, 70–80 days of age) were perfused, brains were collected and processed, as described. Sections were mounted onto gelatin coated slides, air dried and subjected to thionin staining. These brains were used as neuroanatomical reference for identification of PAG columns. 2.3. Single and dual label immunohistochemistry One series of each brain with FG injections was pretreated with 0.3% hydrogen peroxide (H2O2) for 30 min followed by a blocking solution containing 2% normal donkey serum (Jackson Immunoresearch) for 1 h, both diluted in PBS and 0.3% Triton X-100. Sections were incubated in anti-FG primary antisera made in rabbit (1:20,000, Millipore, AB153) overnight at room temperature. After washes in PBS, sections were incubated in biotinylated goat anti-rabbit IgG (1:1,000, Vector Labs) followed by avidin-biotin complex (1:500, Vector Elite Kit) for 1 h at room temperature. Sections were then rinsed and placed in 0.05% diaminobenzidine tetrahydrochloride (DAB; Sigma) and 0.01% H2O2. The reaction was terminated after 4–6 min with two successive rinses in PBS. Sections were mounted onto gelatin coated slides, dried overnight, dehydrated in ethanol, cleared in xylene and coverslipped with DPX (BDH). One series of forebrain sections with FG correctly targeting the PAG was processed for dual label immunohistochemistry. Sections were pretreated as described and incubated in ERα rabbit polyclonal antisera (1:20,000, Millipore, cat# 06-935). This antibody recognizes C-terminal of ERα (accession number NP_036821). Peroxidase reaction was processed with DAB and 0.01% nickel sulfate (Fisher Scientific) resulting in a black staining. Sections were pretreated in 0.3% H2O2 and blocking solution, followed by overnight incubation in FG primary antiserum (1:20,000). Sections were processed as above using only DAB as chromogen resulting in a brown reaction product. Tissue was mounted onto gelatin coated slides, dried overnight, dehydrated in ethanol, cleared in xylene and coverslipped with DPX.

2. Methods 2.1. Animals Adult female Wistar rats (180–200 g) were housed one or two per cage in a light- and temperature-controlled environment (12 h on/off) with food and water available ad libitum. All animals were bilaterally ovariectomized on postnatal days 65–80 to improve the detection of estrogen receptor immunoreactivity (Yamada et al., 2009; Frazao et al., 2014). Experiments were carried out in accordance with the guidelines of the National Institute of Health Guide for the Care and Use of Laboratory Animals and the Institutional Committee for Research and Animal Care of the University of São Paulo, Brazil.

2.4. Data analysis and production of Photomicrographs Brain sections were analyzed using an Axio Imager M2 microscope (Zeiss). The distribution of single-labeled FG immunoreactive (FG-ir) neurons was initially assessed in series of the entire forebrain using the “The Rat Brain in Stereotaxic Coordinates”, by Paxinos and Watson (1997) (Table 1). Sites associated with reproductive control showing apparent differences on pattern of distribution or density of retrograde labeled cells were quantified. These included the intermediate subdivision of the lateral septal nucleus (LSi), the posterior subdivision of the medial bed nucleus of the stria terminalis (BSTMp), the medial preoptic nucleus (MPO), the striohypothalamic nucleus (Sthy), the VMHvl, the posterodorsal subdivision of the medial nucleus of the amygdala (MeApd), the medial tuberal (MTu) and ventral premammillary nuclei (PMV). Only one side from one section of each rat brain using the same area of interest for all sections of the same nuclei was used for quantification to avoid double counting. We considered single-labeled neurons those with brown cytoplasm (FG) and dual-labeled neurons those with brown cytoplasm and black nucleus (ERα) detected under bright field illumination. The mean numbers of FG-ir, of FG-ir + ERα-ir neurons and mean percentage of colocalization comparing total FG-ir neurons were calculated and expressed as mean ± standard error of the mean (SEM). Statistical comparison was not performed due to differences in size and site of injections. Photomicrographs were produced by capturing images with a

