Expression of type one cannabinoid receptor in different subpopulation of kisspeptin neurons and kisspeptin afferents to GnRH neurons in female mice

The endocannabinoids have been shown to target the afferents of hypothalamic neurons via cannabinoid 1 receptor (CB1) and thereby to influence their excitability at various physiological and/or pathological processes. Kisspeptin (KP) neurons form afferents of multiple neuroendocrine cells and influence their activity via signaling through a variation of co-expressed classical neurotransmitters and neuropeptides. The differential potency of endocannabinoids to influence the release of classical transmitters or neuropeptides, and the ovarian cycle-dependent functioning of the endocannabinoid signaling in the gonadotropin-releasing hormone (GnRH) neurons initiated us to study whether (a) the different subpopulations of KP neurons express CB1 mRNAs, (b) the expression is influenced by estrogen, and (c) CB1-immunoreactivity is present in the KP afferents to GnRH neurons. The aim of the study was to investigate the site- and cell-specific expression of CB1 in female mice using multiple labeling in situ hybridization and immunofluorescent histochemical techniques. The results support that CB1 mRNAs are expressed by both the GABAergic and glutamatergic subpopulations of KP neurons, the receptor protein is detectable in two-thirds of the KP afferents to GnRH neurons, and the expression of CB1 mRNA shows an estrogen-dependency. The applied estrogen-treatment, known to induce proestrus, reduced the level of CB1 transcripts in the rostral periventricular area of the third ventricle and arcuate nucleus, and differently influenced its co-localization with vesicular GABA transporter or vesicular glutamate transporter-2 in KP neurons. This indicates a gonadal cycle-dependent role of endocannabinoid signaling in the neuronal circuits involving KP neurons.

Very few data are available, however, about the physiological/pathophysiological conditions and target system of an enhanced endocannabinoid signaling, which may recruit other than the GABAergic afferents. This could be similar to the outcome of treatments with CB1 agonists resulting in a reduction both the inhibitory and excitatory postsynaptic currents (IPSCs and EPSCs) in target neurons (Hentges et al. 2005).
Kisspeptin (KP)-producing neurons are known to form afferents of multiple neuroendocrine cells (Clarkson and Herbison 2006). They are located in three major sites, i.e., the preoptic area (POA), the arcuate nucleus (ARC), and the medial amygdaloid nucleus (ME) (Clarkson et al. 2009;Lehman et al. 2013), where they establish local connections (Comninos et al. 2016;Stephens and Kauffman 2017;Krajewski et al. 2010;Qiu et al. 2018), as well as project to distant target areas including the POA (Qiu et al. 2018), supraoptic (SON), and paraventricular (PVH) nuclei of the hypothalamus (Yeo et al. 2016). Their processes play a key role in mediating the positive and negative estrogen feedback to GnRH neurons (Ohkura et al. 2009), which is based on their direct genomic and non-genomic (Mittelman-Smith et al. 2012) responses to estrogen, and direct connections to GnRH neurons. They are also implicated in conveying circadian Williams et al. 2011), metabolic (Clarke and Arbabi 2016;Wahab et al. 2013), and limbic (Comninos et al. 2016;Stephens and Kauffman 2017) signals to GnRH neurons. In addition, they provide a rich innervation of OT (Seymour et al. 2017;Liu and Herbison 2016;Scott and Brown 2013), VP neurons (Liu and Herbison 2016) in the SON and PVH, and POMC (Higo et al. 2017;Qiu et al. 2018), and tyrosine hydroxylase (TH) neurons (Sawai et al. 2012) in the ARC, and the functional significance of these neuronal connections, however, is incompletely understood.
Besides producing various neuropeptides (neurokinin B, dynorphin, galanin) (Goodman et al. 2007;Murakawa et al. 2016;Kallo et al. 2012;Porteous et al. 2011) or biogenic amines (dopamine) (Clarkson and Herbison 2011;Skrapits et al. 2015; Bardoczi et al. 2018), subpopulations of kisspeptin neurons use classical neurotransmitters like glutamate and GABA, which contribute to the excitatory and/or inhibitory innervation of the neuroendocrine cells. Contrasting the peptide-type transmitters, these classical neurotransmitters are released spontaneously, as well as at an evoked manner, which raises a potential role of endocannabinoid signaling to discretely influence the release of neurotransmitters/neuromodulators from the different kisspeptin subpopulations. The regulation could happen with different efficacy in the GABAegic and glutamatergic subpopulations of KP neurons, similarly to those observed for the G-protein-dependent signaling of CB1 in cortical principal versus interneurons (Steindel et al. 2013).
Electrophysiological recordings indicate ovarian cycledependent alterations in the GABAergic and glutamatergic inputs to GnRH neurons (Balint et al. 2016;Farkas et al. 2018). It was also reported that the endocannabinoid signaling operates in a phase-dependent manner; in metestrus, endocannabinoids suppress the postsynaptic currents (PSCs) in GnRH neurons, whereas in proestrus, they do not seem to contribute to the increase of these events (Balint et al. 2016;Farkas et al. 2018). By establishing many critically important estrogen-sensitive pathways, it is of interest whether (a) GABAergic and/or glutamatergic subpopulations of KP neurons express CB1 mRNAs, (b) the expression is influenced by estradiol (E2), and (c) CB1-immunoreactivity is present in the KP afferents to GnRH neurons.
These questions prompted us to investigate the site-and cell-specific expression of CB1 in female mice using multiple labeling in situ hybridization and immunofluorescent histochemical techniques. Ovariectomized and estradiolreplaced models were used in the experiments, supplemented with mice genetically altered to maximize the visualization of KP afferents to GnRH neurons.

