Introduction: Postweaning social isolation (PWSI) in rodents is an advanced psychosocial stress model in early life. Some psychosocial stress, such as restrain and isolation, disrupts reproductive physiology in young and adult periods. Mechanisms of early-life stress effects on central regulation of reproduction need to be elucidated. We have investigated the effects of PWSI on function of arcuate kisspeptin (ARCKISS1) neurons by using electrophysiological techniques combining with monitoring of puberty onset and estrous cycle in male and female Kiss1-Cre mice. Methods: Female mice were monitored for puberty onset with vaginal opening examination during social isolation. After isolation, the estrous cycle of female mice was monitored with vaginal cytology. Anxiety-like behavior of mice was determined by an elevated plus maze test. Effects of PWSI on electrophysiology of ARCKISS1 neurons were investigated by the patch clamp method after intracranial injection of AAV-GFP virus into arcuate nucleus of Kiss1-Cre mice after the isolation period. Results: We found that both male and female isolated mice showed anxiety-like behavior. PWSI caused delay in vaginal opening and extension in estrous cycle length. Spontaneous-firing rates of ARCKISS1 neurons were significantly lower in the isolated male and female mice. The peak amplitude of inhibitory postsynaptic currents to ARCKISS1 neurons was higher in the isolated mice, while frequency of excitatory postsynaptic currents was higher in group-housed mice. Conclusion: These findings demonstrate that PWSI alters pre- and postpubertal reproductive physiology through metabolic and electrophysiological pathways.

One of the most common environmental stimuli in modern society is stress. It is described as a behavioral and physiological response derived when an organism is faced with a threat (stressor) to its homeostasis [1, 2]. The stress response may be either through the sympathetic-adrenal-medullary axis in which the “flight or fight” is triggered by norepinephrine or epinephrin release [3], resulting in change in the heart rate, blood pressure, and breathing rate [4]. It may also be through the hypothalamic-pituitary-adrenal (HPA) axis which is responsible for the neuroendocrine adaptation of the physical or psychological stress by combining the central nervous system and the endocrine system [5, 6]. Stress response by the HPA axis is characterized by hypothalamic release of corticotrophin-releasing hormone (CRH) and hence release of adrenocorticotropic hormone from the anterior pituitary causing secretion of glucocorticoids (GC) from the adrenal cortex [7, 8]. While the sympathetic-adrenal-medullary activation is considered to regulate the acute stress response (short term), the HPA axis regulates both acute and chronic (long-term and psychological) response [9]. The HPA axis is essential for a successful adaptive response to any stressor [2]. Psychological and chronic stress are known to reduce hypothalamic-pituitary-gonadal (HPG) axis activity in males and females, resulting in suppression of luteinizing hormone (LH) secretion and ovulation, impairment in testosterone synthesis, and spermatogenesis [10‒13].

Kisspeptin neurons, expressing the Kiss1 gene, which are mainly located in the hypothalamic arcuate nucleus (ARC) and the anteroventral periventricular nucleus (AVPV) have the initiator role in the regulation of the HPG axis [14]. Both ARC and AVPVKISS1 neuron populations regulate pulsatile LH secretion and preovulatory LH surge, respectively. Kisspeptin affects gonadotropin-releasing hormone (GnRH) neurons by binding to Kiss1 receptor (Kiss1R) on the soma in the preoptic area and on axon terminals of the same GnRH neurons in the median eminence [15‒18]. Arcuate kisspeptin (ARCKISS1) neurons co-localize neurokinin-B and dynorphin neuropeptides [19]. Thus, ARCKISS1 neurons are named as KNDy neurons which also express receptors for neurokinin B (NK3R) and dynorphin (KOR) [20, 21]. The pulsatile LH secretion is accomplished by driving the episodic release of GnRH into hypophyseal portal vessels with synchronous firing of the KNDy neurons. This phenomenon is called as KNDy hypothesis [22‒25]. Briefly, it proposes that (1) neurokinin-B acts on KNDy neurons to initiate pulse and (2) kisspeptin is the output signal to GnRH neurons to release LH, and dynorphin acts on KNDy neurons to terminate the pulse [25]. ARCKISS1 neurons serve as GnRH pulse generator according to this hypothesis which has been tested in several species using various techniques. In the recent years, advanced neuroscience technologies such as in vivo calcium imaging in freely moving animals have been utilized to test this hypothesis in conscious animals [26, 27].