2.2. Retrograde tracer injection Forty female rats (180–220 g, 65–80 days of age) were anaesthetized with subcutaneous injection of ketamine (1 mg), xylazine (5 mg) and acepromazine (0.2 mg) cocktail (0.2 mL/100 g) and received unilateral stereotaxic delivery of the retrograde tracer Fluorogold (FG, Fluorochrome) into the PAG with the intent to target the dorsolateral, the ventrolateral or the lateral columns. The tracer was injected iontophoretically from a glass micropipette (internal diameter = 10–20 μm) by applying +5 μAmp current pulsed at 7 s intervals over 10 min. The stereotaxic coordinates for FG injections into the PAG ranged from −7.5 to −7.8 mm (anteroposterior from the Bregma), 0.6 to 0.7 mm (mediolateral from the saggital vein), and −5.0 to −5.5 mm (dorsoventral from the dura mater). Following 15 days, rats were deeply anesthetized with intraperitoneal 35% chloral hydrate (1 mL) and perfused via the ascending aorta with saline followed by 4% formaldehyde (obtained from paraformaldehyde) in 0.1 M borate buffer 34

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Table 1 Distribution of forebrain neurons projecting to different columns of the periaqueductal gray matter (PAG). Subjective analysis comparing number of FG immunoreactive (FG-ir) cells (n = 3–4/column). +, low; ++, moderate; +++, high; ++++, very high. Abbreviations: DL, dorsolateral; L, lateral; VL, ventrolateral. Forebrain Sites

FG-ir Neurons ipsilateral

contralateral

DL

VL

L

DL

VL

L

+ + − −

− − − −

+ + ++ +

− − − −

− − − −

+ − ++ −

−

−

+

−

−

+

+ + − − +

− ++ − + +

+ + + + +

− − − − −

− − − − −

+ + + − +

Bed Nucleus of Stria Terminalis/Amygdala Bed Nu. of Stria Terminalis Medial, ventromedial Bed Nu. of Stria Terminalis Medial, lateral ventral Bed Nu. of Stria Terminalis Medial, posteromedial Central Nucleus of the Amygdala Medial Nucleus of the Amygdala, posterodorsal Cortical Nucleus of the Amygdala

− − + ++ + −

++ +++ +++ + ++ −

+++ +++ ++ ++ + +

− − − + − −

+ − + − − −

++ − + + + +

Hypothalamus Medial Preoptic Nucleus, medial Medial Preoptic Nucleus, lateral Lateral Preoptic Nucleus StrioHypothalamic Nucleus Periventricular Nucleus Paraventricular Nucleus, parvicellular anterior Paraventricular Nucleus, parvicellular dorsal Paraventricular Nucleus, parvicellular ventral Paraventricular Nucleus, parvicellular posterior Anterior Hypothalamic Area Lateroanterior Hypothalamic Nucleus Incertohypothalamic Nu. (Medial Zona Incerta) Retrochiasmatic Area Arcuate Nucleus Lateral Hypothalamic Area, anterior Lateral Hypothalamic Area, tuberal Perifornical Area Ventromedial Nu. of the Hypothalamus, dorsomedial Ventromedial Nu. of the Hypothalamus, ventrolateral Ventromedial Nu. of the Hypothalamus, central Dorsomedial Nu. of the Hypothalamus, dorsal Dorsomedial Nu. of the Hypothalamus, ventral Medial Tuberal Nucleus Dorsal Tuberomammillary Nucleus Ventral Premammillary Nucleus Dorsal Premammillary Nucleus Supramammillary Nucleus

+++ + + + + ++ ++ − + ++ ++ ++ ++ ++ ++ ++ − +++ + ++ ++ ++ + ++ − +++ −

+ +++ ++ +++ ++ + ++ + + + +++ ++ + + +++ + ++ − ++ + ++ ++ + ++ + + + +

++ ++ + ++ + ++ ++ + + ++ ++ ++ ++ ++ + ++ ++ ++ ++ ++ ++ ++ ++ ++ + ++ +

+ + + − − + − − − +

+ + + + − − + + − +

++ ++ + + + + + + + ++

+ − − + − − ++ + + + + + − − − −

+ − + − + + − + + + − + + + − +

+ − + + + + + ++ ++ + + + + + + +

+ − − − +

− + − − +

++ + + ++ +++

+ − − − −

− ++ − − +

+ − − + −

− +

+ +

+ +

− −

− −

− −

Cerebral Cortex/Subcortical Nuclei Cingulate Cortex Motor Cortex Insular Cortex Claustrum Dorsal Endopiriform nucleus Septum/Basal Forebrain Lateral Septum, dorsal Lateral Septum, intermediate Lateral Septum, ventral Diagonal Band of Broca Substancia Innominata