Tissue preparation for in situ hybridization
Four hours after the second EB or oil vehicle injections, the animals were sacrificed, and the brains were removed and frozen on dry ice. 14 µm thick coronal sections were cut on a Leica CM 3050 S cryostat (Leica Microsystems, Vienna, Austria), and mounted consecutively onto groups of ten Super Frost Ultra Plus glass slides (Thermo Fisher Scientific, Budapest, Hungary). Two coronal sections of OVX + EB mice (n = 5) 126 µm apart from each other were paired with two corresponding sections of OVX + oil mice (n = 5) on each slide. The slides were stored at −80 °C until processed.

Capturing and analyzing the RNAScope signals
The quadruple-labeled sections were scanned in a Nikon C2 confocal microscope (Nikon, Japan) using the 20 × objective. Multiple stacks of optical slices (1024 × 1024 pixels, z-steps 0.6 µm) were obtained from the −0.6 to −2.4 µm layer of the sections by scanning the full RP3V and ARC regions on one side. The fluorochromes were excited with laser lines 488, 561, and 641 nm. The DAPI nuclear staining was also detected using the 405 nm laser. Laser intensities and other acquisition parameters were kept the same during the whole scanning. Using the Image J software, the different channels in the image stack were combined into maximum intensity projections, and binarized and merged into single TIFF images. The DAPI channel was also saved as separate TIFF images. Lit pixels were counted within regional and cellular borders, which were determined manually based on the DAPI image and kisspeptin signals. A cell was deemed as positive for a given mRNA when the number of lit pixels within the cell area were higher (containing more than five pixels) than the number of lit pixels in identical sized area of the white matter (maximum five pixels). Data were analyzed by one-way ANOVA, and significant difference was determined by the Holm-Sidak method.

Tissue preparation for detecting the viral fluorescent tracer and immunofluorescence
The animals were perfused transcardially with phosphatebuffered saline (PBS 0.1 M) containing 4% paraformaldehyde (PFA). The brains were removed, post fixed for 24 h, and transferred into 30% sucrose for cryoprotection, and then, 30 µm thick coronal sections were cut on a freezing microtome by collecting every third sections into the same well.