Individuals improve their cognitive, emotional, and social skills during transition from childhood to adulthood, as well as having sexual maturity [28, 29]. Puberty is defined as the activation of the HPG axis and attainment of reproductive competency, followed by appearance of secondary sex characteristics and onset of mating behaviors [30, 31]. Energy homeostasis also develops prior to puberty onset, and in that, hypothalamic neuropeptides and peripheral hormones play an important role [32, 33]. Puberty and adolescence both are used to describe transition from childhood to adulthood, but they are not equivalent as adolescence includes puberty. During this developmental period, individuals are more sensitive to the environmental stimuli [34, 35]. Especially, stressful stimuli cause behavioral physiological alterations in adulthood [36, 37]. Perturbations of the developing adolescent brain may result in psychological disorders such as anxiety, depression, and drug addiction [38] or behavioral disorders such as dysfunctions in reproductive behaviors [39].

Double-labeling immunohistochemistry studies have shown that ARCKISS1 and AVPVKISS1 neurons express CRH receptor (CRH-R), and GC receptor (GC-R) is expressed in most of AVPVKISS1 neurons and in a few ARCKISS1 neurons in female rat hypothalamus [40]. Administration of CRH or corticosterone (CORT) decreases Kiss1 mRNA expression in both ARCKISS1 and AVPVKISS1 neurons in female rats [41]. The effects of different stressors on reproductive behavior and physiology have been investigated in different species in various behavioral studies [42‒44]. Suppressive effects of chronic or acute stress have been studied to understand roles of GnRH and gonadotropin-inhibiting hormone (RFRP-3) neurons in the regulation of reduced LH secretion [13, 45‒47]. However, effects of social isolation, especially early-life social isolation, on the kisspeptin neurons are still unclear. Therefore, we examined the effects of early-life social isolation on reproductive physiology in female mice and ARCKISS1 neurons’ electrophysiology in male and female Kiss-Cre mice in the current study. We established postweaning social isolation (PWSI) which is the most common model as a social stressor in the adolescence period [29]. We first evaluated puberty onset and estrous cycling in female mice and anxiety-like behavior induced by PWSI and then observed spontaneous-firing frequency of ARCKISS1 neurons and excitatory and inhibitory postsynaptic currents to ARCKISS1 neurons in both adult male and female mice.

Animals and Experimental Design

Kiss1-Cre mice were reported by Palmiter and colleagues [48]. Heterozygote Kiss1-Cre male mice were purchased (Jax Lab 033169) and mated with wild-type female mice. Mice were housed and maintained on 12-h light-dark cycle with food and water ad libitum during pregnancy and weaning. Pups were genotyped for the Cre gene between postnatal day (P) 10 and P15. Care and experimental manipulations of animals were followed in Yeditepe University Faculty of the Medicine Experimental Research Center (YUDETAM), Istanbul, Turkey, and approved by the Yeditepe University Experimental Animal Research Ethics Committee. Thirty-four male and female Kiss1-Cre+ mice were used for anxiety and reproductive physiology monitoring experiments and 24 male and female Kiss1-Cre+ mice for electrophysiology experiments following intracranial injections after the isolation period.

Postweaning Social Isolation

Mice were housed in isolation as 1 animal per cage or in groups of 3-4 per cage. Animals were isolated postweaning for 3 weeks between P21 and P43 [29, 49]. Isolated mice were housed in small (10*10 cm) and opaque cages with limited light access, to visually block the external environment and neighboring mice [50]. The grouped mice were housed in standard (30*15 cm) cages. Mice were housed at 21°C on 12:12 h light:dark cycle with ad libitum access to food and water. The weight of mice was recorded at the beginning and end of the isolation period. In order to avoid extra stress stimulation, weight was not chronically monitored.

Vaginal Opening and Estrous Cycle Monitoring

The onset of vaginal opening was determined by inspecting the visual appearance of opening starting at P22 and ending at P36. The vaginal status was checked daily, every morning (09:00 a.m.) during the isolation period, to minimize stress and uncomforting of mice. The age and weight at which appearance of vaginal opening was first detected were recorded [51‒53].

The estrous cycle was monitored by the vaginal smear method for at least two consecutive cycles. Vaginal cytology screening started when 3 weeks of isolation was completed, and isolated mice were housed in the same conditions during the estrous cycle monitoring. Vaginal smears were placed on a microscope slide to dry and stained with trypan blue (0.5% v/v). The method has previously been described [53‒56].

Elevated plus Maze

Elevated plus maze (EPM) testing was performed as described previously [57] at P43 for male. Diestrus female mice were tested following cycle examination. Briefly, mice were placed in the behavioral testing room 1 h before testing for habitation. The EPM platform has two open arms (30*5 cm) and two closed arms (30*5*14 cm). Mice were placed in the center of the EPM, and their behavior was tracked for 5 min using a video recorder system. Time spent in each arm was analyzed with software (EthoVision XT11, Noldus Technology, Netherlands). A heat map was created using the same software. EPM experiments were performed between 4:00 and 6:00 p.m. [53]. Female mice were evaluated on the day of diestrus.