Thalamus/Subthalamus Paraventricular Nucleus of the Thalamus Medial Habenula Reticular Thalamic Nucleus Parasubthalamic Nucleus Precomissural Nucleus Circumventricular Organs Vascular Organ of the Lamina Terminalis Subfornical Organ

35

++ +

+ + + + + + +

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Fig. 1. Fluorogold (FG) injection sites in distinct columns of the periaqueductal grey matter (PAG). (A–C) Bright field images showing examples of FG injections (arrows) targeting the dorsolateral PAG (PAG-dl, A), the ventrolateral PAG (PAGvl, B) and the lateral PAG (PAG-l, C). (D-E), Bright field images showing cytoarchitecture of the PAG in thionin staining as reference. Abbreviations: 4, throclear nucleus; Aq, aqueduct; DR, dorsal raphe; mlf, medial longitudinal fascicle. Scale bar: 400 μm.

was also found in the BSTMp (Fig. 4A). In the amygdala, moderate number of FG-ir neurons was observed in the central nucleus and a reduced number in the MeApd. In the hypothalamus, we found strong projections from the medial subdivision of the MPO (MPOm, Fig. 4B), the dorsomedial subdivision of the VMH (VMHdm, Fig. 4C) and the dorsal premammillary nucleus. Moderate numbers of FG-ir neurons were observed in the anterior and dorsal parvicellular subdivisions of the paraventricular nucleus of the hypothalamus (PVH), the anterior and lateroanterior hypothalamic nuclei, the retrochiasmatic area, the arcuate nucleus, the lateral hypothalamic area, the dorsomedial nucleus of the hypothalamus and the dorsal tuberomammillary nucleus. Few retrograde labeled neurons were observed in the lateral MPO (MPOl, Fig. 4B), and in the lateral preoptic, the striohypothalamic, the periventricular and the medial tuberal (MTu) nuclei. Outside the hypothalamus, a small number of retrograde labeled neurons were observed in the subfornical organ, in the paraventricular nucleus of the thalamus, and in the precomissural nucleus.

digital camera (Axiocam, Zeiss) mounted directly on the microscope using the Zen software (Zeiss). Adobe Photoshop CC image-editing software was used to integrate photomicrographs into plates. For data illustration, only sharpness, contrast and brightness were adjusted. 3. Results 3.1. Retrograde tracer injection Of 40 females injected with FG, 10 had injections restricted to one column of the PAG, i.e., the dorsolateral (PAG-dl, n = 3), the ventrolateral (PAG-vl, n = 3) and the lateral (PAG-l, n = 4) columns (Figs. 1 and 2). Eight females had injections centered in the PAG-l but also contaminating one or more PAG columns. Those cases were analyzed in separate and used as control. The remaining 22 females had either no injections (n = 8) or had injections outside the PAG, i.e., in the aqueduct (n = 6) or in the adjacencies of the PAG (n = 8). Brains with injections centered in PAG columns (dl, vl and l) were mapped for distribution of FG-ir neurons and further evaluated for coexpression of FG and ERα immunoreactivity.

