Sexual dimorphism in histamine regulation of striatal dopamine

Abstract

Dopamine modulation of the basal ganglia differs in males and females and is implicated in numerous neuropsychiatric conditions, including some, like Tourette Syndrome (TS) and attention deficit hyperactivity disorder (ADHD), that have marked sex differences in prevalence. Genetic studies in TS and subsequent work in animals suggest that a loss of histamine may contribute to dysregulation of dopamine. Motivated by this, we characterized the modulation of striatal dopamine by histamine, using microdialysis, targeted pharmacology, and shRNA knockdown of histamine receptors. Intracerebroventricular (ICV) histamine reduced striatal dopamine in male mice, replicating previous work. In contrast, and unexpectedly, ICV histamine increased striatal dopamine in females. ICV or targeted infusion of agonists revealed that the effect in males depends on H2R receptors in the substantia nigra pars compacta (SNc). Knockdown of H2R in SNc GABAergic neurons abrogated the effect, identifying these cells as a key locus of histamine's regulation of dopamine in males. In females, however, H2R had no discernible role; instead, H3R agonists in the striatum increased striatal dopamine. Strikingly, the effect of histamine on dopamine in females was modulated by the estrous cycle, appearing only in estrus/proestrus, when estrogen levels are high. These findings confirm the regulation of striatal dopamine by histamine but identify marked sexual dimorphism in and estrous modulation of this effect. These findings may shed light on the mechanistic underpinnings of sex differences in the striatal circuitry, and in several neuropsychiatric conditions.

Figure 1.. Timeline and methods for experiments.

A. Timeline of events for each of the five different experimental types described in this manuscript. B. Cannula placements and drugs for male and female mice used in ICV Histamine experiments. C. Cannula placements and drugs for male and female mice used in ICV Histamine agonist infusion and open field experiments. D. Cannula placements and drugs for male and female mice used in Targeted Histamine Agonist experiments. E. AAV target, cannula placements, and drugs for male mice used in ICV Histamine Infusion with shRNA knock down of histamine receptor H2 (Hrh2) experiments. F. Estrus cycle groups, cannula placements, and drugs for female mice used in Targeted Histamine Agonist Infusion with Estrus Cycle Tracking.

Figure 2.. Histamine regulates striatal dopamine differently in males and females.

A. Schematic and representative image for placement of the microdialysis guide cannula in the striatum. Guide cannula placement was counterbalanced between left and right striatum. B. Schematic and representative image for guide cannula placement for ICV infusion. ICV guide cannulae were placed contralaterally to microdialysis guide cannulae. C. ICV infusion of HA significantly decreases striatal DA, compared to saline control (sal), in male mice (RM-ANOVA: main effect of treatment, F[1,13]=5.668, *p=0.033). D. Combining data over the 20–60 minute interval post infusion confirmed a significant decrease in striatal DA after HA relative to saline (Student’s t-test (two-tailed): T[29]=2.961, **p=0.006). E. ICV infusion of HA significantly increased striatal DA, relative to saline-infused controls, in female mice (RM-ANOVA: main effect of drug, F [1,13]=5.633, *p=0.034). F. This was confirmed when data were collapsed over 20–60 min post -infusion (Shapiro-Wilk test for Normality, Histamine W=0.734, p=0.004 [no pass], Mann-Whitney: *p=0.024). Dashed lines indicate baseline dopamine levels, determined for each animal during the 50 min before HA or saline infusion.

Figure 3.. Striatal dopamine is regulated by activation of histamine H2 receptors in the SNc of male mice.