Increasing time in closed arms is defined as anxiety-like behavior, while more spent time in opened arms is indicative for lower anxiety-like behavior. The number of entries to closed arms and total distance of mice in the arena was also represented.

Stereotaxic Surgeries

Intracranial injection was performed as described previously [39]. AAV-CAG-Flex-GFP was purchased from Vector Core (University of North Carolina, USA). Kiss1-Cre+ mice were mounted in a stereotaxic instrument (Kopf Instruments, USA) under anesthetization with 1–1.5% isoflurane. Two small holes were opened using a drill. Using a hydraulic microinjection system (Narishige, Japan) integrated with a capillary glass pipet, 100 nL of AAV was injected bilaterally into ARC coordinates which are 1.48 mm posterior to Bregma, ± 0.27 mm to midline, and 5.75 mm ventral to dura. To avoid surgery stress during puberty transition, AAV-GFP virus was injected after completion of the isolation period at P44. After surgery, mice were returned to the same isolation conditions. Mice were hosted in the same isolation conditions for 10 days for expressing GFP in kisspeptin neurons.

Electrophysiology

Recording slices and electrophysiology solutions were prepared as described previously [58, 59]. For ex vivo recordings, P55-60 male and diestrus female mice after estrous cycle monitoring were deeply anesthetized with isoflurane and perfused transcardially with N-methyl-d-glucamine-HEPES containing artificial cerebrospinal fluid (aCSF) solution. Coronal brain slices (250 µm) containing ARC were sectioned using a vibratome (Leica VT1000S, UK). Slices were prepared in chilled cutting solution containing (in mM) 92 N-methyl-d-glucamine, 2.5 KCl, 1.2 NaH2PO4, 30 NaHCO3, 20 HEPES, 25 d-glucose, 5 sodium ascorbate, 2 thiourea, 3 sodium pyruvate, 10 MgSO4, and 0.5 CaCl2, aerated with 95% O2/5% CO2. Slices were incubated in the same solution at 32°C for recovery for 15 min. Slices were transferred to aCSF containing (in nM) 124 NaCl, 2.5 KCl, 1.2 NaH2PO4, 24 NaHCO3, 5 HEPES, 12.5 d-glucose, 2 MgSO4, and 2 CaCl2, ph 7.4, aerated with 95% O2/5% CO2. Slices were incubated for 30 min at room temperature and recorded under the same conditions. The slice placed on the recording bath was perfused with aCSF at room temperature. The recordings were obtained using borosilicate glass capillary pipettes (Sutter Instrument, USA) pulled on a vertical pipet puller (Narishige, Japan). GFP-expressing neurons in ARC were visualized with LED illumination (CoolLED, UK) integrated with an electrophysiology rig (Scientifica, UK). Electrophysiological signals were recorded using the MultiClamp 700A (Molecular Devices, USA) and Clampex 10.4 software (Molecular Devices, USA). Spontaneous action potential firing was recorded in voltage-clamp mode in the loose seal (8–12 MΩ seal resistance) with holding current maintained at zero in the absence of any blocker. Whole-cell voltage-clamp recordings were performed for spontaneous excitatory postsynaptic currents (sEPSCs) and spontaneous inhibitory postsynaptic currents (sIPSCs) by adding GABAA (PTX 10 µm) and glutamate receptor blockers (CNQX 10 µm + AP5 50 µm), respectively, into perfusing aCSF. Pipette solution contained (in mM) 125 CsCl, 5 NaCl, 10 HEPES, 0.6 EGTA, 4 Mg-ATP, 0.3 Na2GTP, 10 lidocaine N-ethyl bromide, pH 7.35, and 290 mOSM. The holding potential was set to −60 mV. Recorded data were analyzed using Clampfit 11.0.3 software (Molecular Devices, USA).

Statistical Analysis

The statistical analysis was conducted by using GraphPad Prism 8.0 (GraphPad Software, CA, USA). All results were presented as mean ± SEM. Normally distributed data were analyzed with parametric tests (unpaired t test for group comparisons and two-way ANOVA followed by Holm-Sidak’s multiple comparisons). Non-normally distributed data were analyzed with nonparametric tests (Kolmogorov-Smirnov D test for group comparison). In all analyses, p < 0.05 indicated statistical significance.

