Author response: The cryptic gonadotropin-releasing hormone neuronal system of human basal ganglia (2021)

Article Figures and data Abstract Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Human reproduction is controlled by ~2000 hypothalamic gonadotropin-releasing hormone (GnRH) neurons. Here, we report the discovery and characterization of additional ~150,000–200,000 GnRH-synthesizing cells in the human basal ganglia and basal forebrain. Nearly all extrahypothalamic GnRH neurons expressed the cholinergic marker enzyme choline acetyltransferase. Similarly, hypothalamic GnRH neurons were also cholinergic both in embryonic and adult human brains. Whole-transcriptome analysis of cholinergic interneurons and medium spiny projection neurons laser-microdissected from the human putamen showed selective expression of GNRH1 and GNRHR1 autoreceptors in the cholinergic cell population and uncovered the detailed transcriptome profile and molecular connectome of these two cell types. Higher-order non-reproductive functions regulated by GnRH under physiological conditions in the human basal ganglia and basal forebrain require clarification. The role and changes of GnRH/GnRHR1 signaling in neurodegenerative disorders affecting cholinergic neurocircuitries, including Parkinson’s and Alzheimer’s diseases, need to be explored. Introduction Mammalian reproduction is controlled by a few hundred/thousand preoptic/hypothalamic neurons, which release the decapeptide neurohormone gonadotropin-releasing hormone (GnRH) into the hypophysial portal circulation. GnRH promotes fertility via increasing the synthesis and secretion of luteinizing hormone and follicle-stimulating hormone in the anterior pituitary (Herbison, 2018). Unlike other neurons of the central nervous system, GnRH neurons are born in the olfactory placodes and migrate into the forebrain prenatally (Casoni et al., 2016; Schwanzel-Fukuda and Pfaff, 1989; Wray et al., 1989). Recent developmental studies on embryos/fetuses determined the detailed spatio-temporal profile of this process in the human (Casoni et al., 2016). Approximately 2000 neurons were observed along a ventral migratory path whereby GnRH neurons reach the hypothalamus to regulate reproduction after puberty. In addition, a previously unknown dorsal migratory route has been identified whereby ~8000 GnRH neurons migrated towards pallial and/or subpallial structures. The fate of these neurons at later stages of pre- and postnatal development has been unexplored so far. While GnRH neurons in adult laboratory rodents are mostly preoptic/hypothalamic and serve reproductive functions (Merchenthaler et al., 1980), a handful of anatomical studies on primates identified additional GNRH1 mRNA expression and/or GnRH immunoreactivity in extrahypothalamic regions unrelated to reproduction. These included several basal ganglia and the basal forebrain (Krajewski et al., 2003; Quanbeck et al., 1997; Rance et al., 1994; Terasawa et al., 2001). Initial enthusiasm to study these neurons further faded after suggestions that extrahypothalamic GnRH neurons in monkeys contain the GnRH degradation product GnRH1-5, instead of the bona fide GnRH decapeptide (Quanbeck et al., 1997; Terasawa et al., 2001). Here, we study human extrahypothalamic GnRH neurons in adult postmortem brains with immunohistochemistry (IHC), in situ hybridization (ISH), single-cell transcriptomics (RNA-seq), and high-performance liquid chromatography/tandem mass spectrometry (HPLC-MS/MS). We report and characterize a previously unexplored large GnRH neuron population with ~150,000–200,000 cell bodies scattered in different basal ganglia and the basal forebrain. Extrahypothalamic GnRH neurons, most of which are found in the putamen (Pu), contain bona fide GnRH decapeptide, as shown by HPLC-MS/MS. Deep transcriptome analysis reveals that these neurons express GnRH biosynthetic enzymes, GNRHR1 autoreceptors, and the molecular machinery of cholinergic and GABAergic co-transmission. Somewhat unexpectedly, hypothalamic GnRH neurons also exhibit cholinergic phenotype in the embryonic and adult human brains. Altogether, these data indicate that GnRH is synthesized as a co-transmitter of many cholinergic neurons in the human basal ganglia and basal forebrain. At least in the Pu, GnRH appears to act on GnRHR1 autoreceptors to regulate higher-order