Rapamycin, but not minocycline, significantly alters ultrasonic vocalization behavior in C57Bl/6J pups in a flurothyl seizure model

Abstract

Epilepsy is one of the most common neurological disorders, with individuals having an increased susceptibility of seizures in the first few years of life, making children at risk of developing a multitude of cognitive and behavioral comorbidities throughout development. The present study examined the role of PI3K/Akt/mTOR pathway activity and neuroinflammatory signaling in the development of autistic-like behavior following seizures in the neonatal period. Male and female C57BL/6J mice were administered 3 flurothyl seizures on postnatal (PD) 10, followed by administration of minocycline, the mTOR inhibitor rapamycin, or a combined treatment of both therapeutics. On PD12, isolation-induced ultrasonic vocalizations (USVs) of mice were examined to determine the impact of seizures and treatment on communicative behaviors, a component of the autistic-like phenotype. Seizures on PD10 increased the quantity of USVs in female mice and reduced the amount of complex call types emitted in males compared to controls. Inhibition of mTOR with rapamycin significantly reduced the quantity and duration of USVs in both sexes. Changes in USVs were associated with increases in mTOR and astrocyte levels in male mice, however, three PD10 seizures did not result in enhanced proinflammatory cytokine expression in either sex. Beyond inhibition of mTOR activity by rapamycin, both therapeutics did not demonstrate beneficial effects. These findings emphasize the importance of differences that may exist across preclinical seizure models, as three flurothyl seizures did not induce as drastic of changes in mTOR activity or inflammation as observed in other rodent models.

Graphical Abstract

1. Introduction

Epilepsy is a chronic neurological disorder that affects 1 to 4% of the general population (Berg et al., 2010; Hauser, 1995). It is characterized by recurrent, unprovoked seizures and can be the result of diverse genetic and acquired etiologies. Epilepsy is associated with numerous neurobiological consequences, especially long-term impairments in cognition and behavioral abnormalities (Stafstrom, 2002; Vingerhoets, 2006). Despite the multitude of available pharmacological therapies, approximately one third of individuals with epilepsy are resistant to anti-epileptic treatments and continue to suffer from seizures (Lerche, 2015; Ventola, 2014). In addition, the majority of currently available treatments are symptomatic and function to primarily suppress seizures by decreasing neuronal excitability (Goldenberg, 2010; Rogawski & Loscher, 2004). The development of novel therapies most likely relies on targeting alternative mechanisms in order to modulate the underlying pathological changes caused by seizures and to prevent epileptogenesis.

The incidence of seizures is significantly higher in the first few years of life, making these children at risk for developing a multitude of cognitive and behavioral comorbidities (Baca, Vickrey, Caplan, Vassar, & Berg, 2011; Hauser, 1994; Kramer, 1999). One of the most common comorbid disorders diagnosed in children with epilepsy is Autism spectrum disorder (ASD), which occurs in approximately 30% of children with epilepsy (Talos et al., 2012; Tuchman, Moshe, & Rapin, 2009; Tuchman & Rapin, 2002). The core behavioral impairments associated with ASD, including social and communication deficits, along with repetitive or stereotypical behavior, have been studied extensively in rodent models of epilepsy (Bernard & Benke, 2015; Keller, Basta, Salerno, & Elia, 2017). While previous studies have provided evidence for how early-life seizures can impact long-term behavioral outcomes in adulthood, few studies have examined how seizures could impact the development of autistic-like behaviors in an acute time period.

There has been substantial evidence that epilepsy is a disorder beyond solely neuronal excitation, and that other components such as glial cells and the release of inflammatory signals may play a critical role in the pathogenesis of seizures (van Vliet, Aronica, Vezzani, & Ravizza, 2017; Vezzani, French, Bartfai, & Baram, 2011). Several cytokines and inflammatory molecules are released following seizures and can initiate cascades that ultimately increase neuronal excitability and decrease the threshold for subsequent seizures (Shimada, Takemiya, Sugiura, & Yamagata, 2014; Turrin & Rivest, 2004; Viviani, Gardoni, & Marinovich, 2007). Pharmacological therapies that reduce seizure-induced neuroinflammation have shown to be promising for the treatment of epilepsy in both humans and rodent models (Aronica et al., 2017; van Vliet et al., 2017). While inhibiting inflammatory processes post-seizures is one potential therapeutic target to prevent epileptogenesis, it is most likely not the only factor in what converts the brain into a chronic epileptic state.

Components of the PI3K/Akt/mTOR pathway have also been shown to influence epileptogenesis and neuronal excitability in the brain following seizures (Ostendorf & Wong, 2015). Dysregulation of mammalian target of rapamycin (mTOR) signaling activity is evident in several genetic and acquired epilepsies, and inhibiting mTOR with rapamycin has demonstrated beneficial effects in reducing seizure frequency and associated seizure-induced neurological damage (Wong, 2013). For example, administration of rapamycin can suppress seizures and delay epileptogenesis in rodents, in part by decreasing mossy fiber sprouting and neuronal death (Buckmaster, Ingram, & Wen, 2009; Sunnen et al., 2011; Zeng, Rensing, & Wong, 2009). In addition, inhibition has shown to protect against seizure-induced learning and memory impairments in rats (Brewster et al., 2013). Dysregulated mTOR signaling activity is also associated with autistic-like phenotypes, as mutations in certain components of the pathway serve as monogenic causes of ASD (Onore, Yang, Van de Water, & Ashwood, 2017; Sato, 2016).

Neuroinflammatory signaling, specifically proinflammatory cytokines, and PI3K/Akt/mTOR signaling are known to interact to influence disease states (Dello Russo, Lisi, Tringali, & Navarra, 2009). However, it is unknown whether seizure-induced neuroinflammatory processes interact with mTOR signaling following early-life seizures to result in behavioral impairments characteristic of the autistic-like phenotype. In the present study, we examined the impact of inhibiting neuroinflammation and mTOR pathway activity separately, as well as with a combined treatment, following an early-life flurothyl seizure paradigm on postnatal day (PD) 10 in mice. To determine whether concomitant inhibition of these factors could prevent the development of autistic-like behaviors, we examined communication via measuring ultrasonic vocalizations on PD12. Future therapeutics aimed at targeting the pathological brain abnormalities that underlie epileptogenesis can hopefully reduce the percentage of individuals with medically refractory epilepsy, as well as minimize commonly associated comorbid behavioral and cognitive impairments.

2. Methods

2.1. Animals

Subject mice included male and female C57BL/6J pups (PD10–12). All mice were bred and group housed at Baylor University in standard laboratory conditions at an ambient temperature of 22°C (12-hour light/12-hour dark diurnal cycles). Mice were provided with food and water ad libitum. All procedures were conducted in compliance with the Baylor University Institutional Animal Care and Use Committee and the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.