PWSI Delays Onset of Puberty and Alter Estrous Cycling in the Female Mice

Different stressors during early life impact reproductive physiology by delaying puberty onset and causing alterations in estrous cycling in adolescence in female rodents [53]. The puberty onset has been shown to correlate with body weight [60]. It has been shown that lower body weight delays or suppresses sexual maturation [61, 62]. There was no significant difference between groups for their weight at the vaginal opening day (shown in Fig. 1a). No significant difference was observed among the body weights of groups at the beginning of isolation (shown in Fig. 1b). The body weight of PWSI mice was significantly higher than group-housed mice at the end of the isolation period (shown in Fig. 1b; p < 0.001, two-way ANOVA). We examined vaginal opening during PWSI and estrous cycle after PWSI in female mice to evaluate effects of early-life social isolation on reproductive physiology. Vaginal opening was first observed at P24 in group-housed mice and at P29 in isolated mice. The age of vaginal opening in the isolated mice was significantly higher than the group-housed mice (shown in Fig. 1c; p < 0.0001, unpaired t test). Appearance of vaginal opening in all group-housed mice was completed before first observation of vaginal opening of isolated mice (shown in Fig. 1d; p < 0.05, Kolmogorov-Smirnov test). When estrous cycling was observed after the 3-week isolation, it was seen that isolated mice exhibited significantly longer estrous cycles (shown in Fig. 1e; p < 0.005, unpaired t test) which was caused by significantly increased spent time in metestrus (shown in Fig. 1f; p < 0.05, two-way ANOVA).

Fig. 1.

PWSI delays onset of puberty and alters estrous cycling in the female mice. a Weights of mice on the day of vaginal opening. b Weights of mice at the beginning and end of the isolation period. c First vaginal opening appearance. d Cumulative percentage of mice who had vaginal opening onsets by a given age. e Average of two complete estrous cycles of mice. f Average of each estrous stage of mice in two complete cycles. Data were presented as mean ± SEM and analyzed by a two-tailed unpaired t test for a, c, and e; an unpaired Kolmogorov-Smirnov D test for d; and two-way ANOVA followed by Holm-Sidak’s multiple comparison test for b and f, n = 10 per group in a and b, n = 8 per group in c-f, *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001.

Fig. 1.

PWSI delays onset of puberty and alters estrous cycling in the female mice. a Weights of mice on the day of vaginal opening. b Weights of mice at the beginning and end of the isolation period. c First vaginal opening appearance. d Cumulative percentage of mice who had vaginal opening onsets by a given age. e Average of two complete estrous cycles of mice. f Average of each estrous stage of mice in two complete cycles. Data were presented as mean ± SEM and analyzed by a two-tailed unpaired t test for a, c, and e; an unpaired Kolmogorov-Smirnov D test for d; and two-way ANOVA followed by Holm-Sidak’s multiple comparison test for b and f, n = 10 per group in a and b, n = 8 per group in c-f, *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001.

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PWSI Increases Anxiety-Like Behavior

Baseline anxiety-like behavior was evaluated following 3 weeks of isolation or group-housing after weaning with EPM. Consistent with the previous studies [29], isolated female and male mice showed more anxiety-like behavior than group-housed mice. Isolated male mice spent significantly more time in closed arms and less time in opened arms of the plus maze (shown in Fig. 2a, b; p < 0.005 and p < 0.01 unpaired t test, respectively). The number of entrances of isolated male mice to the closed arms was significantly higher than group-housed mice (shown in Fig. 2c; p < 0.001, unpaired t test). Isolated female mice also spent significantly more time in closed arms and less time in opened arms of the plus maze (shown in Fig. 2d, e; p < 0.0001, unpaired t test, respectively). The number of entrances of isolated and group-housed female mice to the closed arms was not significantly different (shown in Fig. 2f). There was no significant difference in total distance moved between groups in both male and female (shown in Fig. 2g, h).

Fig. 2.

PWSI increases anxiety-like behavior. a, b Spent time of male mice in arms of EPM. c Number of entries of male mice to closed arms of EPM. d, e Spent time of female mice in arms of EPM. f Number of entries of female mice to closed arms of EPM. g, h Total traveled distance of male and female mice, respectively, in total arena. i Heat map created from cumulative PWSI (i) and group-housed (ii) video data. Data were presented as mean ± SEM and analyzed by a two-tailed unpaired t test for each graph, n = 7 per group, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Fig. 2.

PWSI increases anxiety-like behavior. a, b Spent time of male mice in arms of EPM. c Number of entries of male mice to closed arms of EPM. d, e Spent time of female mice in arms of EPM. f Number of entries of female mice to closed arms of EPM. g, h Total traveled distance of male and female mice, respectively, in total arena. i Heat map created from cumulative PWSI (i) and group-housed (ii) video data. Data were presented as mean ± SEM and analyzed by a two-tailed unpaired t test for each graph, n = 7 per group, **p < 0.01, ***p < 0.001, ****p < 0.0001.