3.2.2. Ventrolateral column of the PAG (PAG-vl) Virtually no retrograde labeled neurons were observed in the cerebral cortex, and only few FG-ir neurons were found in the diagonal band of Broca and substantia innominata. Compared to the PAG-dl, we found higher numbers of retrograde labeled neurons in the LSi and in the vascular organ of the lamina terminalis (OVLT). Moderate to high numbers of FG-ir were observed in several subdivisions of the BSTM, including the ventromedial, the lateral ventral and the BSTMp (Fig. 4D). Higher numbers of FG-ir neurons were observed in the MeApd, but lower numbers were found in the central nucleus of the amygdala compared to PAG-dl. In the hypothalamus, differences between the density of neurons projecting to PAG-vl and those projecting to the PAG-dl were evident. Moderate to higher numbers of FG-ir neurons were observed in the lateral MPO (MPOl, Fig. 4E), in the StHy, in the ventrolateral VMH (VMHvl, Fig. 4F) and in the PMV. On the other hand, fewer retrograde labeled neurons were found in the MPOm (Fig. 4E), in the anterior hypothalamic nucleus, in the retrochiasmatic area, arcuate nucleus, in

3.2. Distribution of retrograde labeled neurons projecting to distinct columns of the PAG The analysis of the distribution of retrograde labeled neurons was performed by comparing the innervation pattern of distinct columns of the PAG. The subjective analysis is summarized in Table 1. The forebrain nuclei showing apparent differences were subjected to a quantitative analysis described in Table 2 and Fig. 3. In general, we found that the hypothalamus is the main source of descending afferents to the PAG. 3.2.1. Dorsolateral column of the PAG (PAG-dl) Very few FG-ir neurons were observed in the cerebral cortex and subcortical nuclei, including the cingulate and motor cortex, the dorsal and the intermediate subdivisions of the lateral septal nucleus (LS) and the substantia innominata. A small number of retrograde labeled neurons 36

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Fig. 2. Schematic illustration of fluorogold (FG) injection sites targeting different columns of the periaqueductal grey matter (PAG). (A–C), injections of FG (dark area) into the dorsolateral (A), ventrolateral (B) and lateral (C) columns of the PAG. Abbreviations: Aq, aqueduct; CLi, central linear nucleus; cp, cerebral peduncle; IP, interpeduncular nucleus; ml, medial lemniscus; Pn, pontine nuclei.

the VMHdm (Fig. 4F) and in the dorsal premammillary nucleus. Small to moderate numbers of retrograde labeled neurons were also observed in the lateral preoptic nucleus, in the periventricular nucleus, in several

subdivisions of the parvicellular PVH, in the anterior and lateroanterior hypothalamic nuclei, in the lateral hypothalamic and perifornical areas, in the dorsomedial nucleus of the hypothalamus, in the MTu and in the

Table 2 Distribution of FG-ir neurons coexpressing estrogen receptor alpha (ERα) projecting to different columns of the periaqueductal grey matter (PAG), i.e., dorsolateral (dl), ventrolateral (vl) and lateral (l). Numbers in first column correspond to atlas levels in “The Rat Brain in Stereotaxic Coordinates” (Paxinos and Watson, 1997). Abbreviations: BSTMp, posterior subdivision of the bed nucleus of stria terminalis medial; ctr, control; LSi, lateral septum, intermediate; MeApd, posterodorsal subdivision of the medial nucleus of the amygdala; MPO, medial preoptic nucleus; MTu, medial tuberal nucleus; OVLT, vascular organ of lamina terminalis; PMV, ventral premammillary nucleus; VMHvl, ventrolateral subdivision of the ventromedial nucleus of the hypothalamus. Forebrain Sites

LSi (17) BSTMp (22) OVLT (18) MPO (19) StHy (21) VMHvl (30) MeApd (31) MTu (31) PMV (34)

Total FG (mean ± SEM)

FG-ir + ERα-ir (mean ± SEM)

% doubles/total FG (mean ± SEM)

dl

vl

l

dl

vl

l

dl

vl

l

1 ± 0.4 2 ± 0.81 0 18 ± 6.53 39 ± 8.32 85 ± 8.15 3.5 ± 0.4 16.5 ± 1.21 2 ± 0.81

25.5 ± 9.36 33.5 ± 6.13 27 ± 7.34 63.5 ± 3.64 139 ± 7.34 123.5 ± 9.36 33.5 ± 18.38 62.5 ± 3.64 28.5 ± 2.83