Evaluation of viral tracing of KP fibers
A group of sections were mounted from each brain with RP3V (n = 5), ARC (n = 8), or MEA (n = 7) injections of the viral tracer. Brains showing successful labeling of KP neurons in consecutive sections were selected for subsequent multiple-label immunofluorescence staining of YFP in KP cells, GnRH, and CB1.

Confocal microscopy and 3-D reconstruction of KP afferents to GnRH-IR cells
The triple-labeled sections were scanned in a Nikon A1R confocal microscope (Nikon, Japan) using the × 20 and the 60 × oil immersion objectives. Multiple stacks of optical slices (1024 × 1024 pixels, z-steps 0.15 µm) were generated, which contained the KP-IR fibers in apposition to the cell bodies and the processes of GnRH-IR neurons. The separately recorded green, red, and far-red channels were merged and displayed with the ImageJ software running on an IBM compatible personal computer. Orthogonal views from different planes (x/y, x/z or y/z) of the confocal microscopic images were used to analyze the KP-immunoreactive fibers for apposition to GnRH neurons and CB1-immunoreactivity. To enable three-dimensional (3D) analyses, the images were further processed using the software Amira (6.0, Visual Imaging Group). The stacks of the optical slices were loaded into the visualization program and rendered in three dimensions with surfaces generated from above threshold immunoreactivity. The threshold was set individually for each image and color channel to minimize any noise, while maintaining the proper cellular boundaries. The surfaces generated from the three channels in the same optical volume were visualized to check for cell-to-cell contacts, and the presence of CB1-immunoreactivity in KP fibers associated with GnRH neurons. This enabled verification of the findings from the two-dimensional confocal image analyses.
GnRH neurons (n = 6 per brains with successful viral labeling of RP3V kisspeptin neurons) located in the medial preoptic area and seen to be a candidate of receiving KP-IR afferents at 20 × magnifications were selected for 60 × scans and analyses. An axon was positive for CB1 when immunoreactivity was detected inside or in association with the cell membrane of the YFP-positive KP processes. No such signal was detected in CB1-KO mice (kindly provided by A. Zimmer, University of Bonn and bred at the Medical Gene Technology Unit of the Institute of Experimental Medicine).

Expression of KP, CB1, VGLUT2, and VGAT in the RP3V and ARC regions: effect of estrogen
The RNAscope in situ hybridization technique detected mRNA signals for KP, CB1, VGLUT2, and VGAT concurrently in ovariectomized mouse models treated for 2 days either with EB or oil vehicle. In agreement with the previous reports ), the KP mRNA signal showed an estrogen-dependent alteration in the RP3V (Fig. 1) and ARC (Fig. 2), and this has validated the animal models used in this study. Thus, there was a profound and area-specific effect of estrogen-treatment on the level of KP mRNAs in the RP3V and ARC (Fig. 3A). While EBtreatment significantly increased the mRNA levels for KP in the RPRV region (from 1.83 ± 0.41 to 4.51 ± 0.8, p < 0.02), the same treatment reduced it in the ARC (from 1.95 ± 0.56 to 0.63 ± 0.05, p = 0.02). The expression levels of VGAT and VGLUT2 were relatively low in both regions compared to the neighboring medial preoptic area (MPA) and/or the ventromedial hypothalamic nucleus (VMH) (Figs. 1 and 2). No significant differences were detected between the two animal models for VGAT mRNAs in the ARC and VGLUT2 mRNAs in both the RPRV and ARC. The VGAT mRNA signal was, however, significantly lower (p = 0.028) in the RP3V region of OVX-EB animals (0.29 ± 0.26) than in the OVX + OIL group (0.59 ± 0.07) (Fig. 3A). The CB1 mRNA was also relatively low in the RP3V and ARC compared to the neighboring MPA and VMH regions ( Figs. 1 and 2), respectively. Additionally, CB1 mRNA showed an estrogen-sensitivity with signal being lower both in the RP3V (0.35 ± 0.14 vs 1.2 ± 0.19, p = 0.02) and ARC (0.33 ± 0.05 vs 0.16 ± 0.04, p = 0.05) of EB-treated mice compared to mice receiving vehicle only (Fig. 3A).