A. ICV infusion of HA H2R agonist dimaprit is the only treatment to reduce striatal dopamine over the 20–60 minute post drug infusion interval (RM-ANOVA: Effect of Treatment, F[3,43]=4.093, p=0.012, Post-Hoc Bonferroni **Saline vs Dimparit (H2) p=0.019, Saline vs FMPH (H1), p>0.999, Saline vs RAMH (H3) p>0.999). B. When collapsed over the 20–60 minute time period, ICV infusion of the HA H2R agonist dimaprit significantly reduces striatal DA in male mice, compared to saline controls and all other agonist treatments. (LMEM: Effect of Treatment, F[3, 36]=3.625, p=0.022; Post-Hoc Bonferroni test: Sal vs Dimaprit **p=0.032). No effect of the H3R agonist RAMH or H1R agonist FMPH was seen (Bonferroni’s’s (sal vs RAMH): p>0.99, (sal vs FMPH): p>0.999) C. Locomotion in male mice following treatment with HA agonists was reduced by dimaprit relative to saline control (ANOVA: Effect of Treatment, F[3, 24]=2.467, p=0.044; Tukey’s (sal vs dimaprit): p=0.054). Representative traces for the open field are shown to the right. D. Schematic and representative image for guide cannula placement of microdialysis guide cannula in the striatum and infusion guide cannula above the substantia nigra; drugs targeting the striatum in this experiment were delivered through the same cannula using reverse microdialysis. Guide placement was counterbalanced between left and right striatum. SN infusion guides were placed ipsilateral to microdialysis cannula. E. Infusion of histamine agonists into either the striatum or SNc shows a strong trend towards effect of brain region over time, with post-hoc testing indicating that infusion of dimaprit H2 agonist into the SNc is the only treatment to significantly reduce dopamine (RM-ANOVA: Brain Region, F[2,51]=3.691 p=0.062, Post-Hoc Bonferroni: *SN Sal vs SN H2 p=0.033, SN H2 vs Str H2 p=0.002, all other treatments/regions N.S.) F. When collapsed over the 20–60 minute post infusion interval, infusion of dimaprit into the SN significantly reduces DA levels in the striatum, compared to saline controls and the H3R agonist RAMH (LMEM: Interaction Treatment x Brain Region F[6, 51]=3.508, p=0.037, effect of Brain Region F[2,51]=6.361, p=0.015, Bonferroni (SN Sal vs SN H2 p=0.005, Str Sal vs Str H2 p=0.579, SN H2 vs Str H2 p<0.001, SN Sal vs SN H3 p=0.538, Str Sal vs Str H3 p=0.397).

Figure 4.. GABAergic interneurons in the substantia nigra mediate the effects of H2 activation on striatal DA in males.

A, B. Representative image of GFP expressing GABAergic interneurons (green) and H2R (red) in the SN of a mouse that received control scramble shRNA AAV (A) or AAV-shH2 (B). Higher resolution images of selected regions demonstrate reduced expression of H2R on GFP-expressing GABAergic interneurons. Scalebars: 20μm. C. Quantification of colocalization of red fluorescent H2R puncta with GFP-expressing GABAergic interneurons shows a significant decrease in the quantity of colocalized H2R in mice that received the shH2 AAV compared to controls (Student’s t-test (two-tailed), T[44]=4.349, ****p<0.001). D. H2R knockdown in GABAergic interneurons in the SN blocks the decrease of striatal dopamine by histamine ICV infusion over the 20–60 minute time interval post injection (RM-ANOVA: Interaction Virus x Treatment, F[4,23]=4.518, p=0.045, Post-Hoc Bonferroni: Gad-Cre GFP CTRL (sal vs HA) p=0.035, Gad-Cre shH2 (sal vs HA) p=0.575). E. H2R knockdown in GABAergic interneurons in the SN blocks the reduction of striatal DA after ICV HA infusion [2-Way ANOVA: interaction Virus x Treatment: F[1, 23]=7.337 p=0.014; Post-Hoc Bonferroni: Gad-Cre GFP CTRL (sal vs HA), p=0.014, Gad-Cre shH2 (saline vs HA), p=0. 333, HA (GFP vs shH2) p=0.014]. F. H2R knockdown in GABAergic interneurons in the SN blocks the effects of ICV dimaprit infusion on striatal DA levels (2 Way ANOVA: interaction Virus x Treatment: F[1, 11] = 4.068, p=0.022; Tukey’s: Gad-Cre GFP CTRL (sal vs dimaprit): *p=0.044, shH2 (sal vs dimaprit): p=0.189). Dashed lines indicate baseline striatal DA levels, prior to drug vs saline infusion.

Figure 5.. H3R activation in the striatum increases striatal DA in females, but only during the estrus phase of the estrous cycle.

