Animal experiments conformed to the policies and guidelines of the Canadian Council on Animal Care and were approved by the University Health Network Animal Use Committee.

Male and female (8 week-old) C57BL/6 J were purchased from Jackson Laboratory. Animals were housed in a temperature-controlled room at 21 ± 1°C and 55 ± 5% relative humidity under a 12-h light/dark cycle. Food and water were provided ad libitum. Female mice were trained on gavage using water for 1 week prior to mating to reduce gavage-related stress during pregnancy. Two female virgin mice were mated with an experienced male. Mating was confirmed by the presence of vaginal plug. Plugged mice were randomly assigned to one of the three treatment arms: ABC/3TC + ATV/r (100/50 mg/kg/day +50/16.6 mg/kg/day), TDF/FTC + ATV/r (50 mg/33.3/kg/day +50/16.6 mg/kg/day), or water as control. Dosing was calculated based on previous pharmacokinetic studies in mice that optimized doses to yield Cmax plasma levels similar to levels reported in humans (Kala et al., 2018; Sarkar et al., 2020; Gilmore et al., 2021; Balogun and Serghides, 2022). Mice received daily treatment by oral gavage in a total volume of 100 μL, starting on day of plug detection until time of delivery of litter. Pups were kept with their mothers until weaning on postnatal day 21. Pups were then sexed and divided into three arms based on their maternal treatment – a total of 55 pups (27 female, 28 male) from 17 litters in the control arm, 48 pups (27 female, 21 males) from 14 litters in the ABC/3TC + ATV/r arm, and 43 pups (21 female, 22 male) from 12 litters from the TDF/FTC + ATV/r arm were included in the behavioral testing.

Animals were tested in the following battery of tests in the order presented in Figure 1, beginning at 6 weeks and ending at 8 months of age (N = 21–28 for all treatment groups). All testing took place in an empty behavioral testing room absent of spatial or visual cues. Mice were brought to the testing arena in their home cage and were tested individually. Between tests, the testing cage was thoroughly cleaned with Virox5 (Johnson Diversey, Inc., Sturevant, WI, United States) to eliminate odor cues.

The Open Field Test: The open field test was used to assess exploratory and locomotor behavior, and to assess response to open field stress. Animals were placed in a clean, empty open field (40 cm x 40 cm x 50 cm) made of plywood painted white on the inside floor and walls and black on the outside walls with non-toxic acrylic paint. Animals freely explored the maze for 30 min and their behavior was recorded using ANYMAZE^® and ODLog^™ software. Lighting levels were 330 lux. The following behaviors were coded: immobility; grooming (engaged in self-cleaning behavior), rearing (both front paws raised off of the floor), locomotion (total distance traveled and speed of travel), time spent in the center, and time spent by the wall (thigmotaxis zone).

The Zero Maze Test: Mice were removed from their home cage in the testing room and placed at the entrance of an enclosed region of the maze. Each mouse was allowed to freely explore the maze for a 5-min period after which it was returned immediately to its home cage. Tests were video recorded using ANYMAZE^® software. Lighting levels were 330 lux. The total time spent in the enclosed and unenclosed arms were coded and a percentage of time spent in the open arms was calculated.

Light/Dark Box Test: This test assesses unconditioned anxiety responses in mice. The light/dark box has 2 compartments separated by a 7 cm door - a light area (2/3 of the area) and a dark area (1/3 of the area; Bourin and Hascoët, 2003). Each mouse was placed in the light area and allowed to freely explore for 10 min. The time spent in each of the areas was recorded. Tests were video recorded using ANYMAZE^® software. Lighting levels in the light box were 580 lux.