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PWSI Decreases Electrophysiological Activity of ARC Kisspeptin Neurons

ARCKISS1 neurons are one of the most critical neuron populations which regulate HPG axis functions in the brain [18]. It was shown that they play important roles in puberty timing [63]. Development of the ARCKISS1 neurons and circuits is ongoing during the postnatal and postweaning period [64, 65]. Thus, we investigated the effect of another common early-life stressor, social isolation, on ARCKISS1 neuron function. To do that, we used ex vivo electrophysiology recording. We labeled ARCKISS1 neurons via injection of a cre-dependent GFP-expressing virus. Ten days after AVV injections, we prepared coronal brain slices and performed electrophysiological recordings from ARCKISS1 neurons (shown in Fig. 3a). We first evaluated spontaneous action potentials with 60–90 s loose-seal recordings (shown in Fig. 3b). ARCKISS1 neurons in isolated groups showed reduced firing frequency than group-housed mice. This significant difference is for both male and female mice (shown in Fig. 3c, d; p < 0.01 and p < 0.0001, unpaired Kolmogorov-Smirnov D and t tests, respectively). To understand the reduction of spontaneous action potential ratio in isolated groups, we examined postsynaptic currents to ARCKISS1 neurons using whole-cell patch clamp technique. PWSI significantly increased frequency and peak amplitude of sIPSCs to ARCKISS1 neurons in group-housed male mice (shown in Fig. 4a, b; p < 0.05 and p < 0.005, unpaired Kolmogorov-Smirnov D and unpaired t tests, respectively). In female mice, there was no significant difference between frequency of sIPSCs and ARCKISS1 neurons in both groups (shown in Fig. 4d), while peak amplitude of sIPSCs of isolated female mice was significantly higher than group-housed female mice (shown in Fig. 4e; p < 0.0001, unpaired Kolmogorov-Smirnov D test). Frequency of sEPSCs to ARCKISS1 neurons of group-housed male mice was significantly higher than that of the isolated group (shown in Fig. 5a; p < 0.001, unpaired Kolmogorov-Smirnov D test), but there was no significant difference for the peak amplitudes of sEPSCs between isolated and group-housed male mice groups (shown in Fig. 5b). As with male mice, sEPSCs to ARCKISS1 neurons of group-housed female mice were significantly higher than that of the isolated group (shown in Fig. 5d; p < 0.0001, unpaired Kolmogorov-Smirnov D test), while there was no significant difference for the peak amplitudes of sEPSCs between isolated and group-housed female mice groups (shown in Fig. 5e).

Fig. 3.

PWSI suppresses spontaneous-firing frequency of ARCKISS1 neurons. a Representative labeling of ARCKISS1 neurons in Kiss1-Cre mice and GFP expression in ARCKISS1 neurons containing brain slice. b Representative loose-seal recording traces for each group. c Spontaneous-firing frequency of isolated and group-housed male mice; each point represents individual neurons (PWSI, n = 42 neurons; group-housed, n = 56 neurons from 5 mice per group). d Spontaneous-firing frequency of isolated and group-housed female mice; each point represents individual neurons (PWSI, n = 69 neurons; group-housed, n = 41 neurons from 5 mice per group). Data were presented as mean ± SEM and analyzed by an unpaired Kolmogorov-Smirnov D test for c and a two-tailed unpaired t-test for d, **p < 0.01, ***p < 0.001.

Fig. 3.

PWSI suppresses spontaneous-firing frequency of ARCKISS1 neurons. a Representative labeling of ARCKISS1 neurons in Kiss1-Cre mice and GFP expression in ARCKISS1 neurons containing brain slice. b Representative loose-seal recording traces for each group. c Spontaneous-firing frequency of isolated and group-housed male mice; each point represents individual neurons (PWSI, n = 42 neurons; group-housed, n = 56 neurons from 5 mice per group). d Spontaneous-firing frequency of isolated and group-housed female mice; each point represents individual neurons (PWSI, n = 69 neurons; group-housed, n = 41 neurons from 5 mice per group). Data were presented as mean ± SEM and analyzed by an unpaired Kolmogorov-Smirnov D test for c and a two-tailed unpaired t-test for d, **p < 0.01, ***p < 0.001.

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Fig. 4.