2.2. Seizure induction

On PD10, pups were randomly assigned to receive either seizure or control procedures. Prior to the first seizure on PD10, toes of all pups were clipped for identity purposes throughout the experiment and placed back into the home cage for 30 min prior to seizure induction. Seizure mice received 3 seizures, each 2 hours apart, on PD10 for a total of 3 seizures. For each seizure, mice were placed in groups of 2 or 3 littermates in a clear acrylic inhalation chamber (29 cm × 16 cm × 15 cm) located within a fume hood. The chemoconvulsant flurothyl (bis-2,2,2-trifluroethyl ether) obtained from Sigma-Aldrich (St. Louis, MO, USA) was used to induce seizures. Flurothyl was administered at a rate of 50μl/minute, using a Harvard Apparatus syringe pump (Model 11 Plus), until all mice experienced a generalized tonic-clonic seizure, displayed by tonic extension of the forelimbs and hindlimbs. Pups receiving the control procedure were handled similarly and placed in an identical acrylic chamber placed on a lab bench outside the fume hood, with no syringe pump inserted or infusion of flurothyl administered. Following seizure or control procedures, pups were placed with their same treatment counterpart(s) in individual containers containing clean bedding, warmed with a heating pad to approximately 35°C for the entirety of seizure or control procedures. All pups were monitored and kept in the warmed containers until 15 min following the end of drug administration (1 hour following 3^rd seizure) on PD10. Pups were separated from their home cages for a total of 5h and 15 min. Following the 1^st and 3^rd seizures, both seizure and control pups received a subcutaneous injection between the shoulder blades of 0.1mL 0.9% physiological saline in order to account for time when pups were unable to suckle in the home cage. The seizure and control chambers were cleaned with a 30% isopropanol solution in between each seizure or control administration.

2.3. Treatment administration

One hour following the third (final) seizure on PD10, mice were randomly assigned to receive 1 of 4 treatments. All mice received two intraperitoneal (i.p.) injections of a combination of 0.9% physiological saline, minocycline (Sigma-Aldrich #M9511, St. Louis, MO, USA), or rapamycin (LC Laboratories, Woburn, MA, USA). The treatment groups were as follows: saline/saline, minocycline/saline, rapamycin/saline, minocycline/rapamycin. With the combined administration of both minocycline and rapamycin, the drug that was given first alternated between each mouse in order to counterbalance injection location. Minocycline was dissolved and diluted in saline for a concentration of 12.5mg/mL and administered at a dose of 50mg/kg. For the rapamycin preparation, a vehicle solution containing 5% polyethylene glycol 400 (Sigma-Aldrich) and 5% Tween 80 (Sigma-Aldrich) was first dissolved in saline. Rapamycin was then dissolved in 4% ethanol and the vehicle solution for a final concentration of 0.75mg/mL and was administered at a dose of 3mg/kg. Minocycline and rapamycin were administered so each pup received approximatively 0.02 mL per injection. Saline was administered at a similar dose, with each mouse also receiving approximately 0.02 mL per saline injection. Following administration of all treatments, pups were left in their respective containers on the warmed heating pad (∼35°C) to be monitored for 15 min prior to being returned to the home cage.

2.4. Ultrasonic vocalization recording paradigm

We examined ultrasonic vocalizations (USVs) on PD12 using an isolation-induced paradigm to assess changes in vocalization production following seizure and treatment administration on PD10. Prior to the recording phase, all pups were transferred to a new housing cage with fresh bedding that was warmed with a heating pad to an ambient temperature of ∼35°C. During recording, one at a time pups were placed into a separate housing pan placed within an acrylic sound-attenuating chamber. Vocalizations were recorded for 2 min for each pup. The recording apparatus consisted of a condenser microphone (CM16/CMPA, Avisoft Bioacoustics, Germany) which was connected to an ultrasound-recording interface (UltraSoundGate 116Hb, Avisoft Bioacoustics) and recorded all USVs on a continuous spectrum from 0 to 125kHz. Following recording, pups were placed back into the warmed housing pan with littermates. This procedure was repeated in sequence until all pups were recorded, and upon completion all pups were returned to their original home cage. The total number of recording sessions allowed for pups to not be separated from their home cage and mothers for longer than 30 minutes.

2.5. Ultrasonic vocalization analysis

Avisoft SASLab Pro software (Avisoft Bioacoustics, Germany) was utilized to convert all USV files into spectrograms, by automatically detecting calls via threshold-based algorithms and programmed hold time mechanisms. Spectrograms were created using a fast Fourier transformation (FFT) with the following parameters held constant for all USV files: FFT length = 1024, time window overlap = 75% (100% Frame, Hamming Window), frequency resolution = 488 Hz, time resolution = 1 ms. Duration, peak frequency, fundamental frequency, and amplitude were collected and analyzed for each group of mice. All calls were also manually identified by an experimenter blind to group using a previously described classification scheme (Scattoni, Gandhy, Ricceri, & Crawley, 2008). Calls were designated as 1 of 10 distinct types based on internal pitch changes, lengths, and shapes of individual calls (complex, harmonics, two-syllable, upward, downward, chevron, shorts, composite, frequency steps, flat).

2.6. RNA isolation and qRT-PCR for cytokine analysis

Hippocampal expression of cytokines was examined by isolating RNA and conducting quantitative reverse transcription-polymerase chain reaction (qRT-PCR). Following euthanization via rapid decapitation on PD12 (4 hours following USV testing), the hippocampus was dissected from the brain, rinsed in 1X PBS, placed on dry ice, and stored at −80°C until processed. Left hippocampal samples from each animal were individually homogenized and total RNA was isolated from samples using the RNeasy Mini Kit according to established protocols (Qiagen, Hilden, Germany). Samples were run on a NanoDrop ND-1000 Spectrophotometer (Thermo Fisher Scientific, NanoDrop Products, Wilmington, DE) to ensure adequate concentration (> 40ng/uL) and purity (260/280 = 1.9–2.1) of all samples.

Isolated RNA was reverse transcribed into single-stranded complementary DNA using the High-Capacity cDNA Reverse Transcription Kit according to manufacturer’s instructions (Applied Biosystems, Carlsbad, CA). The thermocycler protocol was as follows: 25°C for 10 min, 37° for 120 min, 85°C for 5 min, and held at a temperature of 4°C until further processing. Gene expression was determined by qRT-PCR utilizing TaqMan probe and primer chemistry on a QuantStudio 5 Real-Time PCR System (Applied Biosystems, Carlsbad, CA). Gene-specific measurements of each cDNA sample were run in triplicate in a 384-well plate, along with the endogenous control gene (β-actin) used for normalization. Males and females were analyzed separately. All groups were compared to control/saline-saline animals. The relative expression levels of each gene (IL-1β, IL-6, TNFα) were quantified using the comparative threshold (2^-ΔΔCt ) method of quantification.