Expression of CB1 in RP3V and Arc KP neurons: effect of estrogen
The same sections were analyzed at cellular levels in the RP3V and ARC for co-expression of KP with CB1 and either VGAT or VGLUT2 (Fig. 3B-E). In the RP3V, 93.4 ± 2.7% of KP neurons expressed CB1 in the OVX + OIL group, and 32 ± 6.9% of them in the OVX + EB group, whereas in the  In contrast, the VMH shows a much stronger CB1 mRNA signal in co-distribution almost exclusively with the VGULT2 mRNA signal (B and D). Scale bar 100 µm ARC, these values were 15.5 ± 2 and 49.3 ± 4.4%, respectively (Fig. 3F).

Expression of CB1 in GABAergic and glutamatergic subpopulations of KP neurons
KP neurons express VGAT and VGLUT2 mRNAs both in the RP3V and the Arc. In the RP3V, 48.4 ± 10.6% of KP neurons expressed VGAT in the OVX + OIL group, and 69.2 ± 13.4% of them in the OVX + EB group, whereas in the ARC, these values were 76.5 ± 2.8 and 73.5 ± 5.9%, respectively. Concerning the co-expression of VGLUT2 with KP, the value was 23.5 ± 1.9% in the RP3V of OVX + OIL group, and 31.4 ± 2.5% in the same region of the OVX + EB group, whereas these values were in the ARC 83.7 ± 2.4 and 88.3 ± 3.8%, respectively. The estrogen level of the animals had no significant influence on the percentage of KP neurons co-expressing VGAT or VGLUT. The percentage of VGLUT2-positive KP neurons was, however, significantly higher in the ARC than in the RP3V (p < 0.001) (data not shown). When CB1 mRNA expression was analyzed in the VGAT-or VGLUT2-positive KP neurons, a profound sitespecific effect of EB was revealed in the RP3V and ARC. Thus, the percentage of the CB1 expressing VGLUT2-positive KP neurons was significantly increased in the ARC of estrogen-treated OVX mice, compared to the oil-treated animals (43.5 ± 5 vs. 14.8 ± 2.4%, p < 0.005). The same treatment resulted in a significant decrease of CB1 expression in VGAT-positive KP neurons in the RP3V region of the brain (19.1 ± 3.5 vs. 67.3 ± 10.9%, p < 0.05) (Fig. 3F).

CB1-immunoreactivity in KP afferents to GnRH neurons
The presence of CB1 in KP afferents of GnRH neurons was studied in CD1 and KP-CRE mice (Fig. 4) by immunohistochemical labeling of preoptic sections for CB1, GnRH, and KP ( Fig. 5A-C) or alternatively YFP (Fig. 5D-F), expressed after transmission of its gene by viral-infection of KP-CRE neurons. Because of its dominant membrane localization, CB1 appeared rarely in overlap with KP-immunoreactivity marking primarily secretory granules in the axon terminals ( Fig. 5A-C). Therefore, axonal projections of KP-CREexpressing neurons were traced with yellow fluorescent proteins, which also marked the borders of axon terminals in contact with GnRH neurons (Fig. 5D-F).
The viral construct (AAV-EF1a-DIOhChR2 (H134R)-EYFP) was delivered to all major KP-populations of the mouse brain, where it was translated to EYFP and transported together with the ChR2 to the processes of Fig. 3 RNAscope in situ hybridization signals detected at area (A) and cellular (B-G) levels in the RP3V and Arc of OVX mice treated with EB or oil vehicle. Co-distribution of signals for KP (red), CB1 (blue), and VGAT (green) in association with a preoptic cell nucleus (grey-white) (B-E). Mean level of signals (A, determined by the number of positive pixels/ROIs in the RP3V and Arc). Expression of CB1 mRNA in KP neurons (F, determined by the presence of pixels identifying CB1 in KP mRNA-positive cells) and co-localization of CB1 mRNA signal with VGAT or VGLUT2 mRNA signals in KP mRNApositive cells of the RP3V and Arc (G determined by the presence of pixels identifying CB1 and VGAT or VGLUT2 mRNA signals in KP mRNA-positive cells). Scale bar in B-E 5 µm p < 0.05, significant difference labeled with asterisk KP-CRE cells including the cell membrane (Fig. 4). YFPpositive processes in apposition to GnRH neurons were rarely seen in the POA of mice, if the viral construct was injected into the ARC or the MePD. In contrast, such afferents were seen more often in mice, which were targeted with the viruses in the RP3V region.
The YFP expressed by AAV-infected RP3V neurons identified the cellular borders of KP neuronal processes in the MPA, including those, which were in juxtaposition to GnRH-IR perikarya and processes (Figs. 5D-F). CB1-IR was found in about two-thirds of these KP neuronal processes (Table 2, 70 ± 2.9 and 59 ± 4.1% of them (n = 187) were in apposition to GnRH perikarya and processes, respectively).