2 ± 0.87 16.7 ± 8.67 18.5 ± 2.83 64.6 ± 3.76 74 ± 4.62 137 ± 2.66 7 ± 2.51 38.3 ± 4.45 24.3 ± 2.31

0 0 0 1.5 ± 0.40 8 ± 2.43 13 ± 1.62 3±0 4±0 0

3 ± 1.62 8.5 ± 1.21 3.5 ± 1.21 5.5 ± 1.21 43.5 ± 6.13 33.5 ± 2.02 10.5 ± 4.45 10.5 ± 2.02 3±0

0 7.6 ± 4.34 1.5 ± 0.40 18 ± 6.53 23 ± 3.18 45.6 ± 10.98 2.6 ± 0.87 6.6 ± 1.16 1.3 ± 0.29

0 0 0 8.8 ± 0.92 19.8 ± 2.08 15.2 ± 0.40 87.5 ± 10.17 24.4 ± 1.79 0

10.3 ± 2.60 25.6 ± 0.98 12.5 ± 1.10 8.5 ± 1.39 31.1 ± 2.77 27.2 ± 3.99 37 ± 6.88 16.6 ± 2.25 10.7 ± 1.04

0 32.3 ± 16.18 5.2 ± 2.72 22 ± 5.72 31.4 ± 5.03 33.5 ± 7.98 39.4 ± 10.52 18.1 ± 4.16 5.8 ± 2.02

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3.2.3. Lateral column of the PAG (PAG-l) The injections centered in the PAG-l invariably contaminated the PAG-dl and/or the PAG-vl. Cases with more restricted injections (n = 4, Fig. 1C and 2C) were evaluated. The distribution of FG-ir neurons was an overlap of those described in previous sections, except that we also found retrograde labeled neurons in the insular cortex, claustrum, dorsal endopiriform nucleus, ventral LS and cortical nucleus of the amygdala. Higher density of retrograde labeled neurons was observed in most of the hypothalamic sites, including the MPOm, anterior hypothalamus, incerto-hypothalamic area, lateral hypothalamic and perifornical areas (Table 1). Outside the hypothalamus, we found low to moderate numbers of FG-ir neurons in the paraventricluar nucleus of the thalamus, medial habenular nucleus, reticular thalamic and parasubthalamic nuclei, and high density in the precomissural nucleus. 3.3. Distribution of FG-ir neurons expressing ERα immuoreactivity The distribution of ERα-ir in rodent brains has been described by several laboratories (Simerly et al., 1990; Merchenthaler et al., 2004; Shughrue et al., 1997, 1998). In agreement with previous studies, we observed dense ERα-ir neurons in the LS, in several subdivisions of the BSTM, in the preoptic area, the periventricular nucleus, StHy, the VMHvl, the MeApd and MTu. Moderate to small numbers of ERα-ir neurons were also observed in the PMV (Fig. 5). In all sites analyzed, we found higher numbers of dual labeled neurons in cases with injections centered in the PAG-vl and PAG-l (Table 2 and Fig. 6). Striking differences were observed in the distribution of FG-ir and of dual labeled neurons in the MPO and VMH. Neurons in the MPOm preferably project to the PAG-dl and PAG-l, whereas neurons in the MPOl mostly target the PAG-vl. A similar pattern was observed for the VMH. The VMHdm targets the PAG-dl and PAG-l, whereas the VMHvl innervates the PAGvl and PAG-l. The coexpression pattern mirrors the FG-ir distribution pattern, i.e., higher numbers of dual labeled neurons were observed in the MPOl and VMHvl projecting to the PAG-vl and PAG-l and moderate numbers of dual labeled neurons were observed in the MPOm projecting to the PAG-dl (Fig. 7). 4. Discussion In the present study, we describe the forebrain sites that innervate distinct columns of the PAG. We show that the afferent projections originate mainly in hypothalamic sites and they are largely segregated considering the PAG-dl vs. PAG-vl. We also show that, in the female rats, retrograde labeled neurons coexpressing ERα preferentially target the PAG-l and the PAG-vl. Because of our focus is the lordosis circuitry, males were not used in this study. Whether a sexually dimorphic circuitry exists needs further evaluation. The PAG is a complex structure organized in columns of physiological domains (Behbehani, 1995; Bandler and Shipley, 1994; Beitz, 1985, 1982). Studies from different laboratories using markers of neuronal activation (e.g., Fos immunoreactivity), anterograde tracers from distinct brain sites and transneuronal viral mapping have contributed to the understanding of the functional organization of the PAG (Lonstein and Stern, 1997; Daniels et al., 1999; Veening et al., 2014; Canteras and Goto, 1999; Canteras et al., 1992, 1994; Canteras et al., 1995; Elias and Bittencourt, 1997; Calizo and Flanagan-Cato, 2003). The present study expanded previous findings by revealing the sources of ERα expressing neurons to the PAG in addition to the medial hypothalamus. As an initial step, we performed a systematic evaluation of forebrain projections to individual columns of the PAG. The rat was elected as the most adequate model for our purpose due to the well-characterized role of PAG in female sexual behavior and the bigger size compared to mouse and hamsters (Lonstein and Stern, 1997; Arendash and Gorski, 1983a; Daniels et al., 1999; Calizo and Flanagan-Cato, 2002; Tsukahara and Yamanouchi, 2001). The target of defined PAG columns in rats

