A GABAergic cell type in the lateral habenula links hypothalamic homeostatic and midbrain motivation circuits with sex steroid signaling

Abstract

The lateral habenula (LHb) has a key role in integrating a variety of neural circuits associated with reward and aversive behaviors. There is limited information about how the different cell types and neuronal circuits within the LHb coordinate physiological and motivational states. Here, we report a cell type in the medial division of the LHb (LHbM) in male rats that is distinguished by: (1) a molecular signature for GABAergic neurotransmission (Slc32a1/VGAT) and estrogen receptor (Esr1/ERα) expression, at both mRNA and protein levels, as well as the mRNA for vesicular glutamate transporter Slc17a6/VGLUT2, which we term the GABAergic estrogen-receptive neuron (GERN); (2) its axonal projection patterns, identified by in vivo juxtacellular labeling, to both local LHb and to midbrain modulatory systems; and (3) its somatic expression of receptors for vasopressin, serotonin and dopamine, and mRNA for orexin receptor 2. This cell type is anatomically located to receive afferents from midbrain reward (dopamine and serotonin) and hypothalamic water and energy homeostasis (vasopressin and orexin) circuits. These afferents shared the expression of estrogen synthase (aromatase) and VGLUT2, both in their somata and axon terminals. We demonstrate dynamic changes in LHbM VGAT+ cell density, dependent upon gonadal functional status, that closely correlate with motivational behavior in response to predator and forced swim stressors. The findings suggest that the homeostasis and reward-related glutamatergic convergent projecting pathways to LHbMC employ a localized neurosteroid signaling mechanism via axonal expression of aromatase, to act as a switch for GERN excitation/inhibition output prevalence, influencing depressive or motivated behavior.

Fig. 1. A novel type of GABAergic neuron with bi-functional output, was identified using in vivo juxtacellular single-cell labeling, immunostaining, and anatomical reconstruction methods.

Three cells are reported in this study, one per each nucleus of the medial division of the lateral habenula (the superior, LHbMS, the central, LHbMC, and the marginal, LHbMMg) are depicted in one schematic coronal view of the habenula aiming to give a general idea about their spatial relationship with the region and among them, although they were from three different rats. Camera-lucida reconstruction from serial sections of the neuronal somata, dendrites, and axons were superimposed manually in a 2-dimension (2D) projection drawing from a coronal view of the rat habenula. The soma and dendrites were represented in black and axonal segments were color-coded as blue for cell A (located in the LHbMS), and green for cell B (located in the LHbMC) (see the third cell in SI Fig. 1). A₁: The soma of the cell A is immunopositive to ERα. A₂, A₃: photomicrographs of labeled soma and proximal dendrites revealed by avidin-biotin-peroxidase diaminobenzidine reaction, at low and high magnifications, red arrowhead in A₃ shows the emergence site of the main axon. A₄: photomicrograph of a rare axonal terminal-like arborization observed at the squared region in the reconstruction. A₅: the main axon emitted a single collateral that coursed ventrally and branched in the medial central region of the LHb (the branching point is indicated by an asterisk, also shown in the inset and in the reconstruction). Note in the reconstruction that the projecting axon entered to the fr and was found in further caudal sections. A₆, A₇: neurobiotin/VGAT double labeling at axon terminals (white solid arrows). Note that some neurobiotin-labeled axonal VGAT (hollow white arrows). A₈: upper traces. extracellular recording of low spontaneous firing pattern and lower trace shows that when low intensity (<10 nA) positive current pulses were injected by way of the microelectrode, the neuron firing pattern was modulated, a requirement to yield a successful labeling. Scale bars: A₁: 25 µm; A₂: 500 µm; A₃: 50 µm; A₅: 100 µm; A₇, A₈: 10 µm. B₁: Immunohistochemical detection of ERα expressed in the neurobiotin-labeled cell. B₂: a compound photomicrograph made up by photomicrographs taken from 11 consecutive sections showing the neurobiotin-labeled soma and the main axon projecting to the fr. In a proximal point of the main axon, an axon-collateral was emitted (indicated by a red asterisk and a circle). Inset shows a higher magnification photomicrograph of the collateral origin point. B₃: shows the intrahabenular branched axon segments and ATs labeled with neurobiotin (NB). B₄: confocal images show the GABAergic nature of this cell (ATs immunopositive to VGAT). B₅: Axon terminals found in caudal sections at the level of sustantia nigra pars reticulata, in close apposition with parvalbumin-expressing dendrites. B₆: upper traces are extracellular recording of spontaneous firing patterns (7.3 Hz before electrical modulation applied for juxtacellular labeling purpose, lower trace). Scale bars: 20 µm except B₃: 100 µm and B₅: 10 µm