Discussion
This study provides evidence that (a) CB1 mRNA is expressed by both GABAergic and glutamatergic subpopulation of kisspeptin neurons, (b) the receptor protein is present in KP afferents of GnRH neurons, and (c) the expression of CB1 mRNA shows estrogen-dependent regulation. The applied estrogen-treatment, known to induce proestrus in mice, reduced the level of CB1 transcripts in the RP3V and ARC, and differently influenced its co-localization with VGAT or VGLUT2 in kisspeptin neurons.

Experimental model to study whether CB1 is involved in presynaptic regulation of kisspeptin afferents
Levels of CB1 expression are highly variable among different brain locations and cell types. While the cerebral cortex and hippocampus contain a very high level of CB1 protein, expression levels are relatively low in hypothalamic regions (Wittmann et al. 2007). Despite its low levels, a large body of evidence supports strong cannabinoid-dependent signaling in the regulation of hypothalamic functions (Gammon et al. 2005;Pagotto et al. 2006;Tasker 2006;Brents 2016). Concerning the reported role of CB1 in the regulation of reproduction, and the critical functions of kisspeptin neurons in this regard, in the current study, the KP cell-specific expression of CB1 was investigated using multiple labeling in situ hybridization and immunofluorescent histochemical techniques. During the ovarian cycle, the hypothalamic regulatory circuits operate under the influence of estrogens' negative and positive feedback effects. Therefore, we have used the animal models with reduced serum ovarian hormone levels achieved by 3-week time gonadectomy (Baumgartner et al. 2019), and the hormone-replaced pairs treated subsequently with a priming and a surge-inducing dose of estrogen (Bosch et al. 2013). As expected, this treatment regime resulted in a significant difference between the uterine weights of the two models by increasing the weights fourfold compared to controls. In addition, the appearance of KP transcript levels showed high estrogen-dependency in the Fig. 4 Illustration of the brain areas containing Cre expressing KP neurons (A and B), which underwent a virus-based identification in the preoptic (C), arcuate (D), and medial amygdala (E) regions, respectively. The expressed YFP has been immunohistochemically amplified to show membranes of KP neurons. Modifications of the atlas images from the mouse brain atlas of Paxinos and Franklin's (2012). Scale bar 200 µm. Arc arcuate nucleus, AVPe anteroventral periventricular nucleus, ME median eminence, MePD posterodorsal subdivision of medial amygdala, och optic chiasm, opt optic tract, Pe periventricular hypothalamic nucleus, st stria terminalis, VMH ventromedial hypothalamic nucleus RP3V and ARC regions, as it was reported earlier (Lehman et al. 2010, Smith et al. 2005. Thus, in the RP3V, KP transcript levels were high in the estrogentreated animals, whereas strong signals were detected in the ARC in the absence of estrogen (Fig. 3A). The estradioltreated model was chosen also to investigate the afferents on GnRH neurons based on the observation of Chan et al. (2011). They reported that the positive feedback levels of estradiol stimulate a robust increase in spine density and most likely in synaptic inputs of GnRH neurons.