Fig. 3. Number of fluorogold immunoreactive (FG-ir) neurons projecting to distinct columns of the periaqueductal grey matter (PAG). (A–C), bar graphs showing the number of FG-ir neurons in distinct forebrain sites projecting to the dorsolateral PAG (PAG-dl, A), to the ventrolateral PAG (PAG-vl, B) and to the lateral PAG (PAG-l, C). Abbreviations: BSTMp, posterior subdivision of the medial bed nucleus of the stria terminalis; LSi, intermediate subdivision of the lateral septum; MeApd, posterodorsal subdivision of the medial nucleus of the hypothalamus; MnPO, median preoptic nucleus; MPO, medial preoptic nucleus; MTu, medial tuberal nucleus; OVLT, vascular organ of the lamina terminalis; PMV, ventral premammillary nucleus; StHy, striohypothalamic nucleus; VMHvl, ventrolateral subdivision of the ventromedial nucleus of the hypothalamus.

dorsal tuberomammillary nucleus. Outside the hypothalamus, small numbers of retrograde labeled neurons were observed in the subfornical organ, in the medial habenular nucleus and in the precomissural nucleus. 38

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Fig. 4. Distribution of retrograde labeled neurons projecting to distinct columns of the periaqueductal grey matter (PAG). (A–I) darkfield images showing FGir cells in the posterior subdivision of the medial bed nucleus of the stria terminalis (BSTMp, A, C and G), in the medial preoptic nucleus (MPO, B, E, H) and in the ventromedial nucleus of the hypothalamus (VMH, C, F, I). Abbreviations: 3V, third ventricle; f, fornix; ic, internal capsule; MPOl and MPOm, lateral and medial MPO; PAG-dl, PAG-l and PAG-vl, dorsolateral, lateral and ventrolateral columns of the PAG; VMHdm and VMHvl, dorsomedial and ventrolateral VMH. Scale bar: 500 μm.

Fig. 5. Distribution of estrogen receptor α immunoreactive (ERα-ir) neurons in brain areas that innervate the periaqueductal grey matter (PAG). (A–F) Bright field images showing ERα-ir in the intermediate subdivision of the lateral septal nucleus (LSi, A), in the posterior subdivision of the medial bed nucleus of stria terminalis and striohypothalamic nucleus (BSTMp and StHy, B), in the medial and lateral subdivisions of the medial preoptic nucleus (MPOm and MPOl, C), in the ventrolateral subdivision of the ventromedial nucleus of the hypothalamus and in the medial tuberal nucleus (VMHvl and MTu, D and E) and in the posterodorsal subdivision of the medial nucleus of the amygdala. Abbreviations: 3V, third ventricle; Arc, arcuate nucleus; f, fornix; ic, internal capsule; LPO, lateral preoptic nucleus; LSv, ventral subdivision of the lateral septal nucleus; MS, medial septal nucleus; sm, stria medularis; VMHdm, dorsomedial subdivision of the VMH. Scale bar: 500 μm.