non-reproductive functions associated with the cholinergic system. Results Human extrahypothalamic GnRH-immunoreactive neurons occur in the basal ganglia and the basal forebrain The primate central nervous system contains extrahypothalamic GnRH cell populations, which have unknown functions (Krajewski et al., 2003; Quanbeck et al., 1997; Rance et al., 1994; Terasawa et al., 2001). An earlier ISH study of adult human brains identified ~6000–7000 GNRH1 mRNA-expressing neurons in the Pu and the nucleus basalis magnocellularis of Meynert (nbM), among other sites (Rance et al., 1994). Here, we used IHC to address the presence and map the distribution of GnRH-immunoreactive (IR) neurons in extrahypothalamic sites of three adult human brains (#1–3). Every 24th 100-µm-thick coronal section between Bregma levels −22.5 and 33.1 (Mai et al., 1997) was immunostained using a well-characterized guinea pig antiserum (#1018) against GnRH decapeptide (Hrabovszky et al., 2011; Figure 1A, B). This experiment revealed numerous extrahypothalamic GnRH-IR neurons in the Pu, moderate numbers in the nucleus accumbens (nAcc) and the head of the nucleus caudatus (Cd), and lower numbers also in the nbM (Figure 1C). Labeled neurons were also scattered in the globus pallidus (GP), the ventral pallidum (VP), and the bed nucleus of the stria terminalis (BnST). The immunostained perikarya showed round or oval shape, with a mean diameter of 29 µm in the Pu (Figure 1B, C). Preabsorption of the working solution of this antiserum with 0.1 µg/ml GnRH decapeptide eliminated all labeling in control experiments using sections of three subjects (#17–19) (Figure 2A). Figure 1 with 1 supplement see all Download asset Open asset Anatomical approaches unveil the distribution, number, fine structure, and cholinergic phenotype of extrahypothalamic gonadotropin-releasing hormone (GnRH) neurons in the adult human brain. (A) Extrahypothalamic GnRH-immunoreactive (GnRH-IR) neurons were mapped with immunohistochemistry (IHC) and quantified in the brain of three adult human individuals (#1–3). (B) Representative coronal section of a 31-year-old female subject (#1) illustrates the caudate nucleus (Cd), putamen (Pu), claustrum (Cl), insular cortex (Ins), anterior limb of the internal capsule (aic), external capsule (ec), extreme capsule (ex), corpus callosum (cc), lateral medullary lamina (lml), lateral ventricle (LV), nucleus accumbens (nAcc), and olfactory tubercle (Tu). High-power insets (1–3) reveal extrahypothalamic GnRH neurons many of which can be found in the Pu. (C) The majority (82.2%) of the 163,168 ± 36,223 extrahypothalamic GnRH neurons occurred in the Pu, followed by the nAcc, Cd, nucleus basalis magnocellularis (nbM), globus pallidus (GP), ventral pallidum (VP), and bed nucleus of the stria terminalis (BnST). (D) To visualize the fine structure of dendrites, the immunofluorescent (IF) detection of GnRH was combined with cell membrane labeling using DiI delivered with a Gene Gun. (E) 3-D reconstruction of the DiI-labeled (magenta) GnRH-IR (green) neurons revealed large multipolar cells, which exhibited only few dendritic spines. (F) Depth color coding, where colors represent distance from the section surface, allowed better distinction between DiI-labeled processes of the GnRH neuron (upper inset; GnRH+) from other DiI-labeled neuronal elements many of which belonged to medium spiny GABAergic projection neurons (lower inset; GnRH-). (G) Double-IF experiments addressed the presence of the cholinergic marker enzyme choline acetyltransferase (ChAT) in GnRH neurons. Nearly all GnRH neurons in the Pu contained ChAT signal. (H) The GnRH neuron population also overlapped with cholinergic projection neurons of the nbM. (I) With few exceptions, GnRH neurons were ChAT-immunoreactive (green columns), whereas they represented relatively small subsets of cholinergic cells (magenta columns) being the highest in the Pu (~35%). Scale bar (shown in H): 3.5 mm in low-power photomicrograph (B), 50 μm in (B, C, G and H) (insets in G and H: 25 μm), 12.5 μm in (E) and (F) (insets: 3.7 μm). Figure 2 Download asset Open asset Combined evidence from immunohistochemistry (IHC), in situ hybridization (ISH), and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) indicates that extrahypothalamic gonadotropin-releasing hormone (GnRH) neurons synthesize bona fide GnRH decapeptide