Medial Hypothalamic 5-Hydroxytryptamine (5-HT)_1A Receptors Regulate Neuroendocrine Responses to Stress and Exploratory Locomotor Activity: Application of Recombinant Adenovirus Containing 5-HT_1A Sequences

5-HT_1A promoter controls the expression of 5-HT_1A receptors. Recombinant adenoviruses containing 5-HT_1A sense sequence controlled under CMV promoter (Ad-CMV-1Asense) or 5-HT_1A promoter (Ad-1AP-sense) were injected into the striatum (A) or cerebellum (B) as described in Materials and Methods. Arrows indicate the expression of adenovirus (GFP) or 5-HT_1A receptors and their function (see Materials and Methods and Results). 4V, Fourth ventricle.

¹²⁵I-MPPI-labeled 5-HT_1A receptors in SERT knock-out mice injected with Ad-1AP-sense in the hypothalamus. A, Autoradiography of ¹²⁵I-MPPI binding in the hypothalamus of mice that were injected with a recombinant adenovirus-containing control (left column) or 5-HT_1A sense (right column). B, ¹²⁵I-MPPI-labeled 5-HT_1A receptors in the hypothalamus of mice injected with a recombinant adenovirus containing a control vector or 5-HT_1A sense. AH, Anterior hypothalamus; DMN, dorsomedial hypothalamus; LH, lateral hypothalamus; PVN, paraventricular hypothalamus; VMN, ventromedial hypothalamus. Data are presented as mean ± SEM for 9-11 mice. Two-way ANOVA for AH: main effect of adenoviral treatment, F_(1,37) = 26.3, p < 0.0001; main effect of genotype, F_(1,37) = 76.8, p < 0.0001; interaction of adenoviral treatment and genotype, F_(1,37) = 1.2, p = 0.27. Two-way ANOVA for PVN: main effect of adenoviral treatment, F_(1,37) = 18.9, p = 0.0001; main effect of genotype, F_(1,37) = 24.1, p < 0.0001; interaction of adenoviral treatment and genotype, F_(1,37) = 0.63, p = 0.43. Two-way ANOVA for LH: main effect of adenoviral treatment, F_(1,37) = 8.5, p < 0.006; main effect of genotype, F_(1,37) = 40.6, p < 0.0001; interaction of adenoviral treatment and genotype, F_(1,37) = 0.18, p = 0.67. Two-way ANOVA for DMN: main effect of adenoviral treatment, F_(1,37) = 26.4, p < 0.0001; main effect of genotype, F_(1,37) = 15.4, p = 0.0004; interaction of adenoviral treatment and genotype, F_(1,37) = 0.24, p = 0.63. Two-way ANOVA for VMN: main effect of adenoviral treatment, F_(1,37) = 10.2, p = 0.0028; main effect of genotype, F_(1,37) = 14.6, p= 0.0005; interaction of adenoviral treatment and genotype, F_(1,37) = 0.003, p = 0.96. ^*Significant difference from Ad-track-injected SERT^+/+ mice (p < 0.05). ^#Significant difference from Ad-track-injected SERT^-/- mice (p < 0.05) by Student-Newman-Keuls post hoc test.

ACTH response to saline injection (a minor stress) in the Ad-1AP-sense- and Ad-track-treated SERT^+/+ and SERT^-/- mice. The mice were injected with saline (5 ml/kg, s.c.) 15 min before decapitation. Data are presented as the mean ± SEM for 10-11 mice. Two-way ANOVA: main effect of adenoviral treatment, F_(1,37) = 9.7, p = 0.0036; main effect of genotype, F_(1,37) = 24.4, p < 0.0001; interaction of adenoviral treatment and genotype, F_(1,37) = 1.2, p = 0.29. ^*Significant difference from Ad-track-injected SERT^+/+ mice (p < 0.05). ^#Significant difference from Ad-track-injected SERT^-/- mice (p < 0.05) by Student-Newman-Keuls post hoc test.