2.7. Western blotting analysis

Western blotting was performed to examine the effect of seizures and subsequent treatment administration on inflammatory markers and mTOR pathway protein expression. Animals were euthanized via rapid decapitation on PD12 and the hippocampus dissected with identical procedures as described for qRT-PCR. The right hippocampus from each animal was utilized for all western blotting. Tissue was processed as previously described to extract whole homogenate samples and crude synaptosomes (Lugo et al., 2008). Briefly, individual right hippocampal samples were homogenized in ice-cold homogenization buffer containing 0.32M sucrose, 1mM EDTA, 5mM HEPES, and a protease inhibitor cocktail (P8340, Sigma, USA). Total homogenate or crude synaptosomes were run through 8–12% SDS-PAGE gels and transferred overnight to Hybond-P polyvinyl difluoride membranes (GE Healthcare, Piscataway, NJ, USA). The membranes were incubated for 1 h at room temperature in a blocking solution consisting of 5% nonfat milk diluted in 1X Tris buffered saline (50mM Tris-HCl, pH = 7.4, 150mM NaCl) with 0.1% Tween (1X TTBS) and 1mM Na₃VO_4. Membranes were then incubated overnight at 4°C on a Hoeffer rocker II with primary antibodies (phosphorylated Akt, Akt, p70S6K, S6, phosphorylated S6[s235/236], phosphorylated S6[s240/244], ionized calcium binding adaptor molecule 1 [Iba1], glial fibrillary acidic protein [GFAP]) in 5% milk in 1X TTBS (See supplementary table 1 for antibody specifics). Following incubation in primary antibodies, membranes were washed in 1X TTBS 3 times (5 min each wash), and then incubated with horseradish peroxidase-labeled secondary antibodies in a milk solution (1:20,000) for 1hr. Secondary antibodies were either anti-mouse immunoglobulin G (IgG) or anti-rabbit IgG (Cell Signaling Technology, Boston, MA, USA). Membranes were washed again (3 × 5 min) in 1X TTBS, and then incubated in GE ECL Prime (GE Healthcare, Piscataway, NJ, USA) for 5 min at room temperature.

Chemiluminescent immunoreactive bands were imaged with a digital western blot imaging system (ProteinSimple, Santa Clara, CA, USA) and the optical density of immunoreactive bands were subsequently measured with ProteinSimple AlphaView software. Measurements from protein bands of interest were normalized to endogenous actin levels for each tissue sample, with all groups being normalized to the control group per blot (control/saline-saline group). To obtain measurements of % total phosphorylated S6 (pS6[235,236], pS6[240,244]), the ratio of pS6 at each phosphorylation site in relation to total hippocampal S6 protein levels was quantified.

2.8. Statistical analysis

All statistical analyses were performed using GraphPad Prism 7 software (La Jolla, CA, USA) or SPSS 25.0 (IBM, USA). The male and female sample sizes for USVs, qRT-PCR, and western blotting are in supplementary tables 2 and 3, respectively. Changes in weight from PD10 until the time of tissue collection on PD12 was analyzed with a repeated-measures analysis of variance (ANOVA) with a within subject factor of timepoint (PD10, PD12). Seizure latency was examined with a repeated-measures ANOVA with a within subject factor of “seizure” (3 seizures on PD10) and between-subject factors of treatment and sex. Quantitative USV results were evaluated using a two-way (Seizure administration [control, seizure] x Treatment [saline-saline, minocycline-saline, rapamycin-saline, minocycline-rapamycin]) ANOVA. Qualitative differences in the types of USVs emitted between all male and female groups was analyzed using a Pearson Chi-Square, followed by individual z-tests to examine individual group differences between the control and seizure groups within each treatment group. For analysis of cytokines with qRT-PCR, the comparative threshold method of quantification was utilized to determine relative gene expression levels, with all groups normalized to the control/saline-saline group followed by analysis with two-way ANOVAs. For western blotting, two-way ANOVAs were utilized, with all groups being normalized to the control/saline-saline group average per blot. For USV, gene, and protein analysis, males and females were analyzed separately. Any significant interactions were followed by creating unique group identifiers for each group combination and examined using Tukey’s HSD post-hoc comparisons. Differences in lettering on all graphs indicate significance between groups at the level of at least p < 0.05 for all comparisons. All data are expressed as mean ± standard error of the mean (SEM).

3. Results

3.1. Animal weights

Measurements of weight were obtained at the time of injection on postnatal day (PD) 10 and prior to tissue collection on PD12. A repeated-measures analysis of variance (ANOVA) with a within subject factor of “timepoint” (PD10, PD12) was utilized to analyze weight, with analyses divided by sex. In males, there was a significant within subject effect of timepoint, with mice weighing more at the time of tissue collection on PD12 (F[1,72] = 261.75, p < 0.001). In addition, treatment significantly interacted with timepoint (F[3,72] = 22.71, p < 0.001). Tukey’s post-hoc analyses demonstrated that treatment significantly impacted weight measurements on PD12, specifically with mice treated with rapamycin or the combined treatment having reduced weight compared to saline-treated mice, as well as rapamycin-treated mice having reduced weight compared to minocycline-treated mice (p < 0.05). Seizure administration also interacted with timepoint (F[1,72] = 22.54, p < 0.001), however, Tukey’s post-hoc analyses revealed no discernable effects on weight at either timepoint. No three-way interaction between treatment, seizure administration, and timepoint was detected (F[3,72] = 1.40, p = 0.25). Between-subjects effects revealed a significant main effect of treatment on weight (F[3,72] = 3.33, p < 0.05). Tukey’s post-hoc analyses demonstrated that mice treated with rapamycin or the combined treatment had reduced weight compared to saline-treated mice, as well as rapamycin-treated mice had reduced weight compared to minocycline-treated mice (p < 0.05). There was no significant between-subjects main effect of seizure administration on weight (F[1,72] = 0.87, p = 0.36). A two-way interaction between treatment and seizure administration was detected (F[3,72] = 3.15, p < 0.05). Given the significant interaction, animals were subdivided into 8 groups for post-hoc analyses: control/saline-saline, control/minocycline-saline, control/rapamycin-saline, control/minocycline-rapamycin, seizure/saline-saline, seizure/minocycline-saline, seizure/rapamycin-saline, seizure/minocycline-rapamycin. Tukey’s post-hoc analyses revealed that control/saline-saline male mice had significantly increased weight compared to several groups (seizure/saline-saline, control/rapamycin-saline, seizure/rapamycin-saline, control/minocycline-rapamycin) (Fig. 1A).

Figure 1. Weights of male and female mice.

At the time of tissue collection on PD12, male mice that were administered rapamycin or the combined treatment weighed less than saline-treated mice, as well as rapamycin-treated mice had reduced weight compared to minocycline-treated male mice (A). In female mice, those given the combined treatment weighed significantly less than saline-treated mice on PD12 (B). There was a significant effect of time in both sexes, as both male and female mice gained weight from PD10 to PD12 (A,B). Data are expressed as mean ± standard error of the mean (SEM), * p < 0.05. Differences in lettering indicate significance between groups at the level of p < 0.05.