seemed possible and proved achievable, although contamination of adjacent columns may not be completely ruled out. In agreement with previous findings (Beitz, 1982), we observed that the hypothalamus is the main conveyor of inputs to the PAG. Differences in pattern and density of afferent neurons were evident comparing individual columns. The PAG-l and PAG-vl receive a dense ERα-ir projections from the VMHvl. This is in agreement with previous studies focused on sexual behaviors (Daniels et al., 1999; Akesson et al., 1994; Beitz, 1982; Calizo and Flanagan-Cato, 2003, 2002; Tsukahara and Yamanouchi, 2001). The interest of the scientific field on VMHvl is highly justified due to its well-defined actions in lordosis (Pfaff and Sakuma, 1979b, c; Davis and Barfield, 1979; Hennessey et al., 1990; Sakuma and Pfaff, 1980; Calizo and Flanagan-Cato, 2003). However, other brain sites also play a role. Among them, the MPO, a sexually dimorphic site (Gorski et al., 1980; Jacobson and Gorski, 1981; Simerly et al., 1984), is of particular relevance due to the dense concentration of sex steroids responsive neurons and its actions in reproductive physiology (Simerly et al., 1990; Merchenthaler et al., 2004). The MPO ERα neurons have a clear function in male sexual behavior (Arendash and Gorski, 1983b; Murphy and Hoffman, 2001; Coolen et al., 1997), but their role in lordosis is

controversial since either inhibition or facilitation of lordotic reflex were reported following MPO interventions (Arendash and Gorski, 1983b; Coolen et al., 1997; Been and Petrulis, 2010; Coolen et al., 1996; Caldwell and Clemens, 1986; McCarthy et al., 1990; Acosta-Martinez and Etgen, 2002). These differences suggested that a heterogeneous population of neurons may differentially affect sexual behavior in each sex (Kohl et al., 2018; Moffitt et al., 2018; Wu et al., 2014; Kataoka et al., 2001). Moreover, distinct MPO subdivisions may exert different effects in lordosis making the data obtained from the use of unspecific lesions and/or stimulation difficult to interpret. The MPO sends estrogen-responsive inputs to both PAG-l and PAG-vl, and most of the projecting neurons are located in the MPOl. Whether these projections are directly associated with lordosis needs further evaluation. Together with the central nucleus of the amygdala and parvicellular subdivisions of the PVH, the MPOm preferentially innervates the PAGdl. These sites have well-defined roles in autonomic control, in agreement with the actions of PAG-dl in cardiovascular responses, mostly inducing hypertension and tachycardia (Behbehani, 1995; Bandler and Shipley, 1994). The PAG-dl is also a prime site of pain modulation. It receives ascending projections from the spinothalamic tract carrying sensorial information on tactile and adverse skin stimuli (Behbehani, 39

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Fig. 6. Retrograde labeled neurons coexpressing estrogen receptor α (ERα). (A–I) bright field images showing retrograde labeled neurons (brown cytoplasm) coexpressing ERα immunoreactivity (arrows, black nucleus) in the posterior subdivision of the medial bed nucleus of the stria terminalis (BSTMp, A and D), in the lateral subdivision of the medial preoptic nucleus (MPOl, B and E) and in the ventrolateral subdivision of the ventromedial nucleus of the hypothalamus (VMHvl, C, F, G, I). G is a higher magnification of C (box); H is a higher magnification of E (box), and I is a higher magnification of F (box). Scale bar: A–F = 100 μm, G–I = 50 μm.