Elevated plus-maze test on Ad-1AP-sense-injected SERT knock-out mice. Top, The parameters indicate locomotor activity. Bottom, The parameters indicate exploratory anxiety-like behavior. Data are presented as mean ± SEM for 9-12 mice. Two-way ANOVA for total entries: main effect of adenoviral treatment, F_(1,39) = 3.7, p = 0.06; main effect of genotype, F_(1,39) = 17.1, p = 0.0002; interaction of adenoviral treatment and genotype, F_(1,39) = 5.3, p = 0.03. Two-way ANOVA for close entries: main effect of adenoviral treatment, F_(1,39) = 5.5, p = 0.02; main effect of genotype, F_(1,39) = 4.6, p = 0.04; interaction of adenoviral treatment and genotype, F_(1,39) = 1.3, p = 0.25. Two-way ANOVA for open entries: main effect of adenoviral treatment, F_(1,39) = 0.04, p = 0.85; main effect of genotype, F_(1,39) = 8.9, p = 0.005; interaction of adenoviral treatment and genotype, F_(1,39) = 2.8, p = 0.1. Two-way ANOVA for percentage open entries: main effect of adenoviral treatment, F_(1,39) = 0.31, p = 0.58; main effect of genotype, F_(1,39) = 5.0, p = 0.03; interaction of adenoviral treatment and genotype, F_(1,39) = 2.6, p = 0.1. Two-way ANOVA for percentage open time: main effect of adenoviral treatment, F_(1,39) = 2.0, p = 0.16; main effect of genotype, F_(1,39) = 9.5, p = 0.004; interaction of adenoviral treatment and genotype, F_(1,39) = 1.3, p = 0.25. ^*Significant difference from Ad-track-injected SERT^+/+ mice. ^#Significant difference from Ad-track-injected SERT^-/- mice (p < 0.05) by Student-Newman-Keuls post hoc test.

¹²⁵I-MPPI binding for 5-HT_1A receptors in the hypothalamic Ad-1AP-antisense-injected SERT normal mice. A, Photography of 5-HT_1A binding, GFP expression, and Nissl staining of hypothalamic sections that received Ad-track and Ad-1AP-antisense adenovirus. GFP and Nissl staining sections were adjacent sections with ¹²⁵I-MPPI binding of Ad-1AP-antisense. The black box indicates the area presented in the photograph of GFP expression. The white box represents the area in the Nissl staining. 3V, Third ventricle; AH, anterior hypothalamus; DMN, dorsomedial hypothalamus; LH, lateral hypothalamus; PVN, paraventricular hypothalamus; VMN, ventromedial hypothalamus. B, ¹²⁵I-MPPI binding in the hypothalamus of Ad-1AP-antisense- and Ad-track-injected mice. Data are presented as mean ± SEM for 14-22 mice. One-way ANOVA for AH: F_(1,34) = 4.8, p = 0.03; for PVN: F_(1,34) = 8.0, p = 0.008; for DMN: F_(1,34) = 9.5, p = 0.004; for VMN: F_(1,34) = 6.0, p = 0.02; for LH: F_(1,34) = 0.13, p = 0.58. ^*Significant difference from Ad-track-injected mice (p < 0.05) by Student-Newman-Keuls post hoc test.

Hormonal responses to 8-OH-DPAT in Ad-1AP-antisense- and Ad-track-injected mice. Data are presented as mean ± SEM for 8-10 mice. Top, Oxytocin response to 8-OH-DPAT. Two-way ANOVA: main effect of adenoviral treatment, F_(1,32) = 11.8, p = 0.017; main effect of 8-OH-DPAT injection, F_(1,32) = 56.7, p < 0.0001; interaction of adenoviral and 8-OH-DPAT treatment, F_(1,32) = 1.5, p = 0.23. Bottom, ACTH response to 8-OH-DPAT. Two-way ANOVA: main effect of adenoviral treatment, F_(1,35) = 6.07, p = 0.019; main effect of 8-OH-DPAT injection, F_(1,35) = 117.9, p < 0.0001; interaction of adenoviral and 8-OH-DPAT treatment, F_(1,35) = 4.94, p = 0.03. ^*Significant difference from saline-treated group with same treatment of adenovirus (p < 0.05). ^#Significant difference from Ad-track-treated mice with same challenge treatment (p < 0.05) by Student-Newman-Keuls post hoc test.

Elevated plus-maze test on the Ad-1AP-antisense-injected mice. Top, The parameters indicate locomotor activity. Bottom, The parameters indicate exploratory anxiety-like behavior. Data are presented as mean ± SEM for 15-20 mice. One-way ANOVA for total entries: F_(1,33) = 13.4, p = 0.0009; for close entries: F_(1,33) = 15.5, p = 0.0004; for rearing: F_(1,33) = 20.4, p < 0.0001; for percentage open entries: F_(1,33) = 1.28, p = 0.27; for percentage open time: F_(1,33) = 0.68, p = 0.41. ^*Significant difference from Ad-track-injected mice (p < 0.05) by Student-Newman-Keuls post hoc test.