Distribution of CB1 in hypothalamic GABAergic and glutamatergic neurons in previous studies
Our previous mapping studies in male mice ) revealed a differential expression of mRNAs for CB1, the GABAergic marker GAD65, and the glutamatergic marker VGLUT2 in the RP3V and ARC regions, where the two major subpopulations of KP neurons are located. Relatively low abundance of the hybridization signal and high-to-moderate-to-low number of labeled cells were found for CB1 in the AVPe, the Pe, and the ARC nucleus, respectively. CB1 mRNA was detected in both GABAegic and glutamatergic neurons in these regions. In the current study, the mRNA signals for CB1, VGAT and VGLUT2 were relatively low in both the RP3V and ARC of female mice and their relative abundance compared to the neighboring brain areas (i.e., MPA or the VMH) was similar to the one detected previously in male mice ).

Estrogenic regulation of hypothalamic CB1, VGAT, and VGLUT2 expression
Estrogen treatment resulted in a down-regulation of CB1 expression in both regions (Fig. 3A). This agrees with the observation of Riebe and Gorzalka, (Riebe et al. 2010), showing that OVX females have higher amounts of hypothalamic cannabinoid receptor binding relative to both cycling and OVX + E2 females. The current result, therefore, might indicate a reduced involvement of CB1 mediating suppression in local circuit activity, which generate higher frequency postsynaptic events in target cells in the presence of estrogen (Glanowska and Moenter 2011).
Similarly to CB1, expression of VGAT mRNA was lower in RP3V of the OVX + EB mice compared to the oil-treated ones. This seems to be congruent with the observation of Ottem et al. (Ottem et al. 2004) showing a decrease of the VGAT-containing vesicles in the RP3V of female rats at the time of the surge.
Gonadal hormones were reported to alter also the VGLUT2 expression in these regions. E2-treatment was shown to increase the VGLUT2 immunoreactive vesicles in the afferents of GnRH neurons in ovariectomized rats (Ottem et al. 2004). An increase of VGLUT2 mRNAs by estrogen-treatment was reported also in the ARC of ovariectomized mice (Qiu et al. 2018). The positive effect of gonadal hormones on VGLUT2 expression is, however, not unambiguous, since gonadectomy led to a significant elevation of VGLUT2 mRNA in male mice, compared to the levels observed in gonad intact conditions .
The comparison of current RNAscope signals of the EB or oil-treated animals, however, did not reveal a significant difference for VGLUT2 signals neither in the RP3V nor in the ARC. This may indicate the presence of mixed Demonstration of appearance of CB1-immunoreactivity in one of the KP-IR afferent fiber (white arrow) in confocal microscopic Z-stack series. A A single optical slice shows multiple KP-immunoreactive (IR) varicosities (red and white arrows) in apposition to a GnRH-IR neuron (blue). B is an adjacent optical slice. The boxed area in this image is magnified in C to demonstrate the yellow-colored double-labeled KP-IR varicosity (white arrow). D Yellow fluorescent protein (YFP)positive, green-colored axon varicosities (KP fibers after viral and immunohistochemical detection) in apposition to two adjacent gonadotropin-releasing hormone (GnRH)-IR cells (blue) shown in merged three optical slices. E The 3D rendered view of all optical slices of the same structures. The GnRH neurons are embedded in a tissue showing punctate CB1-IR sites (red), where CB1-IR clusters (dotted circles) and KP-IR fibers (white arrows) are in association with GnRH-IR cell bodies or processes. F The projection image of the 3D reconstructed area of E (white rectangle) is shown at higher power. Some of the CB1-IR sites (white asterisks) are visible only, if the KP-IR fiber is made semi-transparent. The CB1-IR sites in the membrane of the KP-IR fiber (white arrows) turn up in orange-red color marking co-localization. The contact site between the KP fiber and the GnRH dendron is marked by black arrowheads. Scale bar 10 µm (on A, B, D, E) and 5 µm (on C) glutamatergic cell populations in these regions in which expression of VGLUT2 is differently or even antagonistically regulated by estrogen.

