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. 2023 Dec 22:14:1269672.
doi: 10.3389/fendo.2023.1269672. eCollection 2023.

The transcription factor VAX1 in VIP neurons of the suprachiasmatic nucleus impacts circadian rhythm generation, depressive-like behavior, and the reproductive axis in a sex-specific manner in mice

Brooke M Van Loh #  1 , Alexandra M Yaw #  1 , Joseph A Breuer  2 , Brooke Jackson  3 , Duong Nguyen  1 , Krystal Jang  1 , Fabiola Ramos  1 , Emily V Ho  2 , Laura J Cui  2 , Dominique L M Gillette  2 , Lorenzo F Sempere  3 , Michael R Gorman  4   5 , Karen J Tonsfeldt  2   5 , Pamela L Mellon  2   5 , Hanne M Hoffmann  1   2
Affiliations

The transcription factor VAX1 in VIP neurons of the suprachiasmatic nucleus impacts circadian rhythm generation, depressive-like behavior, and the reproductive axis in a sex-specific manner in mice

Brooke M Van Loh et al. Front Endocrinol (Lausanne). .

Abstract

Background: The suprachiasmatic nucleus (SCN) within the hypothalamus is a key brain structure required to relay light information to the body and synchronize cell and tissue level rhythms and hormone release. Specific subpopulations of SCN neurons, defined by their peptide expression, regulate defined SCN output. Here we focus on the vasoactive intestinal peptide (VIP) expressing neurons of the SCN. SCN VIP neurons are known to regulate circadian rhythms and reproductive function.

Methods: To specifically study SCN VIP neurons, we generated a novel knock out mouse line by conditionally deleting the SCN enriched transcription factor, Ventral Anterior Homeobox 1 (Vax1), in VIP neurons (Vax1Vip; Vax1fl/fl:VipCre).

Results: We found that Vax1Vip females presented with lengthened estrous cycles, reduced circulating estrogen, and increased depressive-like behavior. Further, Vax1Vip males and females presented with a shortened circadian period in locomotor activity and ex vivo SCN circadian period. On a molecular level, the shortening of the SCN period was driven, at least partially, by a direct regulatory role of VAX1 on the circadian clock genes Bmal1 and Per2. Interestingly, Vax1Vip females presented with increased expression of arginine vasopressin (Avp) in the paraventricular nucleus, which resulted in increased circulating corticosterone. SCN VIP and AVP neurons regulate the reproductive gonadotropin-releasing hormone (GnRH) and kisspeptin neurons. To determine how the reproductive neuroendocrine network was impacted in Vax1Vip mice, we assessed GnRH sensitivity to a kisspeptin challenge in vivo. We found that GnRH neurons in Vax1Vip females, but not males, had an increased sensitivity to kisspeptin, leading to increased luteinizing hormone release. Interestingly, Vax1Vip males showed a small, but significant increase in total sperm and a modest delay in pubertal onset. Both male and female Vax1Vip mice were fertile and generated litters comparable in size and frequency to controls.

Conclusion: Together, these data identify VAX1 in SCN VIP neurons as a neurological overlap between circadian timekeeping, female reproduction, and depressive-like symptoms in mice, and provide novel insight into the role of SCN VIP neurons.