derived from the GNRH1 transcript. (A) IHC labeling of the human putamen with the guinea pig polyclonal GnRH antiserum #1018 reveals a large number of immunoreactive neurons in control sections (Ctrl) of a 64-year-old male subject (#19). All labeling is eliminated if the working solution of #1018 is preabsorbed overnight with 0.1 µg/ml GnRH. (B) A series of additional primary antisera against the human GnRH-associated peptide (hGAP1) or GnRH also recognize immunoreactive neurons in the human putamen (#6). Such antibodies include the LR-1 rabbit primary antiserum, which was reported previously not to label extrahypothalamic GnRH neurons in the developing monkey brain. (C) Positive control with the combined use of two GnRH antibodies from different host species for dual-immunofluorescence (IF) experiments on sample #5 provides evidence that the antibodies detect the same neuronal elements. (D) Non-isotopic ISH/IF dual-labeling studies reveal that GnRH-immunoreactive neurons express GNRH1 mRNA, indicating that extrahypothalamic GnRH is a GNRH1 gene product (sample #15). (E) As illustrated in representative chromatograms, HPLC-MS/MS ( #21–28) detects bona fide GnRH decapeptide in tissue extracts from the mediobasal hypothalamus (MBH), putamen (Pu), and nucleus caudatus (Cd), but not the claustrum (Cl). (F) The GnRH1-5 degradation product is present in the Pu and Cd and undetectable in the MBH and Cl. (G) Quantitative analysis reveals the highest tissue concentrations of GnRH in the MBH, somewhat lower levels in the Pu and the Cd, and no detectable GnRH decapeptide signal in the Cl. Note that tissue concentrations of GnRH in the Pu and the Cd are 3–4 times higher than those of GnRH1-5. Scale bar (shown in D): 120 μm in (A, B), 50 μm in (C, D). Quantitative analysis detects ~150,000–200,000 extrahypothalamic GnRH neurons in the adult human brain most of which are located in the putamen GnRH neurons develop in the olfactory placodes and migrate to the brain prenatally (Schwanzel-Fukuda and Pfaff, 1989; Wray, 2001). Recent studies from Casoni and colleagues identified 10,000 migrating GnRH neurons in human embryos/fetuses most of which (~8000) followed a previously unknown dorsal migratory route targeting subpallial and/or pallial structures, as opposed to the ~2000 neurons in the ventral route leading to the hypothalamus (Casoni et al., 2016). We addressed the possibility that extrahypothalamic GnRH-IR neurons of the adult brain originate from the 8000 neurons observed in the dorsal pathway. With this aim, we determined the total number of GnRH-IR neurons in the basal ganglia and the basal forebrain. Immunolabeled neurons were counted in every 24th section of a single hemisphere using light microscopy (Figure 1A, B). Cell counts were then multiplied by 24 and 2 (for the two hemispheres) and compensated for overcounting (Abercrombie, 1946; Guillery, 2002; Figure 1—figure supplement 1A). The total number of extrahypothalamic GnRH neurons calculated this way in three subjects was 229,447 (31-year-old female; #1), 155,357 (61-year-old male; #2), and 104,699 (62-year-old male; #3) (163,168 ± 36,223; mean ± SEM). These unexpectedly high GnRH cell numbers made it unlikely that human extrahypothalamic GnRH neurons develop from olfactory placodes and migrate into the brain along the dorsal migratory route (Casoni et al., 2016). The individual variations in total GnRH cell numbers of the three samples may be both biological and technical, which would be difficult to separate. 82.2 ± 1.1% of labeled cells were observed in the Pu, 5.5 ± 0.2% in the nAcc, 4.9 ± 0.7% in the Cd, 3.5 ± 1.1% in the nbM, 1.8 ± 0.5% in the GP, 1.3 ± 0.1% in the VP, and 0.8 ± 0.2% in the BnST (Figure 1C). Extrahypothalamic GnRH neurons synthesize bona fide GnRH decapeptide derived from the GNRH1 transcript Results of previous studies with IHC on embryonic and fetal rhesus monkey brains questioned whether extrahypothalamic GnRH neurons synthesize bona fide GnRH decapeptide (Quanbeck et al., 1997; Terasawa et al., 2001). First, developing GnRH neurons in the septum, stria terminalis, amygdala, striatum, and internal capsule of the monkey brain were not detected by several GnRH antibodies (Quanbeck et al., 1997; Terasawa et al., 2001), including the widely used LR-1 rabbit GnRH polyclonal antiserum (Silverman et al., 1990). Second, these