In female mice, there was also a significant within subject effect of timepoint, with mice weighing more at the time of tissue collection on PD12 (F[1,94] = 355.09, p < 0.001). In addition, treatment significantly interacted with timepoint (F[3,94] = 30.12, p < 0.001). Tukey’s post-hoc analyses demonstrated that treatment significantly impacted weight measurements on PD12, specifically with female mice given the combined treatment weighing less than saline-treated female mice (p < 0.05). Seizure administration also interacted with timepoint in female mice (F[1,94] = 15.52, p < 0.001), however, Tukey’s post-hoc analyses revealed no discernable effects on weight at either timepoint. There was no three-way interaction between treatment, seizure administration, and timepoint for female mice (F[3,94] = 1.31, p = 0.28). No between-subjects effect of treatment (F[3,94] = 1.07, p = 0.37), seizure administration (F[1,94] = 0.73, p = 0.39), or a two-way interaction between treatment and seizure administration (F[3,94] = 1.17, p = 0.33) were detected in female mice (Fig. 1B).

3.2. Seizure latency

To determine whether the time it took mice to undergo a generalized seizure impacted behavioral or molecular findings, we analyzed overall seizure latency between the groups and across the three seizure timepoints on PD10. The average time it took for mice to undergo a generalized seizure was 3 minutes and 47 seconds for all mice. A repeated-measures ANOVA with a within subject factor of “seizure” (3 seizures on PD10) and between-subjects factors of treatment and sex was utilized to analyze seizure latency. There was a significant within-subject effect of seizure timepoint, such that all mice exhibited a reduced latency to seize by the 3^rd seizure on PD10 (F[2,330] = 46.60, p < 0.001). However, sex did not interact with seizure timepoint (F[2,330] = 0.99, p = 0.37), nor did treatment (F[6,330] = 0.36, p = 0.90). There was also no three-way interaction between seizure timepoint, sex, and treatment (F[6,330] = 0.55, p = 0.77). There was no between-subjects effect of sex (F[1,165] = 2.70, p = 0.10) or for treatment (F[3,165] = 0.65, p = 0.59) on seizure latency. There was also no between-subjects interaction between treatment and sex (F[3,165] = 2.67, p = 0.49) (Data not shown).

3.3. Ultrasonic vocalization results

3.3.1. Quantitative USV parameters in male mice

Following seizures and treatment administration on PD10, ultrasonic vocalizations (USVs) of all mice were recorded on PD12 to identify potential deficits in early-life communicative behaviors. A total of 8 male mice did not vocalize and were removed from all USV analysis. The male mice that did not vocalize belonged to the following groups: control/saline-saline: n = 2; control/minocycline-saline: n = 1; seizure/saline-saline: n = 1; seizure/minocycline-saline: n = 1; seizure/rapamycin-saline: n = 2; seizure/minocycline-rapamycin: n = 1. A two-way ANOVA was utilized to examine the impact of seizure and treatment administration on quantitative parameters of the USVs. The USVs emitted from male and female mice were analyzed separately to parallel how gene and protein expression levels were examined and quantified. In male mice, a two-way ANOVA did not detect a significant main effect of seizure administration (F[1,141] = 0.33, p = 0.57) on the quantity of calls emitted. There was a significant effect of treatment on quantity of calls emitted in male mice (F[3,141] = 5.72, p < 0.05), with rapamycin-treated mice emitting significantly reduced calls compared to minocycline-treated mice and those that received the combined treatment of minocycline and rapamycin (p < 0.05) (Fig. 2A). No interaction was detected between seizure administration and treatment for quantity of calls in male mice (F[3,141] = 0.34, p = 0.80).

Figure 2. Quantitative parameters of ultrasonic vocalizations (USVs) in male mice.

Flurothyl seizures did not impact the quantity of USVs emitted in male mice, however, rapamycin-treated male mice emitted significantly less USVs compared to minocycline-treated mice and those that received the combined treatment (A). Rapamycin-treated mice also emitted USVs of significantly reduced duration compared to minocycline-treated mice and those that received the combined treatment (B). No significant effects were detected in the average peak frequency of USVs in male mice (C). Male mice that were treated with rapamycin emitted USVs with significantly reduced fundamental frequency compared to minocycline-treated mice (D). Control mice that received rapamycin emitted calls of significantly increased amplitude compared to control saline-treated mice (E). Data are expressed as mean ± standard error of the mean (SEM). Differences in lettering indicate significance between groups at the level of p < 0.05.

In addition to the quantity of USVs emitted, quantitative spectral properties of calls were examined. A two-way ANOVA did not detect a significant effect of seizure administration on average duration of USVs (F[1,141] = 0.03, p = 0.85). However, there was a significant main effect of treatment (F[3,141] = 3.01, p < 0.05), with rapamycin-treated mice emitting USVs of significantly reduced duration compared to minocycline-treated mice and those that received the combined treatment (p < 0.05) (Fig. 2B). There was not a significant interaction between seizure administration and treatment for the duration of USVs (F[3,141] = 0.44, p = 0.72). No significant main effects for seizure administration (F[1,141] = 0.53, p = 0.47) or treatment (F[3,141] = 1.94, p = 0.13) were detected in the average peak frequency of USVs emitted in male mice. There was also no significant interaction between seizure administration and treatment for average peak frequency of calls (F[3,141] = 1.72, p = 0.17) (Fig. 2C). We also examined the average fundamental frequency of USVs and found that seizure administration similarly had no effect in male mice (F[1,141] = 0.02, p = 0.90). However, there was a significant main effect of treatment for average fundamental frequency (F[3,141] = 3.09, p = 0.03), with rapamycin-treated mice emitting USVs of significantly reduced fundamental frequency compared to mice given minocycline (p < 0.05) (Fig. 2D). No interaction between seizure administration and treatment was detected in the fundamental frequency of USVs in male mice (F[3,141] = 2.00, p = 0.12). There were also no significant main effects for seizure administration (F[1,141] = 1.69, p = 0.20) or treatment (F[3,141] = 0.91, p = 0.44) on the average amplitude of USVs emitted by male mice. There was a significant interaction between seizure administration and treatment for average amplitude (F[3,141] = 3.92, p < 0.05). Given the significant interaction, male mice were subdivided into 8 groups for post-hoc analysis. Tukey’s post-hoc analyses showed that male control mice that received rapamycin treatment had significantly increased amplitude compared to male control mice that received saline (p < 0.05) (Fig. 2E).

3.3.2. Quantitative USV parameters in female mice

A total of 7 female mice did not vocalize and were removed from all USV analysis. The female mice that did not vocalize belonged to the following groups: control/rapamycin-saline: n = 1; control/minocycline-rapamycin: n = 2; seizure/saline-saline: n = 3; seizure/rapamycin-saline: n = 1. In female mice, a two-way ANOVA revealed a significant main effect of seizure administration (F[1,167] = 3.93, p < 0.05), with female seizure mice emitting significantly more USVs than female control mice (p < 0.05). There was also a main effect of treatment for quantity of calls (F[3,167] = 8.66, p < 0.001), with rapamycin-treated mice emitting significantly fewer calls than all other groups (p < 0.05). There was no interaction between seizure administration and treatment for quantity of USVs emitted in female mice (F[3,167] = 0.45, p = 0.72) (Fig. 3A).

Figure 3. Quantitative parameters of ultrasonic vocalizations (USVs) in female mice.