Keywords: arginine vasopressin; circadian rhythm; cortisol; premenstrual disorders; reproduction; suprachiasmatic nucleus; vasoactive intestinal peptide; ventral anterior homeobox 1.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Vax1 is expressed in SCN Vip neurons from the early postnatal period through adulthood in both males and females. RNAscope® ISH representative images (A–F). Images at P2 are displayed with increased contrast, applied to all channels, to compare with P10 and P60. Percent co-expression between Vax1 and Vip, Avp, and Nms-expressing neurons (G, I, K) and cell counts (H, J, L) in P2, P10 and P60 males and females at ZT5. Scale bar is 300 µm. Student’s t-test *, p <0.05.
Figure 2
Figure 2
VIP peptide is sexually dimorphic in the SCN. (A) Example image and (B) quantification of SCN IHC for VIP in adult males and females at ZT3. Scale bar 100µm. Student’s t-test, **, p < 0.01 (C) RT-PCR shows VipCre recombination of the Vax1flox allele in indicated tissues. Abbreviations stand for olfactory bulb (olf), magnocellular preoptic area (MCPO), caudoputamen (CP), supraoptic nucleus (SON), piraform area (Piri), paraventricular nucleus (PVN), cingulate gyrus (CG), and ventral palidum (VP). Blue, underlined text indicates tissues that are known to express Vax1. (D) Example images and quantification (E) of dual IHC for VipCre:Td (red, cytoplasm) and VAX1 (green, nuclear) expressing cells in the adult SCN (DAPI, nuclei). Scale 50 µm.
Figure 3
Figure 3
Vax1 deletion within VIP neurons shortens behavioral free-running period in males and females. (A, B) Male and (C, D) female Ctrl and Vax1Vip mice were single housed with running wheels. (A, C) Data show double plotted actogram activity with 14 days in LD12:12 (LD) followed by 28 days in constant darkness (DD). Data are presented in ClockLab normalized format. Horizontal bar above the actograms indicates lights on (white) and lights off (black) during the LD12:12 cycle. (B, D) Chi2 periodograms during 2 weeks in DD. Matching codes (a1, a2, etc.) on the upper right corner of each actogram and chi2 periodogram indicate data from a particular mouse, with variable scaling indicated in the upper left. 14-day average wheel-running data were used for indicated analysis parameters in LD and DD. Average histogram data for (E) Wheel-running period, (F) Chi2 amplitude, (G) activity amplitude, (H) wheel revolutions, and (I) activity duration (Alpha). Number within the bar indicates number of animals in each group. 3-way ANOVA, ***, p < 0.001; ****, p < 0.0001.
Figure 4
Figure 4
Vax1Vip SCN has a shortened PER2::LUC period in ex vivo culture. Histogram of PER2::LUC SCN (A, B) circadian period and (C, D) amplitude from control and Vax1Vip:PER2::LUC females (combined proestrus and diestrus) and males. Statistical analysis by Student’s t-test, *, p<0.05 **, p < 0.01, n = 7-16. (E) PER2::LUC phase (time of first peak) in the SCN of control and Vax1Vip:PER2::LUC males and (F) females, n = 7-16. Mean times of first peak are indicated by vector lines, and symbols indicate individual data points. Data were analyzed via the Rayleigh Test of Uniformity, where crossing the dotted gray line indicates significant clustering (p < 0.05), and the Watson’s Two-Sample Test of Homogeneity. No significant differences were found in females between estrous stages.
Figure 5
Figure 5
VAX1 binds directly to regulatory regions of molecular clock genes Per2 and Bmal1. (A, C) EMSA assay of COS-1 cells, represents in Lane 1: pCMV-Flag (CMV, empty vector), Lane 2: Vax1-Flag plasmid and Lane 3: Vax1-Flag plasmid + anti Flag antibody. White star indicates super shift. Example gels of n = 3. (B) Transient transfections of NIH3T3 cells with the mouse Bmal1 regulatory region driving luciferase (Bmal1-luciferase) with and without Vax1 overexpression vector (20 ng) or its empty vector (EV, pCMV6, 20 ng). Numbers indicated with the stars on the regulatory regions refer to ATTA sites that have been mutated (see Table 1 ). Statistical analysis by Two-way ANOVA mixed effect model, *, p < 0.05; **, p < 0.01; ***, p < 0.001, n = 4-6 in duplicate or triplicate.
Figure 6
Figure 6
Loss of Vax1 in VIP neurons does not reduce Vip and Bmal1 expression in the SCN. RNAscope® assay at ZT16 in the SCN of proestrus Ctrl and Vax1Vip females. (A) Example images of RNAscope® assay for Vip (blue), Avp (red) and Bmal1 (green). N = 3-4 per group. Scale bar 300 µm. (B) Percentage of cells that co-express Bmal1 with Avp or Vip. Mann-Whitney, p > 0.05. (C) Average cytoplasm dye concentration reflecting mRNA transcripts for Avp, Vip, and Bmal1 Mann-Whitney, p > 0.05. (D) Number of cells expressing indicated combinations of Avp, Vip, and Bmal1. Mann-Whitney, p > 0.05.
Figure 7
Figure 7
Vax1Vip females have a reduction in ovary weight, estrogen, and FSH, as well as an increased sensitivity to kisspeptin. (A) Age at preputial separation (PPS) and (B) million sperm per epididymis in Ctrl and Vax1Vip males, n indicated in graphs, Student’s t-test, *, p<0.05; **, p <0.01. (C) Estrous cycles were evaluated in females, and average estrous cycle length was established. Student’s t-test, *, p < 0.05. (D) Ovary weight, (E) circulating estrogen and (F) circulating FSH of diestrus females. n indicated in graphs, Student’s t-test, *, p < 0.05. (G) Circulating LH levels in diestrus females evaluated over a 45-minute time period in response to an i.p. kisspeptin injection. Mixed Effects analysis, *, p < 0.05. and (H) the resulting area under the curve. Student’s t-test, *, p < 0.05.
Figure 8
Figure 8
Vax1Vip females have increased Avp in the PVN, increased corticosterone, and increased depressive-like behaviors. (A) Example images of RNAscope® detection for Avp in the SCN and paraventricular nucleus (PVN) of proestrus females at ZT16. Scale bar is 600 µm, n=3-5. Quantification of RNAscope® assay by (B) cell counts and (C) dye concentration of Avp in the PVN, n=3-5, Student’s t-test, **, p <0.01. (D) Corticosterone was measured at ZT3-5 in males (M) and females (F), Two-way ANOVA, *, p<0.05. (E) To test depressive-like behavior, metestrus female or male mice were tested at ZT13 by a Porsolt forced swim test, and the % time floating assessed. N indicated in graph, Student’s t-test, *, p<0.05. (F) Corticosterone levels in circulating blood 1 h following the Porsult forced swim test. n = 4-9, Student’s t-test, **, p<0.01.
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