Fig. 2. GABAergic estrogen-receptive neurons (GERNs) localization, mRNA expression by ISH and somatic receptor expression by IHC.

a Serial coronal sections showing the ERα immunolabelling at the Bregma rostro-caudal coordinates (numbers in mm under the photomicrographs). Boxed area in A₆ at higher magnification showing the exclusive nuclear labeling pattern. The bold numbered levels (A₁ and A₈), were chosen to show that no positive labeling was found in either rostral or caudal directions. A₉: a horizontal view of the distribution of estrogen-receptive cells was symbolized by the red oval. A₁₀: sagittal view of rat brain atlas, modified from Paxinos & Watson, at lat. 0.90 mm, where lateral habenula (LHb) is symbolized with a gray shade and A9 plane was symbolized with a horizontal line. b In situ hybridization using multiple RNAscope methods. B₁: multiplex fluorescence method, Esr1, gene that encodes ERα (red punctuated labeling) co-expressed with Slc32a1, gene that encodes VGAT (green punctuated labeling); arrows indicate the double-labeled cells; B₂: duplex method, Esr1 (red punctuated labeling) co-localization with Slc17a6, gene that encodes VGLUT2 (green punctuated labeling); arrows indicate the double-labeled cells; B₃: with duplex method, Slc32a1 encoding VGAT (red punctuated labeling) shows complete co-localization with Slc17a6 encoding VGLUT2 (green punctuated labeling); arrows indicate the double-labeled cells. Inset of B₃, Slc32a1 expression in a sexually active (SA) rat LHb, Br. −3.72 mm (brown labeling, single chromogenic-Brown method-RNAscope). *Note the similarity with ERα expression in A₆. c Indirect immunohistochemistry showing the GABAergic nature of the ERα+ neurons (red) in a SA rat brain. The GABA antibody (green, Sigma, A0310) produced characteristic surface labeling (C₁, C₂: the strip-like image was produced by Vibratome slicing leaving the brain section with an uneven surface). d ERα+ neurons co-express receptor/receptor subtypes for vasopressin, orexin, dopamine, and serotonin. D₁: In situ hybridization using RNAscope-multiplex method targeting Esr1 (red dots), Slc32a1 (white dots) and Hcrtr2, gene encodes the orexin receptor 2 (green dots). D₂, D₃: Indirect immunofluorescence reactions, showing that the ERα-IR cells co-expressed vasopressin receptor V1a (inset showing the V1a antibody labeling pattern in temporal hippocampus CA2 cells body layer. See also SI Fig. 6 for more information about this antibody), dopamine receptor D5R (also called D1Rb) and serotonin receptor 5-HTr2a, respectively. Scale bars: A₅ and b: 500 µm and rest: 10 µm

Fig. 3. Presence of convergent inputs from vasopressinergic, orexinergic, dopaminergic and serotoninergic pathways expressing VGluT2 and P450 aromatase in LHbM where the GERNs cells were located.