neurons exhibited immunoreactivity to EP24.15 (aka thimet oligopeptidase; THOP1), a metalloendopeptidase, which can cleave GnRH at the Tyr5-Gly6 position to generate GnRH1-5 (Terasawa et al., 2001). To investigate the possibility that GnRH neurons in the basal ganglia and the basal forebrain of the adult human brain use GnRH1-5, rather than GnRH decapeptide for signaling, we first tested a series of polyclonal antibodies against human GnRH-associated peptide (hGAP1) or GnRH decapeptide (Supplementary file 2) for their reactivity with GnRH neurons of the human Pu (N = 10; #5, 6, and 12–19). All of the tested antibodies, including the LR-1 antiserum, recognized GnRH-IR neurons (Figure 2B), suggesting that these cells contain the bona fide GnRH. Neurons detected with different antibodies were identical as they were double-labeled (Figure 2C) in dual-immunofluorescence (IF) experiments (N = 2; #4 and 5) using two GnRH antibodies from different host species. Results of further control experiments with the combined use of IF and non-isotopic ISH (N = 5; #15–19) showed that GnRH-IR neurons express GNRH1 mRNA (Figure 2D). Finally, to provide direct evidence for the biosynthesis of the GnRH decapeptide in these cells, tissue samples (#21–28) were microdissected from the mediobasal hypothalamus (MBH; N = 2), Pu (N = 5), Cd (N = 2), and Cl (N = 3). HPLC-MS/MS analysis of the tissue extracts established that the dominant peptide form in the Pu and Cd is the GnRH decapeptide. GnRH1-5 was also present, albeit at 3–4 times lower tissue concentrations (Figure 2E–G). Only GnRH decapeptide was detectable in the MBH (used as a positive control) where hypophysiotropic GnRH neurons occur and neither peptide form was present in the Cl, in accordance with the absence of IHC labeling at this site (Figure 2E–G). Together with observations from the IHC and ISH experiments, HPLC-MS/MS results gave firm support to the notion that extrahypothalamic GnRH neurons mainly produce bona fide GnRH decapeptide derived from the GNRH1 gene. GnRH neurons of the putamen are large multipolar interneurons with smooth-surfaced dendrites The IHC method was unable to visualize the entire dendritic arbor of extrahypothalamic GnRH-IR cells (Figure 1B, C). This limitation could be due to the low amount and/or restricted subcellular distribution of the peptide. To overcome this problem, we labeled the dendritic compartment of GnRH cells with the lipophilic dye DiI for further morphological analysis (Takács et al., 2018; Figure 1D). Following the immunofluorescent (IF) visualization of GnRH neurons in the Pu of a 72-year-old female (#20), DiI-coated tungsten particles were delivered into the sections using a Helios Gene Gun (Bio-Rad) (Figure 1D; Takács et al., 2018). Spreading of this lipophilic dye along the cytoplasmic membrane caused Golgi-like labeling of random-hit neurons, including 12 GnRH-IR cells (Figure 1E, F). Confocal microscopic analysis and 3-D reconstruction of the DiI signal revealed spider-like neurons with rich arborization of poorly spined dendrites. DiI-labeled GnRH neurons were clearly distinct from the main Pu cell type, the densely spined medium spiny GABAergic projection neurons (SPNs) (Figure 1E, F). Extrahypothalamic GnRH cells represent subpopulations of cholinergic neurons SPNs represent 80–98% of striatal neurons, the remainder being made up of cholinergic and different subclasses of GABAergic interneurons (Gonzales and Smith, 2015). DiI-labeled GnRH cells resembled cholinergic interneurons (ChINs) in size and dendritic morphology. Indeed, dual-IF experiments (N = 4; #3–5 and 19) established that GnRH neurons of the Pu contain the cholinergic marker enzyme choline acetyltransferase (ChAT) (Figure 1G). Similarly, GnRH neurons in the nbM (Figure 1H) and other extrahypothalamic sites also exhibited ChAT immunoreactivity. The extent of ChAT/GnRH colocalization was assessed quantitatively in five distinct regions of a 62-year-old male subject (#3). Confocal microscopic analysis of representative dual-labeled sections established that the vast majority of extrahypothalamic GnRH neurons are cholinergic (green bars in Figure 1I). In contrast, GnRH-IR neurons represented only 34.9% of all cholinergic neurons in the Pu, 6.3% in the nAcc, 1.8% in the head of the Cd, 3.6% in the nbM, and 28.4% in the GP (magenta bars in Figure 