A. ICV infusion of the H3R agonist RAMH trended towards increased striatal DA in female mice, compared to saline controls and all other treatments when analyzed over the 20–60 minute time interval post-infusion (RM-ANOVA: F[3,44]=2.968, p=0.042; Post-hoc Bonferroni: Saline vs H3, p=0.077). In contrast to males, infusion of H2 agonist dimaprit had no effect (Bonferroni, p>0.999). B. Collapsing the data for the 20–60 interval post-infusion to account for individual variance over time shows a significant increase in striatal DA with the infusion of RAMH compared to all other treatments (LMEM: Effect of Treatment, F[3, 44] = 3.417, p=0.025; Post-Hoc Bonferroni (sal vs RAMH), p=0.05). In contrast to what we see in males, there is no effect of dimaprit infusion in females (Bonferroni: p>0.999). C. In females, there was a significant increase in locomotion following RAMH infusion compared to saline control (1-way ANOVA: F (1.903, 13.32) = 6.896, p=0.009; Tukey’s (sal vs RAMH): p=0.042, (RAMH vs dimaprit): p=0.027). In contrast to what we see in males, there is no effect of ICV dimaprit infusion [Tukey’s (sal vs H2): p=0.921]. Representative path tracings are shown on the right. D. Infusion of RAMH into the striatum appears to increase levels of striatal dopamine over the 20–60 minute interval following infusion, although this did not reach significance (2-Way RM-ANOVA: Treatment x Brain Region F[4,25]=0.740, p=0.487). It appears that this effect is driven by outlier points and causes a bimodal distribution. E. When collapsed over the 20–60 minute interval post-infusion, RAMH in the striatum appears to increase levels of striatal dopamine in females, with outlier points still apparent, but fails to reach statistical significance (LMEM: F[6, 25]= 0.792, p=0.464). F. When mice are separated into the proestrus/estrus or metestrus/diestrus phases of the estrous cycle, infusion of RAMH into the striatum increases striatal DA only during the proestrus/estrus when measured over the 20–60 minute post infusion interval (3-Way RM-ANOVA: Treatment x Brain Region x Estrus, F[2,87]=2.236, p=0.113, Treatment x Estrus, F[2,87]=2.823, p=0.024, Treatment x Brain Region, F[2,87]=3.436, p=0.037, Brain Region x Estrus, F[1,87]=5.045, p=0.027, Brain Region, F[1,87]=6.507, p=0.012; Fisher’s LSD: P/E Str H3 vs P/E Str Sal, p<0.001, P/E SN H3 vs P/E SN Sal, p=0.698, M/D Str H3 vs M/D Str Sal, p=0.552, M/D SN H3 vs M/D SN Sal, p=0.552). G. When collapsed over the 20–60 minute post infusion interval, RAMH in the striatum during pro/estrus remains the only treatment and timing to significantly increase striatal dopamine (LMEM: 3-way interaction, F[12, 48]=2.516, p=0.086; Treatment x Estrus Phase, F[4,48]=3.918, p=0.023, Brain Region x Estrus Phase, F[4,48]=4.094, p=0.046, Treatment x Brain Region, F[4,48]=0.681, p=0.509; Post-Hoc Fisher’s LSD: P/E Str Sal vs P/E Str H3, p=0.079, P/E Str H3 vs P/E SN H3, p=0.003, P/E Str Sal vs M/D Strl Sal p>0.999, P/E SN Sal vs P/E SN H3, p>0.999). H. Infusion of dimaprit into the striatum during different estrous phases did not alter striatal dopamine over time (3-Way RM-ANOVA: Fisher’s LSD: P/E Str H2 vs P/E Str Sal, p=0.822, M/D Str H2 vs M/D Str Sal, p=0.240, P/E SN H2 vs P/E SN Sal, p=0.315, M/D SN H2 vs M/D SN Sal, p=0.914). I. Collapsed data for infusion of dimaprit into the striatum during different phases of the estrous cycle did not significantly alter levels of striatal DA (LMEM: 3-way interaction N.S.; treatment x brain region: F (1, 61) = 4.377, p=0.041).