a–d Confocal images revealed that axon terminals immunopositive for neurophysin II (NPII, a), orexin (OR, b), serotonin transporter (SerT, c), and tyrosine hydroxylase (TH, d) contained aromatase (ARO) and vesicular glutamate transporter 2 (VGLUT2) inside the LHbMC. Note in c, 3D computer reconstruction of the serial optical slices in Z-stack to show that fibers which expressed SerT were of two types: thick SerT+/VGLUT2+/aromatase+ (yellow arrows) and thin SerT+/VGLUT2−/aromatase− profiles (green arrows). Insets for each confocal photomicrograph group show the global fiber distribution patterns with peroxidase immunoreaction against arginine vasopressin (AVP, a), orexin (OR, b), serotonin transporter (SerT, c), and tyrosine hydroxylase (TH, d) at the LHbM region. A detailed anatomical distribution from serial coronal sections is depicted in SI Fig. 3. e Summary diagram of FG retrograde tracing results presented in SI Fig. 4. The upstream regions identified by FG retrograde tracing experiments are coded by colors: hypothalamic vasopressinergic nuclei in blue; lateral hypothalamic orexinergic cell population in gray; dorsal raphe lateral (DRL) serotonin transporter (SerT) expressing neurons in beige; substantia nigra pars compact (SNpc), and ventral tegmental area (VTA) tyrosine hydroxylase (TH) expressing neurons in pink. The projection distributions of each pathway, in the habenula region, are symbolized with the corresponding color patches. The beige gradient filling symbolizes the predominant distribution pattern of SerT+ fibers observed (for details see SI Fig. 3D). The four pathways to habenula (color-coded arrows) shared a common feature of co-expression of VGLUT2 (symbolized in green) and estrogen synthase/P450 aromatase (symbolized in red). Scale bars: a–d 500 µm and e–h 10 µm

Fig. 4. Androgen receptor (AR) and P450 aromatase (ARO) expression in PVN, LH, VTA/SNpc and DRL, AVP+, OR+, TH+ and SerT+ neurons, respectively.

The series of a–d show the confocal images for co-expression of AR, aromatase and AVP (PVN, a), orexin (LH, b), SerT (DR, c) and TH (VTA, d). Examples of co-expressed cells are indicated with arrows. Scale bars: a 500 µm; A₁–A₄: 50 µm; b 500 µm; B₁–B₄: 20 µm; c 500 µm; C₁–C₄ 25 µm; d 1 mm; D₁–D₄ 50 µm

Fig. 5. Hormonal conditions affect GERN-LHbM input composition and cell density: correlation with depressive vs motivated behaviors.

a Gonadectomy in male rats produced a remarkable reduction of AVP immunoreactivity in lateral habenula medial (LHbM) that is restored with hormone (testosterone) replacement therapy (HRT). A₁: Control; A₂: AVP-IHC performed 60 days after gonadectomy (PGnxD60), and A₃: 60 days after gonadectomy animals received hormone replacement therapy for 30 days (PGnxD60+HRT30). b Photomicrographs showing in situ hybridization experiment using RNAscope 2.5 HD Assay-BROWN method. B₁: Slc32a1 cell density changed in the LHbM under the four hormonal conditions. HRT (**p < 0.01) and sexual activity (*p < 0.05) significantly increased the number of Slc32a1 neurons in LHbM. B₂: No significant differences were found between all groups SI and GNX rats with respect to the Esr1 expression. c Upon cat exposure, rats expressed innate fear-related passive (freezing), and active (rearing, climbing, and displacement) behaviors: rats from 60 days post gonadectomy group (Gnx) showed significant increase of freezing counts (*p = 0.0203) and reduced number of climbing and rearing behaviors (*p = 0.0421). d Quantification of behavioral despair assessed using forced swimming test (FST). Gnx significantly increased the immobility counts in the FST (***p < 0.001). Gnx rats that received HRT recovered to levels comparable to control animals. By contrast, water and food deprivation had the opposite effect, significantly decreasing the immobility counts compared to control (*p < 0.05)