1I). GnRH-positive and GnRH-negative cholinergic neurons often intermingled, without gross morphological differences between the two phenotypes (Figure 1G, H). Hypothalamic GnRH neurons regulating reproduction also exhibit an unexpected cholinergic phenotype The ChAT phenotype emerged as a hallmark of extrahypothalamic GnRH neurons. To confirm this notion by verifying the absence of ChAT in the hypothalamic GnRH neuron population, hypothalamic tissue sections were processed for dual-IF detection of ChAT and GnRH, followed by confocal microscopic analysis. Unexpectedly, 34.6 ± 7.1% of the hypothalamic GnRH neurons also exhibited ChAT signal in seven adult human male and female subjects (Figure 3A, B; #3 and 6–11), a phenomenon not observed in other species before. Figure 3 Download asset Open asset Both hypothalamic and extrahypothalamic gonadotropin-releasing hormone (GnRH) neurons exhibit cholinergic phenotype gained during early fetal development. (A) The cholinergic phenotype is not a hallmark of human extrahypothalamic GnRH neurons because large subsets of GnRH neurons in the adult human hypothalamus (green immunofluorescent signal; subject #3) also exhibit choline acetyltransferase (ChAT; magenta) immunoreactivity. High-power insets show single- (arrowhead) and dual-labeled (arrows) GnRH neurons from framed regions. (B) Quantitative analysis of 97 GnRH neurons from seven subjects (#3; 6–11) reveals the ChAT phenotype in 34.6 ± 7.1% of hypothalamic GnRH neurons. (C–E) The cholinergic phenotype of GnRH neurons is gained during early fetal development. Left panels illustrate coronal views of the fetal head at GW11 (#29). Representative photomicrographs taken from sites indicated by the red arrows show results of dual-immunofluorescence experiments. (C) At this stage of development, a large subset of GnRH neurons (green immunofluorescent signal) migrate in the nasal region toward the brain and do not exhibit ChAT signal. (D, E) In contrast, GnRH neurons migrating through the septal area (D, arrows) or located in the striatum (E, arrow) express ChAT (magenta). Scale bar (shown in E): 50 µm in (A, C, D) (insets in A: 12.5 µm), 20 µm in (E). Cholinergic phenotype of human GnRH neurons develops prenatally To address when GnRH neurons gain the cholinergic phenotype, prenatal co-expression of ChAT and GnRH was explored via dual-IF experiments in coronal sections of two fetal heads (#29 and 30) at gestational week 11 (GW11). At this age, ~20% of GnRH neurons can still be found in the nasal region, whereas the majority have already entered the brain to migrate toward hypothalamic and extrahypothalamic target areas (Casoni et al., 2016). While GnRH-positive neurons within the nasal compartment did not contain ChAT signal (Figure 3C), those in the septum (Figure 3D), the striatum (Figure 3E) and elsewhere in the developing brain were already ChAT-IR. These data suggest that migrating GnRH neurons become cholinergic after entering the brain and continue to express ChAT immunoreactivity in hypothalamic as well as extrahypothalamic regions. Neurons laser-capture microdissected from the postmortem putamen provide sources for high-quality RNA suitable for RNA-seq ChINs, at least one-third of which synthesize GnRH in the human Pu (Figure 1I), communicate with SPNs locally (Ahmed et al., 2019). To localize the receptors mediating the effects of GnRH in the adult human Pu, transcriptome profiling of cellular samples enriched in ChINs and SPNs was carried out. Being the largest cell type, ChINs were readily recognizable in sections subjected to Nissl-staining under RNase-free conditions, whereas the medium-sized SPNs represent the most frequently encountered neuronal phenotype in the Pu (Figure 4A). Laser-capture microdissection (LCM) was used to collect neuronal pools enriched in ChINs and SPNs from cresyl violet-stained Pu sections of two human subjects (#21 and 22). Each ChIN-enriched pool contained ~300 large neurons and each SPN-enriched pool consisted of ~600 medium-sized neurons (Figure 4A). Total RNA was isolated and RNA-seq libraries were prepared from the four cell pools and sequenced with the Illumina NextSeq 500/550 High Output (v2.5) kit. 29.4M and 25.8M reads were obtained from the two ChIN pools and 26.3M and 3.2M reads from the two Approximately and reads from ChIN and and reads from pools were mapped to the