Expression of CB1 in preoptic and arcuate KP neurons
Analyses of the RNAscope data at cellular levels revealed the presence of CB1 transcripts in KP neurons in both the RP3V and ARC regions, but the hormonal conditions had a great impact on the level of co-expression. Nearly all preoptic KP neurons expressed CB1 in the OVX + OIL group (93.4 ± 2.7%), but this value fall to one-third in the OVX + EB group (32 ± 6.9%). The opposite was observable for ARC KP neurons, showing low levels of co-expression in the OVX + OIL group (15.5 ± 2%), whereas CB1 mRNA expression was detectable in nearly half of the KP neurons in the OVX + EB group (49.3 ± 4.4%). By involving data on the mRNA levels for VGAT and VGLUT2 in the analyses, the presence of CB1 mRNA was also examined separately in the GABAergic and glutamatergic subpopulations of KP neurons. Both subpopulations proved to be positive for CB1 mRNA and the co-localization levels varied again according to the hormonal status of the animals. The percentage of the GABAergic KP neurons expressing CB1 was significantly lower in the RP3V of OVX + EB-treated mice, indicating that CB1 signaling might be reduced or suspended in this cell population at high estrogen levels.
Conversely, the percentage of the glutamatergic KP neurons expressing CB1 (Fig. 3F) was significantly higher in the ARC of the estrogen-treated animals compared to controls. The dramatic differences seen in the preoptic versus arcuate co-localization levels can be explained by the antagonistic regulation of KP transcript in these regions, which is combined with the reduced expression of CB1 mRNA in response to estrogen-treatment in both locations. Thus, lower co-localization levels can be found in RP3V in the OVX + EB model and in the arcuate nucleus in the OVX + Oil model, in which models the KP expression is high and the CB1 message is downregulated.

Colocalization of KP and VGAT or VGLUT2 mRNAs
Both VGAT or VGLUT2 mRNA-containing KP cells could be detected in the preoptic and ARC regions, which is in concordance with studies detecting VGAT/or VGLUT2 mRNA in KP-CRE mice (Cravo et al. 2011) or KP-immunoreactivity in VGAT-CRE or VGLUT2-CRE mice (Cheong et al. 2015). Thus, about half to two-third of the KP neurons were positive in the current study for VGAT mRNA, and about one quarter to one-third of KP neurons were positive for VGLUT2 mRNA in the RP3V. Most of the ARC KP neurons were also positive for VGLUT2 mRNA and three quarter of them also expressed the VGAT mRNA. Of note, the level of co-localization of KP and VGAT or VGLUT mRNAs may depend on the animal model used, since quantitative PCR studies were able to find relatively few, or no RP3V KP cells with Slc17a6 (VGLUT2) mRNA or no ARC KP cells with Slc32a1 (VGAT) mRNA (Qiu et al. 2018) in Kiss1Cre:GFP mice (Gottsch et al. 2011). Furthermore, translation of neither amino acid transporter markers has been proved to functional transporter proteins, respectively, in the RP3V or ARC KP cells of these animals (Qiu et al. 2018).