PWSI promotes inhibitory inputs to ARCKISS1 neurons. Firing frequency (a) and peak amplitude (b) of spontaneous inhibitory postsynaptic currents (sIPSCs) to ARCKISS1 neurons of isolated and group-housed male mice; each point represents individual neurons (PWSI, n = 31 neurons; group-housed, n = 30 neurons from 3 mice per group). c Representative whole-cell voltage-clamp recording traces for sIPSCs of male mice. Firing frequency (d) and peak amplitude (e) of spontaneous inhibitory postsynaptic currents (sIPSCs) to ARCKISS1 neurons of isolated and group-housed female mice; each point represents individual neurons (PWSI, n = 30 neurons; group-housed, n = 30 neurons from 3 mice per group). f Representative whole-cell voltage-clamp recording traces for sIPSCs of female mice. Data were presented as mean ± SEM and analyzed by an unpaired Kolmogorov-Smirnov D test for a, b, and d and a two-tailed unpaired t test for e, *p < 0.05, **p < 0.01, ****p < 0.0001.

Fig. 4.

PWSI promotes inhibitory inputs to ARCKISS1 neurons. Firing frequency (a) and peak amplitude (b) of spontaneous inhibitory postsynaptic currents (sIPSCs) to ARCKISS1 neurons of isolated and group-housed male mice; each point represents individual neurons (PWSI, n = 31 neurons; group-housed, n = 30 neurons from 3 mice per group). c Representative whole-cell voltage-clamp recording traces for sIPSCs of male mice. Firing frequency (d) and peak amplitude (e) of spontaneous inhibitory postsynaptic currents (sIPSCs) to ARCKISS1 neurons of isolated and group-housed female mice; each point represents individual neurons (PWSI, n = 30 neurons; group-housed, n = 30 neurons from 3 mice per group). f Representative whole-cell voltage-clamp recording traces for sIPSCs of female mice. Data were presented as mean ± SEM and analyzed by an unpaired Kolmogorov-Smirnov D test for a, b, and d and a two-tailed unpaired t test for e, *p < 0.05, **p < 0.01, ****p < 0.0001.

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Fig. 5.

PWSI attenuates excitatory inputs to ARCKISS1 neurons. Firing frequency (a) and peak amplitude (b) of spontaneous excitatory postsynaptic currents (sEPSCs) to ARCKISS1 neurons of isolated and group-housed male mice; each point represents individual neurons (PWSI, n = 30 neurons; group-housed, n = 30 neurons from 3 mice per group). c Representative whole-cell voltage-clamp recording traces for sEPSCs of male mice. Firing frequency (d) and peak amplitude (e) of spontaneous inhibitory postsynaptic currents (sIPSCs) to ARCKISS1 neurons of isolated and group-housed female mice; each point represents individual neurons (PWSI, n = 31 neurons; group-housed, n = 32 neurons from 3 mice per group). f Representative whole-cell voltage-clamp recording traces for sEPSCs of female mice. Data were presented as mean ± SEM and analyzed by an unpaired Kolmogorov-Smirnov D test for a, b, and d and a two-tailed unpaired t test for e, ***p < 0.001, ****p < 0.0001.

Fig. 5.

PWSI attenuates excitatory inputs to ARCKISS1 neurons. Firing frequency (a) and peak amplitude (b) of spontaneous excitatory postsynaptic currents (sEPSCs) to ARCKISS1 neurons of isolated and group-housed male mice; each point represents individual neurons (PWSI, n = 30 neurons; group-housed, n = 30 neurons from 3 mice per group). c Representative whole-cell voltage-clamp recording traces for sEPSCs of male mice. Firing frequency (d) and peak amplitude (e) of spontaneous inhibitory postsynaptic currents (sIPSCs) to ARCKISS1 neurons of isolated and group-housed female mice; each point represents individual neurons (PWSI, n = 31 neurons; group-housed, n = 32 neurons from 3 mice per group). f Representative whole-cell voltage-clamp recording traces for sEPSCs of female mice. Data were presented as mean ± SEM and analyzed by an unpaired Kolmogorov-Smirnov D test for a, b, and d and a two-tailed unpaired t test for e, ***p < 0.001, ****p < 0.0001.

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Although stress is known to reduce reproductive system by decreasing GnRH and LH secretion [66], response of HPG system to early-life stress (ELS) has not been well studied, and effects of early-life social isolation in kisspeptin signaling remains unclear to understand the mechanism of suppression of the HPG axis after chronic stress conditions. Therefore, we established PWSI which has been used widely as an early-life stressor to investigate its consequences on electrophysiological activity of ARCKISS1 neurons. We monitored the puberty onset and estrous cycling to evaluate the effects of PWSI on reproductive physiology.