1995; Bandler and Shipley, 1994; Vanderhorst et al., 1996). Lordotic posture in sexually receptive females is a reflex response triggered by sensory stimulation of the back and flanks (Kow et al., 1979). Once copulation is initiated, opioids are released in several brain sites, including the PAG, increasing the threshold for pain (Szechtman et al., 1981). Thus, the MPOm innervation of PAG-dl may be a potential path for the modulation of sexual behavior-induced changes in cardiovascular function and/or nociception (Sansone et al., 1997; Catelli et al., 1987). Neurons expressing ERα in the BSTMp and the MeApd preferentially innervate the PAG-l and PAG-vl. These sites are particularly relevant in conspecific odors discrimination. In rodents, olfactory stimulation induces sex hormones release and behavioral responses via projections originated in the vomeronasal organ and main olfactory systems (Halpern and Martinez-Marcos, 2003; Dulac and Torello, 2003; Keller et al., 2009). From the BSTM and MeApd, olfactory cues are transmitted to the MPO and the PMV (Canteras et al., 1995; Halpern and MartinezMarcos, 2003; Scalia and Winans, 1975; Cavalcante et al., 2006; Yoon et al., 2005). The BSTM, the MeApd, the MPO and the PMV all express Fos in response to opposite sex odor stimulation and are, therefore, engaged in odorants-triggered sexual arousal (Coolen et al., 1997, 1996; Cavalcante et al., 2006; Veening et al., 2005; Donato et al., 2010; Kollack-Walker and Newman, 1995; Wersinger et al., 1993). Lesions of the vomeronasal organ or the MeApd decreases female receptivity (Winans and Powers, 1977; Saito and Moltz, 1986; Wood and Newman, 1995; Rajendren et al., 1990), and ablation of PMV neurons disrupts the dynamic changes in hormone levels observed in females across the estrous cycles (Donato et al., 2013). Together with previous findings, our data indicates that the estrogen-responsive neurons in the vomeronasal

circuitry control female receptivity via direct projections to the PAG-l and PAG-vl. Although we have focused our studies on the identification of potential lordosis-associated neural network, it is important to emphasize that the PAG also exerts key effects in other autonomic and behavioral responses potentially unrelated to sexual behavior (Behbehani, 1995). Moreover, estradiol may modulate behavior via other receptors, i.e., ERβ or GPER1 (Antal et al., 2012; Anchan et al., 2014). Therefore, the projecting sites targeting distinct columns described here should be seen as a guiding map for the identification of chemically defined neural circuitry associated with ERα actions in female sexual behavior using more precise neuronal targets and technologies. Studies using molecular mapping with viral tracers, opto- and chemo-genetic approaches to identify specific PAG networks are warranted. Ethical statement Experiments were carried out in accordance with the guidelines of the National Institute of Health Guide for the Care and Use of Laboratory Animals and the Institutional Committee for Research and Animal Care of the University of São Paulo, Brazil. Authors declare no conflict of interest exists. Acknowledgments We would like to thank Joelcimar Martins da Silva for expert technical assistance and José A. Martins for the animal care. Grants from São Paulo Research Foundation (FAPESP-Brazil, 99/03490-0, 00/ 09365-1, 01/02282-6, 00/05530-8) supported this work. JCB is the 40

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Fig. 7. Schematic illustration of the distribution pattern of dual labeled neurons projecting to distinct columns of the periaqueducatal grey matter (PAG). (A–F), drawings illustrating fluorogold immunoreactive (FG-ir, ●) neurons and FG-ir neurons coexpressing estrogen receptor α (ERα, X) in the medial preoptic nucleus (MPO, A–C) and ventromedial nucleus of the hypothalamus (VMH, D–E). Abbreviations: 3V, third ventricle; Arc, arcuate nucleus; BST, bed nucleus of the stria terminalis; DMH, dorsomedial nucleus of the hypothalamus; MPOl and MPOm, lateral and medial MPO; ox, optic chiasm; VMHdm and VMHvl, dorsomedial and ventrolateral VMH.

recipient of a joint CAPES (Agency for the Advancement of Higher Education)-COFECUB (French Committee for the Evaluation of Academic and Scientific Cooperation with Brazil) grant 848/15. JCB is an Investigator with the National Council for Scientific and Technological Development – CNPq (Brazil), grant 426378/2016-4. CFE is funded by NIH grants (R01 HD069702; R21 HD090567; R03 HD092855; P30 DK020572).

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