Administration of flurothyl seizures significantly increased USV quantity compared to control mice. In addition, rapamycin treatment in female mice resulted in significantly reduced USV quantity when compared to all other treatment groups (A). Seizures had no effect on the average duration of USVs, however, rapamycin-treated male mice emitted USVs of significantly reduced duration compared to minocycline-treated mice (B). No significant effects were detected in the average peak frequency (C), average fundamental frequency (D), or average peak amplitude (E) of USVs in female mice. Data are expressed as mean ± standard error of the mean (SEM), * p < 0.05. Differences in lettering indicate significance between groups at the level of p < 0.05.

When examining the average duration of USVs in female mice, there was no significant effect of seizure administration (F[1,167] = 1.97, p = 0.16). There was a significant effect of treatment (F[3,167] = 2.89, p < 0.05), with rapamycin-treated mice emitting USVs of significantly reduced average duration compared to minocycline-treated mice (p < 0.05) (Fig. 3B). No significant interaction between seizure administration and treatment was detected for average duration of USVs in female mice (F[3,167] = 0.02, p = 1.00). For the average peak frequency of USVs, there was no significant main effect of seizure administration (F[1,167] = 0.54, p = 0.47), treatment, (F[3,167] = 0.81, p = 0.49), or a significant interaction between the two factors in female mice (F[3,167] = 0.81, p = 0.49) (Fig. 3C). There were also no significant main effects for average fundamental frequency of USVs for seizure administration (F[1,167] = 0.07, p = 0.79), treatment (F[3,167] = 1.81, p = 0.15), or an interaction between the two factors (F[3,167] = 0.63, p = 0.60) (Fig. 3D). For the average amplitude of USVs in female mice, there was also no significant main effect of seizure administration (F[1,167] = 0.08, p = 0.78), treatment (F[3,167] = 1.64, p = 0.18), or an interaction between seizure administration and treatment (F[3,167] = 0.66, p = 0.58) (Fig. 3E).

3.3.3. Qualitative USV parameters in male mice

In addition to quantitative parameters of USVs, qualitative differences in the types of calls emitted by each group were also examined. Ultrasonic vocalizations were identified as 1 of 10 distinct types based on changes in call length, pitch, and the shapes of individual calls (Scattoni et al., 2008). A Pearson Chi-Square revealed a significant population difference between the types of calls emitted between all 8 groups of male mice (X²[63, N = 14,328] = 467.80, p < 0.001). Individual group differences were examined by performing separate z-tests to analyze the number of calls emitted between male control and seizure mice within each treatment group: control treatment (saline-saline), minocycline-treated (minocycline-saline), rapamycin-treated (rapamycin-saline), combined treatment (minocycline-rapamycin). In the male control treatment group, seizure mice emitted significantly reduced complex, upward, and composite calls and a significantly increased number of harmonics, chevron, and short calls compared to control male mice (p < 0.05) (Fig. 4A). In minocycline-treated male mice, seizure mice emitted significantly reduced complex and harmonic calls, and an increased amount of upward, short, and frequency step calls compared to control minocycline-treated mice (p < 0.05) (Fig. 4B). In rapamycin-treated male mice, seizure mice emitted a decreased amount of upward and downward calls, and an increased amount of complex, chevron, and frequency step calls compared to control rapamycin-treated mice (p < 0.05) (Fig. 4C). Lastly, in male mice that received the combined treatment of minocycline and rapamycin, seizure mice emitted reduced upward, downward, and chevron call types, as well as a significantly increased amount of short call types compared to control mice that received the combined treatment (p < 0.05) (Fig. 4D).

Figure 4. Call type utilization patterns in male mice.

The types of calls emitted different significantly across all 8 groups of male mice (X²[63, N = 14,328] = 467.80, p < 0.001). Differences in call type patterns between seizure and control mice were compared between each treatment group: saline-treated (A), minocycline-treated (B), rapamycin-treated (C), and those that received the combined treatment (minocycline/rapamycin) (D). Significant differences between control and seizure groups at the level of p < 0.05 are denoted by an asterisk (*).

3.3.4. Qualitative USV parameters in female mice

A Pearson Chi-Square also revealed a significant population difference between the types of calls emitted between all 8 groups of female mice (X²[63, N = 16,558] = 770.51, p < 0.001). Similar to the analyses performed in male mice, separate z-tests were conducted to analyze the number of calls emitted between female control and seizure mice within each treatment group: control treatment (saline-saline), minocycline-treated (minocycline-saline), rapamycin-treated (rapamycin-saline), combined treatment (minocycline-rapamycin). In the female control treatment group, seizure mice emitted a significantly reduced amount of frequency step calls, as well as an increased number of harmonics, two-syllable, and short call types compared to female control mice in the control treatment group (p < 0.05) (Fig. 5A). In female minocycline-treated mice, seizure mice emitted a reduced number of downward and short calls, and an increased amount of complex, harmonics, two-syllable, chevron, and frequency step calls compared to minocycline-treated control mice (p < 0.05) (Fig. 5B). In female rapamycin-treated mice, seizure mice emitted a significantly reduced amount of short and composite calls, along with an increased number of complex and harmonic call types compared to control rapamycin-treated mice (p < 0.05) (Fig. 5C). In female mice that received the combined treatment, seizure mice had a significantly decreased amount of complex and composite call types, as well as emitted a significantly increased amount of two-syllable, chevron, and frequency step calls compared to control female mice that received the combined treatment (p < 0.05) (Fig. 5D).

Figure 5. Call type utilization patterns in female mice.

The types of calls emitted different significantly across all 8 groups of female mice (X²[63, N = 16,558] = 770.51, p < 0.001). Differences in call type patterns between seizure and control mice were compared between each treatment group: saline-treated (A), minocycline-treated (B), rapamycin-treated (C), and those that received the combined treatment (minocycline/rapamycin) (D). Significant differences between control and seizure groups at the level of p < 0.05 are denoted by an asterisk (*).

3.4. Cytokine expression results

In male mice, seizures on PD10 did not significantly increase hippocampal gene expression of interleukin-1β (IL-1β) (F[1,50] = 0.42, p = 0.52), interleukin-6 (IL-6) (F[1,50] = 0.48, p = 0.49), or tumor necrosis factor-α (TNFα) (F[1,50] = 0.28, p = 0.60) on PD12. However, there was a significant main effect of treatment for IL-1β (F[3,50] = 7.83, p < 0.001), IL-6 (F[3,50] = 7.10, p < 0.001), and TNFα (F[3,50] = 3.75, p < 0.05) hippocampal gene expression levels in male mice (Fig. 6A-C). Mice that received minocycline or the combined treatment of minocycline and rapamycin had significantly increased hippocampal IL-1β expression compared to mice that received rapamycin or saline (p < 0.05) (Fig. 6A). No differences were detected in IL-β between the minocycline and the combined treatment group, or between the saline and rapamycin groups (p > 0.05). For IL-6, male mice that received minocycline or the combined treatment had significantly higher expression levels compared to saline-treated mice (p < 0.05). In addition, mice that received rapamycin had significantly decreased IL-6 expression compared to mice that received the combined treatment (p < 0.05) (Fig. 6B). Post-hoc analyses revealed no discernable effects between treatment groups for TNFα, apart from a trending increase in mice that received that combined treatment compared to rapamycin-treated mice (p = 0.07) (Fig. 6C). No significant interactions were detected between seizure administration and treatment for IL-1β (F[3,50] = 0.55, p = 0.65), IL-6 (F[3,50] 0.19, p = 0.90), or TNFα (F[3,50] = 0.06, p = 0.98) hippocampal gene expression levels in male mice.