human and identified occurred at in ChIN pools and and in pools (Figure and file 3). Figure Download asset Open asset Deep transcriptome profiling of cholinergic interneurons (ChINs) and spiny projection neurons (SPNs) provides into extrahypothalamic gonadotropin-releasing hormone (GnRH) signaling and the molecular connectome of the human (A) coronal sections were on membrane from putamen samples of two male human subjects (#21 and and with an Neurons were using Nissl-staining and isolated with laser-capture microdissection neurons included in each ChIN-enriched cell pool were recognized on their large The vast majority of the medium-sized microdissected and neurons to the putamen cell Total RNA was isolated and RNA-seq prepared from both cell and sequenced with the Illumina NextSeq 500/550 High Output (v2.5) kit. (B) analysis high of cholinergic in the two ChIN pools and of in the two levels in counts reads and in each areas transcript to the highest (C) elements of GnRH signaling, and GnRH are illustrated in two The GNRH1 and GNRHR1 are present in ChINs only ChINs express all for The which may for GnRH in both cell at higher levels in SPNs than in transcript whereas numbers indicate of subjects and 22). laser-capture microdissection of striatal cholinergic interneurons and medium spiny projection neurons Cholinergic including and were enriched in the ChIN pools from subjects and These were or found at low levels only in the two pools (Figure ChINs from development, the expression of the and in cell into ChINs and These as well as 3 showed and expression in ChINs (Figure The in expressed higher levels of than ChINs, including cholinergic and and several (Figure distribution of the that the ChINs from SPNs for transcriptome high levels of expression of GABAergic marker and in ChINs, in to SPNs (Figure revealed that ChINs use GABAergic as earlier for ChINs of the et al., 2018). Cholinergic interneurons express GNRH1 and GNRHR1 and contain GnRH biosynthetic GNRH1 was expressed in the two ChIN in accordance with the morphological observations (Figure of the with by from which ChINs expressed the of GnRH including and were also present in ChINs (Figure The enzyme for the of including GnRH. This enzyme was expressed in both ChINs and with a higher in the The most of the RNA-seq study was that the GNRHR1 was expressed in This indicated that GnRH in the human Pu on GnRHR1 autoreceptors and also made it unlikely that SPNs are by GnRH derived from Altogether, transcriptome profiling of ChINs and SPNs molecular support to a that GnRH is synthesized by ChINs and locally via GnRHR1 profiling provides into the molecular connectome of the human putamen profiling of ChINs and SPNs revealed a large of that were expressed or in cell in to many other expressed in and identified this way allowed to signaling that act in the between ChINs and receptors to serve as autoreceptors may from neuronal sources within or the from the transcriptome are illustrated as a in Figure A into the molecular connectome of the human Pu can be obtained from the detailed and expression of ChINs and SPNs and from the of expressed in the two and two ChIN cell pools (Supplementary file Figure Download asset Open asset RNA-seq studies reveal the and expression of cholinergic interneurons (ChINs) and spiny projection neurons (SPNs) and provide into the molecular connectome of putamen cell types. signaling are on and peptide expression of the two cell types. ChINs to use and and may at least as autoreceptors in between the two cell is also indicated by receptors from different neuronal sources within or the in transcript expressed as mean counts from subjects and The illustrates receptors that were observed in the cell of both human Discussion Extrahypothalamic GnRH-IR neurons to GnRH neurons detected earlier with in situ hybridization and synthesize the GnRH decapeptide derived from the GNRH1 gene A ISH study by Rance and three of GNRH1 mRNA-expressing neurons in the human brain on shape, and labeling (Rance et al., 1994). The GnRH-IR neurons we detected in study to neurons by shape, large nucleus and and GNRH1 mRNA levels between those of labeled neurons in the MBH and labeled neurons in the septum and the dorsal area (Rance et al., 1994). IHC studies also detected many hypothalamic GnRH neurons but found only few septal neurons, which to the