Quantitative and qualitative sex difference in habenula-induced inhibition of midbrain dopamine neurons in the rat

Abstract

Introduction: Clinically relevant sex differences have been noted in a number of affective, behavioral, cognitive, and neurological health disorders. Midbrain dopamine neurons are implicated in several of these same disorders and consequently are under investigation for their potential role in the manifestation of these sex differences. The lateral habenula exerts significant inhibitory control over dopamine neuronal firing, yet little is known about sex differences in this particular neurocircuit.

Methods: We performed in vivo, single unit, extracellular recordings of dopamine neurons in female and male anesthetized rats in response to single pulse stimulation of the lateral habenula. In addition, we assessed baseline firing properties of lateral habenula neurons and, by immunochemical means, assessed the distribution of estrogen receptor alpha cells in the lateral habenula.

Results: Habenula-induced inhibition of dopamine neuronal firing is reduced in female rats relative to male rats. In addition, male rats had a higher prevalence of rebound excitation. Furthermore, the firing pattern of lateral habenula neurons was less variable in female rats, and female rats had a higher density of estrogen receptor alpha positive cells in the lateral habenula.

Discussion: We found that the dopamine neuronal response to habenular stimulation is both qualitatively and quantitatively different in female and male rats. These novel findings together with reports in the contemporary literature lead us to posit that the sex difference in dopamine inhibition seen here relate to differential firing properties of lateral habenula neurons resulting from the presence of sex hormones. Further work is needed to test this hypothesis, which may have implications for understanding the etiology of several mental health disorders including depression, schizophrenia, and addiction.

Keywords: LHb; RMTg; RPE; SABV; prediction error; tVTA.

Figure 1

Representative histology and PSTH associated with LHb-induced DA inhibition. (A) Representative photomicrograph of an electrolytic lesion (*) from a stimulating electrode placed within the LHb and counterstained with neutral red. (B) Representative photomicrograph of DA area used for recording. The blue-green dye mark (circle) was iontophoretically released at the end of the recording track after having passed through the SN. (C,D) Representative histograms of raw spike counts of DA neurons inhibited by LHb stimulation in both a female and male rat, respectively. 3 V, third ventricle; Hipp, hippocampus; LHb, lateral habenula; MHb, medial habenula; ml, medial lemniscus; SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata; Thal, thalamus; VTA, ventral tegmental area. Scale bar = 1 mm (A,B).

Figure 2

LHb-induced inhibition of DA neuronal firing is of longer duration in male rats. (A) Pie charts displaying the DA neuronal response to LHb stimulation by type in both female and male rats. (B) Cumulative probability graph showing the distribution of the duration of inhibition of DA neuron firing following LHb stimulation. Female rats (red line) had a distribution significantly shifted to the left of male rats (blue line; p < 0.001). Inset bar graph shows the mean duration of inhibition of DA neurons from female rats (red) and male rats (blue; *, p = 0.002). (C) PSTH (mean EWMA ± SEM, see methods for detail) for all recorded neurons relative to LHb stimulation (dashed line), regardless of response type, for both female rats (red) and male rats (blue). Overlap between groups is in purple. (D) Group difference from PSTH in C (black line) with ± three standard deviations (dotted lines) of the baseline difference score overlaid. Both the sex difference in inhibition and rebound excitation exceeded three standard deviations for an extended period (purple shading).

Figure 3

Analysis of rebound excitation. (A) Bar chart comparing duration of inhibition in two response phenotypes (inhibition only, solid; inhibition with rebound excitation, hatched) by sex (female, red; male, blue). Only the main effect of sex was significant (*, p = 0.002). (B) Bar chart comparing duration of rebound excitation in neurons from female (red) and male (blue) rats, which was not significantly different. (C) Scatterplot of duration of inhibition against duration of rebound excitation for neurons from female (red circles) and male (blue squares) rats. Overlaid lines represent regression lines for female (red) and male (blue) neurons, as well as the overall regression (black), none of which were significant.

Figure 4

NeuN+ counts within the RMTg (A) Representative photomicrographs of immunochemistry for NeuN in the RMTg of a female (left) and male (right) rat at approximately bregma −6.7 mm (B) Higher magnification view of the boxed area from A. (C) Line graph showing bilateral NeuN+ counts through the rostral-caudal extent of the RMTg for both female (red) and male (blue) rats. Aq = cerebral aqueduct, IPN, interpeduncular nucleus; ml, medial lemniscus, PAG, periaqueductal gray; RMTg, rostromedial tegmental nucleus. Scale bar = 2 mm (A), 0.5 mm (B).

Figure 5

LHb neuron firing activity in female and male rats. (A) Mean firing rate of LHb neurons in female (red) and male (blue) rats. No significant difference in firing rate by sex was found. (B) Pie charts of tonic (solid) and burst (hatched) firing LHb neurons from female (red) and male (blue) rats. No significant difference in firing pattern by sex was found. (C) Box plots showing CV of ISI for female (red) and male (blue) rats. Female rats had a significantly lower CV of ISI (*, p = 0.02), demonstrating greater LHb firing regularity.

Figure 6

ERα + counts within the LHb. (A) Representative photomicrographs of immunochemistry for ERα in the LHb of a female (left) and male (right) rat. (B) Higher magnification view of the boxed area from A. (C) Line graph showing unilateral ERα + counts through the rostral-caudal extent of the LHb for both female (red) and male (blue) rats. Female rats showed a higher count of ERα + cells overall and the specific sections marked (*, p < 0.05). Scale bar = 500 μm (A), 125 μm (B).

Figure 7

Approximate location of all DA neurons recorded in female (left) and male (right) rats, calculated from the location of dye spots and superimposed on coronal drawings at −5.2, −5.6, and − 6.0 mm from bregma. All neurons were electrophysiologically identified as DA neurons within the bounds of the SN or lateral VTA. Neurons are color coded by their response to LHb stimulation: excitation (green), no change (yellow), or inhibition (orange). The duration of inhibition is represented by the intensity of orange saturation. SNc, substantia nigra pars compacta; SNr, substantia nigra pars reticulata; VTA, ventral tegmental area.

Figure 8

Approximate location of all LHb neurons recorded in female (left) and male (right) rats, calculated from the location of dye spots and superimposed on coronal drawings at −3.3, −3.6, and − 3.8 mm from bregma. All neurons were within the bounds of the LHb. For each neuron, the CV of ISI is represented by the intensity of black saturation. D3V, dorsal third ventricle; LHbL, lateral habenula, lateral portion; LHbM, lateral habenula, medial portion; MHb, medial habenula.