Figure 6. Hippocampal proinflammatory cytokine expression in male mice.

Seizures did not significantly increase the expression of IL-1β, IL-6, or TNFα in male mice (A-C). A significant effect of treatment was detected for IL-1β, with minocycline-treated mice or those that received the combined treatment having significantly increased expression compared to mice that received rapamycin or saline (A). A significant effect of treatment was detected for IL-6, with mice that received minocycline or the combined treatment having significantly higher expression levels compared to saline-treated mice, along with rapamycin-treated mice having reduced IL-6 expression compared to mice that received the combined treatment (B). A significant effect of treatment was also detected for TNFα, however, no individual treatment group differences were detected (C). Gene expression measurements on individual samples were performed in triplicate. Data are expressed as mean ± standard error of the mean (SEM). Differences in lettering indicate significance between groups at the level of p < 0.05.

In female mice, seizures similarly did not result in enhanced hippocampal gene expression of IL-1β (F[1,56] = 0.20, p = 0.66), IL-6 (F[1,56] = 1.71, p = 0.20), or TNFα (F[1,56] = 1.10, p = 0.30). There were also no significant effects of treatment for IL-1β (F[3,56] = 2.27, p = 0.09), IL-6 (F[3,56] = 0.87, p = 0.46), or TNFα (F[3,56] = 1.30, p = 0.28) expression levels in female mice. No interactions were detected between seizure administration and treatment for IL-1β (F[3,56] = 0.59, p = 0.62), IL-6 (F[3,56] = 1.29, p = 0.29), or TNFα (F[3,56] = 0.53, p = 0.67) hippocampal expression levels in female mice (Fig. 7A-C).

Figure 7. Hippocampal proinflammatory cytokine expression in female mice.

Seizures and treatment administration did not result in any changes in IL-1β (A), IL-6 (B), or TNFα (C) expression. Gene expression measurements on individual samples were performed in triplicate. Data are expressed as mean ± standard error of the mean (SEM).

3.5. Western blotting results

3.5.1. Hippocampal expression of mTOR signaling proteins

Western blotting was utilized to examine protein expression in components of the PI3K/Akt/mTOR pathway on PD12, two days after seizure and treatment administration. A two-way ANOVA revealed that seizures in male mice had significantly increased p70S6K expression (F[1,46] = 13.48, p < 0.05) (Fig. 8A). There was a significant main effect of treatment for S6 (F[3,46] = 4.53, p < 0.05), with rapamycin-treatment mice having a trending decrease in S6 expression levels compared to mice administered minocycline (p = 0.07) (Fig. 8B). There was also a significant main effect of treatment for pS6(235,236) (F[3,46] = 47.38, p < 0.001) and % total pS6(235,236) (F[3,46] = 91.57, p < 0.001), with mice administered rapamycin or the combined treatment having significantly reduced expression levels compared to all other groups (p < 0.001) (Fig. 8C,D). In addition, there was a significant main effect of treatment for pS6(240,244) (F[3,46] = 14.49, p < 0.001) and % total pS6(240,244) (F[3,46] = 88.74, p < 0.001), with mice administered rapamycin or the combined treatment having significantly reduced expression levels compared to all other groups (p < 0.001) (Fig. 8E,F). There was also a significant main effect of treatment for pAkt (F[3,46] = 12.15 p < 0.001), with those administered the combined treatment having significantly decreased expression levels compared to saline-treated and minocycline-treated mice (p < 0.05). In addition, rapamycin-treated mice had significantly reduced pAkt expression compared to minocycline mice (p < 0.001) (Fig. 8H). No differences were detected in Akt or % total pAkt expression in male mice (Fig. 8G,I). A summary of the means, SEM, and ANOVA results for western blotting analyses in male mice can be found in supplementary table 4.

Figure 8. Hippocampal expression of mTOR signaling proteins in male mice.

Seizures significantly increased p70S6K expression levels compared to controls (A). No effect of seizure administration or treatment was detected for S6 (B). Mice that received rapamycin or the combined treatment had significantly downregulated levels of pS6(235,236) (C), % total pS6(235,236) (D), pS6(240,244) (E), or % total pS6(240,244) (F). There was no effect of seizures or treatment on Akt (G) or % total pAkt (I) expression levels. There was a significant effect of treatment for pAkt, with those administered the combined treatment having significantly decreased expression levels compared to saline-treated and minocycline-treated mice, and rapamycin-treated mice having reduced expression compared to minocycline-treated mice (H). Data are expressed as mean ± standard error of the mean (SEM). Differences in lettering indicate significance between groups at the level of p < 0.05.

In female mice, seizures did not significantly alter the expression of any of the examined proteins. There was a significant main effect of treatment for S6 protein levels in female mice (F[3,32] = 7.51, p < 0.05), with mice that were administered the combined treatment having significantly decreased expression levels compared to saline-treated and minocycline-treated mice (p < 0.05). In addition, rapamycin-treated mice had significantly reduced S6 expression compared to minocycline-treated mice (p < 0.05) (Fig. 9B). There was also a significant main effect of treatment for pS6(235,236) (F[3,32] = 7.95, p < 0.001) and % total pS6(235,236) (F[3,32] = 7.77, p < 0.001), with mice administered rapamycin or the combined treatment having significantly reduced expression levels compared to all other groups (p < 0.05) (Fig. 9C,D). In addition, there was a significant main effect of treatment for pS6(240,244) (F[3,32] = 59.79 p < 0.001) and % total pS6(240,244) (F[3,32] = 29.42, p < 0.001), with mice administered rapamycin or the combined treatment having significantly reduced expression levels compared to all other groups (p < 0.001) (Fig. 9E,F). No differences were detected in p70S6K, Akt, pAkt, or % total pAkt expression in female mice (Fig. 9A,G,H,I). A summary of the means, SEM, and ANOVA results for western blotting analyses in female mice can be found in supplementary table 5.

Figure 9. Hippocampal expression of mTOR signaling proteins in female mice.

No effect of seizure administration or treatment was detected for p70S6K expression levels (A). There was a significant effect of treatment for S6, with female mice that received the combined treatment having significantly decreased expression levels compared to saline-treated and minocycline-treated mice, and rapamycin-treated mice having reduced expression compared to minocycline-treated mice (B). Mice that received rapamycin or the combined treatment had significantly downregulated levels of pS6(235,236) (C), % total pS6(235,236) (D), pS6(240,244) (E), or % total pS6(240,244) (F). No effect of seizure administration or treatment was detected for Akt (G), pAkt (H), or % total pAkt (I). Data are expressed as mean ± standard error of the mean (SEM). Differences in lettering indicate significance between groups at the level of p < 0.05.