In the present study, body weight of the mice was determined at vaginal opening days to examine if there is a relationship between delayed puberty onset and extension of estrous cycle with regard to weight gain. Navarro et al. [67] and Majarune et al. [68] showed that fasting or calorie restriction causes reduction in the expression of Kiss1, Tac3 (neurokinin B gene), and Pdyn (dynorphin gene) mRNA and delay in puberty onset. Although calorie restriction was not aimed in the present study and isolated mice had access to food, there was no significant difference in the weight at the vaginal opening day between groups (shown in Fig. 1a). This result shows that female mice in the PWSI group grew more slowly than group-housed mice until puberty onset. This slow weight gain during the prepubertal period may be effective on Kiss1 signaling. Nieves [53] showed that induction of ELS by limited bedding between P4 and P11 caused less weight gain than control mice following 60 days. However, weight of ELS-exposed mice at the vaginal opening day was significantly higher than control values. Consistently with our results, ELS also delayed puberty onset in their study. Contrary to our findings, in another ELS study [69], maternal separation in which pups were separated from their mothers for 4 h/day between P2 and P20 interestingly caused acceleration in the onset of vaginal opening in rats. They also found less body weight gain in stressed groups. In these ELS models, pups still have contact with their mothers. In these paradigms, staying in contact with mothers would affect access of pups to food because mothers are under the same stress conditions, possibly resulting in reduced food intake. Metabolism or thermoregulation of pups would potentially be affected under these conditions. We interestingly found that PWSI mice gained more weight when we compared the weights of the first and last days of isolation (shown in Fig. 1b). This may be partly because there may have been competition for food in group-housed cages. Since our stress model covers the pubertal transition period and does not affect the body weight in a negative way, we may propose that the alterations in reproductive physiology of PWSI mice were investigated from the HPG axis and stress mechanism’s perspective. It was previously shown that different stressors such as limited bedding, maternal separation, exposing predator odor in early life [53, 70, 71], or neonatal lipopolysaccharide exposure as an immunological stressor [72] delayed vaginal opening in rodents. In these studies, it was reported that stress in early life caused alterations in the primary components of central reproduction regulators such as GnRH, RFRP-3, or kisspeptin neurons. According to our findings, PWSI significantly delayed onset of vaginal opening of isolated mice (shown in Fig. 1a) consistently with previous reports outlined above. We also observed that the mice exposed to PWSI displayed a significantly longer estrous cycle which is orchestrated by interactions between kisspeptin and GnRH neurons. This extension was caused by increased time spent at the metestrous stage (shown in Fig. 1c, d).

Effects of PWSI on anxiety-like behaviors of rodents, specifically C57BL/6J mice [73], have been tested with different experimental methods including the EPM test [29]. Although some researchers reported no effect of isolation on anxiety [74], the majority of the studies showed that isolation in different times of life span increases anxiety-like behaviors [75, 76]. In the present study, we observed that both isolated female and male mice showed more anxiety-like behaviors, spending significantly more time in the closed arm of EPM (shown in Fig. 2a, d). The number of entrances of isolated mice to the closed arms was significantly higher, and there was no significant difference in total distance moved between groups (shown in Fig. 2c, f, g, h). These findings suggest that isolated mice have no deficiency in exploration and locomotion, and they prefer to stay in closed arms. It has previously been reported that estrous stages do not have an impact on anxiety-like behavior in mice [53, 77]. We performed EPM tests at the diestrus phase to be consistent with the electrophysiology experiments.

Our findings showed that PWSI affected reproductive physiology during pubertal transition and continuation. In female and male rats, decreased Kiss1 activation and increased RFRP-3 activation induced with acute restraint stress was observed by using cfos coexpression, whereas Kiss1 gene expression in the arcuate nucleus did not change [45, 46]. Effects of different stressors on the HPG axis have been investigated for the hormonal and gene expression responses [2, 47, 66]. Kisspeptin neurons are one of the possible targets for stress to suppress reproductive functions, and electrophysiological response of these neurons to stress is unclear. Therefore, we hypothesized that social isolation in the prepubertal term could affect electrophysiological properties of ARCKISS1 neurons.

Our findings have shown for the first time that early-life social isolation suppresses electrophysiological activity of ARCKISS1 neurons. The spontaneous action potential ratio of ARCKISS1 neuron population of isolated group was significantly lower than group-housed mice for female and male mice (shown in Fig. 3c, d).

It is known that GnRH pulse frequency increases during sexual maturation. GnRH pulses occur every 90 min at P5, every 60 min at P15, and every 30 min in juvenile and peripubertal periods in rats, but the plasma LH level remains low until first ovulation, reaching at proestrus levels of LH [78]. Unlike ARCKISS1 neurons, AVPVKISS1 neurons are essential for the preovulatory LH surge receiving positive feedback effect of estradiol (E2) [79, 80]. Furthermore, when the complexity of the neural network to GnRH neurons [2, 78] is considered, it is conceivable to assume that suppression of ARCKISS1 neurons by PWSI is not the only reason for the delay in puberty onset.