Potential role of CB1-mediated signaling in KP-GnRH interactions
KP neurons in all major locations are involved in the regulation of GnRH secretion. We have traced projections of KP neurons located in the RP3V, ARC, and MePD (Fig. 4), and in concordance with the previous observations Pineda et al. 2017;Yeo 2013;Yeo and Herbison 2011;Yeo et al. 2019;Wintermantel et al. 2006), found KP fibers in apposition to GnRH cells to originate primarily from the RP3V cells. CB1-IR was found in the majority of these KP neuronal processes independent of being in apposition to the perikaryon or proximal processes of GnRH neurons ( Fig. 5C and F). This suggests that neurotransmitter release from the KP afferents to GnRH neurons is also under regulation of endocannabinoids.
The endocannabinoid control is inhibitory by reducing the frequency of mPSCs and the firing activity of GnRH neurons in slices of metestrus female mice (Balint et al. 2016). This phase of the cycle is characterized by low serum estrogen levels, which triggers the synthesis and release of 2-AG from GnRH neurons via activating membrane-associated, rapid ERβ-mediated processes in these neurons. Acute administration of a high dose of estrogen to the slice had no effect on this inhibition, indicating that rapid, non-genomic actions of estrogen might not interfere with the CB1 signaling. The inhibition, however, does not seem to operate at high serum estrogen levels in proestrus GnRH neurons (Farkas et al. 2018). A possible explanation for this could be a longer exposure to and most likely genomic action of estrogen seen in the current study, which resulted in the downregulation of CB1 expression in both the RP3V and ARC KP neurons. Whether it has involved both the GABAergic and glutamatergic KP afferents of GnRH neurons needs to be further investigated. However, the down-regulation of CB1 in the glutamatergic KP afferents may have contributed to the appearance of the glutamate receptor mediated PSC-es in the GnRH neurons seen on the proestrus afternoon (Farkas et al. 2018).

Potential role of CB1-mediated signaling in KP-KP interactions
Glutamate released by ARC KP neurons targets KP neurons in this brain region and may contribute to the synchronized activity increase in populations of KP neurons during pulse generation. Glutamate released by ARC KP neurons also targets preoptic KP neurons, which in turn can enhance stimulation of GnRH neurons leading ultimately to a GnRH surge . Therefore, a down-regulation of CB1 mRNA expression in the glutamatergic KP neurons by the preovulatory estrogen rise would facilitate an increased firing of KP afferents to other KP neurons, which in turn could enhance the activity of GnRH neurons.

Potential role of CB1-mediated signaling in KP-POMC interactions
KP neurons also innervate the Arc POMC neurons. The glutamatergic and GABAergic afferents of POMC neurons are under tonic, endocannabinoid-induced inhibition (Hentges et al. 2005), which seems to be differentially regulated by estrogen. The estrogen-treatment, given 24 h prior to experimentation, increased the mEPSC frequency, and markedly decreased the potency of CB1 agonists to decrease mEPSC frequency in POMC neurons. In contrast, estrogen potentiated the cannabinoid-induced decrease in mIPSC frequency (Nguyen and Wagner 2006). The opposing effects of estrogen on the cannabinoid regulation of amino acid neurotransmission lead ultimately to the excitation of POMC neurons. This shows similarities with the effect of estrogen on the tonic endocannabinoid signaling of GnRH neurons. As the GABAergic input of GnRH neurons is facilitatory due to their high intracellular chloride levels (DeFazio et al. 2002), an estrogen-mediated down-regulation of CB1 in both GABAergic and glutamatergic afferents, including those originating from KP neurons, could potentially increase the excitation of GnRH neurons. These needs, however, to be confirmed by electrophysiological recordings.

Conclusions
Based on the current results, estrogen seems to play a fundamental role in the regulation of CB1 expression in the different KP subpopulations of the hypothalamus. Estrogenic regulation of CB1 expression in KP cells may release neurotransmitter release from tonic endocannabinoid suppression and contribute to the modification of GABAergic and glutamatergic input of the different target cells, including the GnRH neurons. was performed on animals under deep anesthesia induced by an intraperitoneally injected cocktail of ketamine (25 mg/kg b.w.), xylavet (5 mg/kg b.w.), and pipolphen (2.5 mg/kg b.w.) in.
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