3.5.2. Hippocampal expression of neuroinflammatory proteins

In male mice, seizures resulted in significantly upregulated GFAP expression (F[1,46] = 6.22, p < 0.05), indicative of increased astrocyte reactivity following seizures on PD10 (Fig. 10B). However, there was no effect of treatment (F[3,46] = 1.66, p = 0.19) or an interaction between the two factors for GFAP expression (F[3,46] = 1.25, p = 0.30). No significant effect of seizure administration (F[1,46] = 2.94, p = 0.09), treatment (F[3,46] = 1.13, p = 0.35), or an interaction (F[3,46] = 0.90, p = 0.45), was detected for Iba1 expression levels in male mice (Fig. 10A).

Figure 10. Hippocampal expression of microglial (Iba1) and astrocyte (GFAP) reactivity in male and female mice.

Hippocampal expression of microglial (Iba1) and astrocyte (GFAP) reactivity in male and female mice. In male mice, no effect of seizure administration or treatment was detected for Iba1 (A). However, seizures significantly increased GFAP levels in male mice (B). In female mice, there was no effect of seizure administration or treatment on Iba1 (C) or GFAP (D) hippocampal expression levels. Data are expressed as mean ± standard error of the mean (SEM). Differences in lettering indicate significance between groups at the level of p < 0.05.

In female mice, no significant effects of seizure administration (F[1,32] = 0.64, p = 0.43), treatment (F[3,32] = 1.89, p = 0.15), or an interaction (F[3,32] = 0.78, p = 0.52) was detected for Iba1 expression levels (Fig. 10C). In addition, there was no significant effect of seizure administration (F[1,32] = 0.81, p = 0.37), treatment (F[3,32] = 0.51, p = 0.68), or an interaction (F[3,32] = 0.59, p = 0.62) for GFAP expression levels in female mice (Fig. 10D).

4. Discussion

Early-life seizures can have profound effects on the developing brain and have been associated with both acute and long-term impairments in cognition and behavior (Bernard & Benke, 2015; Lugo, Swann, & Anderson, 2014; Velíšková, Silverman, Benson, & Lenck-Santini, 2018). Hyperactivity of the mTOR pathway and neuroinflammation contribute to epileptogenesis and could serve as promising therapeutic targets for epilepsy. Overall, three flurothyl seizures on PD10 resulted in minimal quantitative differences in USV behavior. Seizures resulted in females emitting a significantly greater amount of USVs, however, other quantitative parameters of USVs were not impacted by seizures. Qualitatively, male mice that received seizures emitted a reduced amount of complex call types compared to control mice, suggesting that their calls were less spectrographically diverse and complicated. Behavioral changes were associated with increased astrocyte reactivity and p70S6K expression in male mice that received seizures, however, these changes were not detected in female mice. Minocycline or rapamycin treatment alone, or when combined, did not attenuate these changes in male mice, nor did they have beneficial effects on USV behavior.

The behavioral and molecular changes following seizures early in life may be dependent on seizure load, or the quantity of seizures experienced in a critical period of development (Hermann et al., 2002; Nolan et al., 2019). Several factors, including earlier age at seizure onset, lifetime seizure frequency, and longer duration of seizures, has been related to the magnitude of executive functioning and memory deficits in children (Berg, Zelko, Levy, & Testa, 2012; Black et al., 2010; Hermann et al., 2002). Similarly, in rodent models, the extent of behavioral deficits could be associated with a multitude of factors, such that increasing the seizure load or using an alternative seizure induction method may have resulted in more profound molecular and behavioral changes JJ(Keller, Saucier, Sheerin, & Yager, 2004; Lopez-Meraz et al., 2014; Nolan et al., 2019; Reynolds, Nolan, Huebschman, Hodges, & Lugo, 2017; Reynolds, Smith, Jefferson, & Lugo, 2016).

Beyond examination of quantitative parameters of USVs, qualitative aspects of calling behavior are also indicative of changes in early-life communication. Several studies utilizing both seizure and ASD models have found significant changes in call type production patterns (Binder & Lugo, 2017; Nolan et al., 2019; Reynolds et al., 2017; Scattoni et al., 2008; Takahashi et al., 2016). While the significance of the different call types in mice is not entirely elucidated, specific patterns that have shown to be consistent across studies suggest that altered call type utilization may be a better indicator of autistic-like communicative deficits in mice in comparison to quantitative changes in USVs. For instance, we found that seizure male mice emitted significantly fewer complex calls compared to control male mice. Several other studies have found that complex calls are altered following seizures, as well as in several rodent models of ASD (Binder & Lugo, 2017; Hiramoto et al., 2011; Nolan et al., 2019; Reynolds et al., 2017; Scattoni et al., 2008; Takahashi et al., 2016). A reduction in the complexity of USVs has previously been associated with less maternal retrieval in mice (Takahashi et al., 2016). This atypical pattern of USV emission could therefore impact social communication between pups and mothers and influence long-term neurodevelopmental outcomes in seizure pups. However, tt is important to note that while altered USV behavior may be indicative of deficits in communication, which is a component of the autistic-like phenotype, there are other behavioral impairments that would need to be assessed to determine whether early life seizures are leading to a phenotype that resembles ASD. While there is a high comorbidity between epilepsy and ASD in clinical populations, there are many factors that contribute to this complex relationship and further research is needed to determine the mechanisms underlying the comorbidity.