In addition, Moore et al. [26] showed that LH pulses are generated by synchronized activation of KNDy neurons. They used an in vivo calcium imaging technique at single-cell resolution in freely moving female mice. This synchronization occurs in a temporal order with subpopulation of KNDy neurons supporting KNDy hypothesis. These subpopulations were named as “leader” and “followers” which may be responsible for the initiation, maintenance, and termination of the GnRH pulse generator. It was also suggested that another subpopulation of leader cells should reach a threshold to drive activation of leader cells in order to initiate LH pulse. It may be proposed that suppression of firing of “leader” KNDy cell subpopulation can prevent LH release. The suppressed spontaneous-firing rate of ARCKISS1 neurons may be taken into account for the delay in the puberty onset and extension in the estrous cycle of PWSI exposed mice in the present study. Although previous studies showed that stress decreases LH pulses and increases circulating CORT [45], it would be better to support our behavioral and electrophysiological data with LH pulses and CORT levels.

ARCKISS1 neurons receive intensive GABAergic synaptic input from different areas of the brain including the amygdala and hypothalamic paraventricular nucleus which are related with stress response regulation [81, 82]. Therefore, after loose-seal recordings, we performed the voltage-clamp whole-cell electrophysiology methods from GFP-expressing ARCKISS1 neurons to investigate possible changes in postsynaptic currents to ARCKISS1 neuron to explain the decrease in the action potential ratio. In the presence of glutamate receptor blockers, the peak amplitude of sIPSCs to ARCKISS1 neurons was significantly higher in the isolated female and male groups (shown in Fig. 4). sIPSCs frequencies were also higher in the isolated male mice, not in female (shown in Fig. 4a, d). This may be the reason for the low firing frequency of ARCKISS1 neurons in the isolated group.

The amygdala, one of the key brain limbic regions sensitive to stress stimulus, plays a critical role in processing anxiety and emotions as well as regulating pubertal timing and the HPG axis [83, 84]. It is known that 70% of the neuronal outputs originate from the MePD are GABAergic neurons [85], and significant amounts of these projections reach areas related with regulation of reproduction [86]. Optogenetic stimulation of MePD GABAergic neuron terminals in ARC suppressed pulsatile LH secretion [87]. Inhibition of MePD GABA neurons with DREADD prevented suppression effect of predator odor and restraint stress on LH pulse frequency [87]. In addition, McIntyre [82] showed that optogenetic stimulation of CRH-R-expressing GABA neurons and CRH neurons in paraventricular nucleus increases inhibitory inputs to ARCKISS1 neurons and decreases GnRH pulse generator frequency. Increased sIPSC inputs to ARCKISS1 neurons in isolated groups in the present study support the current concept about interactions between GABAergic inputs to ARCKISS1 neurons from the hypothalamic areas related to stress.

Our findings also show that in the presence of the GABAA blocker, frequency of sEPSCs to ARCKISS1 neurons in the isolated female and male mice was significantly lower than that in the group-housed mice (shown in Fig. 5a, d). There was no significant difference in the peak amplitude data (shown in Fig. 5b, e).

In conclusion, the present study showed that PWSI caused delay on puberty onset and alterations in the estrous cycle. The delay may be related to slow growth induced by PWSI in the prepubertal period. Although the body weight of isolated mice catch-up control mice after puberty under isolated conditions, the estrous cycle of isolated mice prolonged. PWSI suppressed electrical activity of ARCKISS1 neurons with increased inhibitory inputs and decreased excitatory inputs to ARCKISS1 neurons. These findings demonstrate that PWSI alters pre- and postpubertal reproductive physiology through metabolic and electrophysiological pathways. This study would be of interest to further investigate the roles of GABAergic and glutamatergic inputs from stress-related areas to ARCKISS1 neurons to understand adaptation of the reproductive system to the ELS conditions.

We thank Dr. Volkan Adem Bilgin for his technical support.

This study protocol was approved by the Yeditepe University Experimental Animal Research Ethics Committee, approval ID 2020/02-18.

The authors have no conflict of interest to declare.

This study was partially supported by the Scientific and Technological Research Council of Türkiye (TUBITAK project # 219S554). All experiments were conducted in the Brain Research Laboratory of Faculty of Medicine at Yeditepe.

S.A. and B.Y. designed the study. Animal genotyping, surgery, and all behavioral experiments were performed by S.A. Electrophysiology experiments were performed by S.A. and Y.Y. The data were analyzed by S.A. and Y.Y. The manuscript was written by S.A. D.A. has helped in planning of the study at the early stage and also contributed to data analysis. All the coauthors reviewed the manuscript and provided comments.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.

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