Several preclinical and clinical studies have demonstrated a role for mTOR signaling in both genetic and acquired models of epilepsy (Wong, 2013). However, despite considerable evidence suggesting that mTOR activity would be increased following seizures, we did not detect significant changes in hippocampal protein expression of components of the pathway (Citraro, Leo, Constanti, Russo, & De Sarro, 2016; Ostendorf & Wong, 2015). In males, seizures significantly increased p70S6K, however, this seizure effect appears to be driven by the impact of treatment in addition to seizures rather than solely a seizure-induced change on its own. In contrast to the strong evidence of mTOR hyperactivity in genetic epilepsy models (Pten, Tsc1/Tsc2 mutations), other studies in addition to ours have demonstrated variable mTOR hyperactivation in acquired models, especially in the neonatal period (Raffo, Coppola, Ono, Briggs, & Galanopoulou, 2011; Talos et al., 2012). Talos et al. (2012) found that following hypoxia-induced seizures in rats on PD10, mTOR pathway activity was elevated in the hippocampus and cortex at 12hrs. and 24hrs. post-seizures, however, these changes returned to baseline by 48hrs. post-seizures on PD12 (Talos et al., 2012). In contrast to the neonatal period, many studies in adulthood have shown evidence for sustained mTOR hyperactivity, suggesting that different mechanisms may regulate mTOR pathway activity in the neonatal period compared to in adulthood (Talos et al., 2018; van Vliet et al., 2012; Zeng et al., 2009; Zhu et al., 2017). While the therapeutic potential of rapamycin has demonstrated efficacy in treating seizures in both preclinical and clinical populations, the side effects resultant from continual treatment is of concern. Some of the more common side effects of rapamycin treatment include chronic immunosuppression, mucositis, skin reactions, and enhanced cholesterol and triglyceride levels (McDaniel & Wong, 2011; Tsang, Qi, Liu, & Zheng, 2007). In addition to these observed side effects in clinical populations, the impact of rapamycin on behavioral phenotypes in mice is variable. Chronic rapamycin treatment in a model of Fragile X syndrome (Fmr1 KO mice) did not reverse the majority of autistic-like behavioral impairments observed in KO mice (Sare et al., 2017). In addition, treatment had an adverse effect on social behavior and sleep duration in both Fmr1 KO and WT mice, as well as increased anxiety in control mice (Sare et al., 2017).In the present study, we found that rapamycin treatment significantly reduced the quantity and duration of vocalizations emitted from both male and female seizure and control mice. In addition, control mice that received rapamycin emitted significantly fewer complex calls when compared to saline-treated control mice. This was the first study to examine the impact of mTOR inhibition on communicative behaviors in a mouse model. The significant impact that rapamycin treatment had on USV production in neonatal mice is concerning, in that it could potentially lead to long-term deficits in communication, a component of the autistic-like phenotype (Ferhat et al., 2016; Mody & Belliveau, 2013). With Everolimus being FDA approved to treat partial epilepsy in individuals with TSC, in addition to several other ongoing trials with mTOR inhibitors, it is critical to investigate whether these deficits in USV emission persist beyond the acute time period following treatment and could impact communication in adulthood (Krueger et al., 2013; Krueger et al., 2016).

There is substantial evidence for enhanced inflammatory signaling following seizures, which has shown to contribute to hyperexcitability and potentially underlie epileptogenesis in the brain (Vezzani, Balosso, & Ravizza, 2019). We found that seizures in male mice resulted in enhanced GFAP expression, indicative of increased astrocyte reactivity, with no evidence of microglia reactivity in either sex following seizures. In addition to the release of inflammatory molecules, reactive astrocytes undergo extensive physiological changes that can alter neurotransmitter homeostasis, including increasing release of glutamate which can further contribute to the excitability underlying seizures (Bezzi et al., 2001; Choi & Koh, 2008; Robel et al., 2015; Shimada et al., 2014). The observed increase in GFAP in only male mice, and not females, could be attributed to differences in immune cell development between the sexes. For example, astrocyte populations differ between the sexes early in life, such that male astrocytes often have increased processes and branches, but less dendritic spines compared to female astrocytes (Schwarz & Bilbo, 2012). Studies have found that microglial activation often precedes astrocyte reactivity after seizures, suggesting that if we had examined Iba1 expression at an earlier timepoint such as at 24hrs. post-seizures we may have found the expected increase (Sano et al., 2019; Shapiro, Wang, & Ribak, 2008; Vargas-Sánchez et al., 2018). Microglial reactivity has found to be model-dependent, suggesting that the time course of reactivity may also differ in the flurothyl model compared to other preclinical models (Benson, Manzanero, & Borges, 2015). This is the first study to examine microglial reactivity in a neonatal flurothyl seizure model in mice, and it could be that flurothyl does not induce as drastic changes in the neuroinflammatory response as is detected in other models.

Minocycline has previously been shown to have anti-convulsant effects in rodent models of epilepsy, by reducing microglial activation and decreasing hippocampal damage post-seizures (Abraham, Fox, Condello, Bartolini, & Koh, 2012; Heo et al., 2006; Nowak et al., 2012; Wang, Englot, Garcia, Lawton, & Young, 2012). In addition, it has been shown to impact communication and language abilities in both preclinical models and clinical populations. In a mouse model of Fragile X syndrome (FXS), minocycline treatment was able to normalize USV deficits in adult Fmr1 KO mice, specifically increasing the USV call rate to WT levels (Rotschafer, Trujillo, Dansie, Ethell, & Razak, 2012). In children with FXS, treatment with minocycline for at least 2 weeks increased the use of expressive language and improved social communication (Utari et al., 2010). Mutations in Fmr1 are the largest genetic cause of ASD, suggesting that anti-inflammatory treatments could also help ameliorate communicative deficits in other models that exhibit autistic-like behavior (Mila, Alvarez-Mora, Madrigal, & Rodriguez-Revenga, 2018). However, we did not find that minocycline treatment had any impact on USV behavior following flurothyl seizures in the neonatal period. The exact mechanism in which minocycline impacts communicative behaviors in rodent models and in humans is unknown and requires further investigation.

While minocycline has shown to be efficacious in reducing inflammation in disease models, our findings suggest that the anti-inflammatory treatment may have diverse effects when administered in the neonatal period versus in adulthood (Elewa, Hilali, Hess, Machado, & Fagan, 2006; Heo et al., 2006; Wang et al., 2012). Male mice that received minocycline or the combined treatment on PD10 had significantly increased IL-1β and IL-6 levels compared to saline-treated and rapamycin-treated mice. Another study has found that minocycline treatment (45mg/kg) in perinatal female mice from embryonic day 18 to PD1 resulted in significantly increased cell death in pups in several brain regions and enhanced microglia reactivity (Strahan, Walker, Montgomery, & Forger, 2017). Increased cell death was also observed in pups when they were administered 5 minocycline treatments from PD3 to PD5 (Strahan et al., 2017). These findings, along with our own, suggest there may be a developmental switch in how neonatal rodents respond to minocycline, which could potentially be due to age-related changes in cell populations early in life (Santos-Galindo, Acaz-Fonseca, Bellini, & Garcia-Segura, 2011; Schwarz & Bilbo, 2012).

Early-life seizures can have a profound impact on the developing brain, producing long-lasting behavioral and cognitive effects (Bernard & Benke, 2015; Holmes, 2016). Several pathophysiological changes occur following seizures that can contribute to the persistent hyperexcitability that underlies the development of chronic epilepsy. The present study emphasizes that the seizure paradigm and model utilized can significantly impact the degree of seizure-induced brain changes. While three flurothyl seizures on PD10 only produced minor increases in mTOR hyperactivity and astrocyte reactivity on PD12, it is possible that increasing the seizure load could induce the more drastic changes other preclinical models have found. However, when determining the best model to use it is critical to consider how it parallels the human condition, specifically the frequency of seizures that are typically experienced in the neonatal period. Flurothyl has been used as a chemoconvulsant in numerous studies, yet the question of the optimal number of seizures and time period of induction still requires investigation.

Highlights.

• Early-life seizures increased ultrasonic vocalization (USV) quantity in female mice

• Rapamycin reduced USV emission and duration in both sexes on postnatal day 12

• Minocycline and rapamycin did not demonstrate beneficial effects on USV behavior

• Seizures increased p70S6K expression and astrocyte reactivity in male mice only

• 3 flurothyl seizures did not result in enhanced proinflammatory cytokine expression