The Novel Object Recognition Test: Mice are habituated to the testing arena (clear plastic mouse cages) for 15 min over 7 daily sessions and were considered successfully habituated when they consumed a small piece of breakfast cereal (Fruit Loops) within 2 min of the entrance to the testing arena. On the novel object recognition test day, each animal was exposed for 10 min to a LEGO^® construct (single LEGO brick; LEGO Group, Billund, Denmark) and a Hot Wheels^® car (model Corvette Z06; Mattel, Inc., El Segundo, CA, United States). The objects were previously determined to be of matched saliency in mice. Objects were fixed to the floor of the mouse cage with magnets. All tests were recorded using ANYMAZE^® software. Time spent exploring the objects was recorded using ODLog^™ software. Exploration was scored when the mouse touched an object with its forepaws or snout, bit, licked, or sniffed the object from a distance of no more than 1.5 cm. Following a 3-h delay, mice were re-exposed for 5 min to one object from the original test pair and to a novel object. The position and shape of the novel object was counterbalanced between treatment groups to control for bias. Lighting levels during all testing were 330 lux.

The 3-Chamber Social Approach Test: The test was done according to the following method (Kaidanovich-Beilin et al., 2011). Lighting levels during testing were 330 lux. Briefly, the sociability test was initiated by habituating the test mouse to an empty 3-room-chamber apparatus for 5 min. An unfamiliar mouse (same sex, background, age, and weight as the test mouse)—stranger 1—was placed in a wire cup in one room of the 3-room chamber. An empty wire cup was placed in the opposite room. The test mouse was released in the middle room and had free access to explore the rooms. The duration and frequency of direct contacts between the test mouse and the empty wire cup or the wire cup housing the unfamiliar mouse were recorded for 10 min. Following the sociability test, the social novelty test was initiated by placing a second unfamiliar mouse—stranger 2—in an identical wire cup in the opposite chamber for 10 min. The duration and frequency of direct contacts between the test mouse and the wire cup housing the familiar mouse (stranger 1) or the wire cup housing the novel mouse (stranger 2) were recorded for 10 min. Normal sociability for a mouse is to prefer to spend time with another mouse rather than the empty cup, and also show preference for social novelty; any deviation from this indicates impaired sociability (Kaidanovich-Beilin et al., 2011). A “sociability index” was calculated as (T_S1-T_WC)/(T_S1 + T_WC), wherein “T_S1” represents the duration spent with stranger 1 and “T_WC” the duration spent exploring empty wire cup. A “social novelty index” was calculated as (T_S1-T_S2)/(T_S1 + T_S2), wherein “T_S1” represents the duration spent with stranger 1 and “T_S2” the duration spent with stranger 2 or novel mouse.

Brain collection: At 8 months of age, following completion of all behavioral testing, mice were sacrificed and brains were collected for use in either magnetic resonance imaging, immunofluorescence imaging, or expression analyses.

Sample preparation: To prepare the brains for neuroanatomical assessment, mice underwent trans-cardiac perfusion (N = 14–24 per treatment group; Cahill et al., 2012). Briefly, mice were anesthetized with ketamine/xylazine and perfused through the left ventricle with 30 mL PBS and 2 mM ProHance (Bracco Diagnostics, Inc., NJ, United States), followed by 30 mL of 4% Paraformaldehyde (PFA) and 2 mM ProHance. The brains within the skull were incubated in 4% PFA containing 2 mM ProHance overnight at 4°C and then stored in a solution containing PBS, 2 mM ProHance, and 0.01% sodium azide for at least 28 days before imaging (de Guzman et al., 2016).

Imaging: A multi-channel 7.0 T magnet (Varian Inc. Palo Alto, CA, United States) and a custom-built solenoid array were used to image 16 samples in parallel (Dazai et al., 2011). An anatomical scan was performed using a T2-weighted, 3D fast spin-echo sequence with a cylindrical k-space acquisition and the following parameters: TR = 350 ms, TE = 12 ms, echo train length = 6, four averages, field-of-view 20 mm x 20 mm x 25 mm, matrix size = 504 × 504 × 630, isotropic image resolution = 40 μm (Spencer Noakes et al., 2017). Five control and one ABC/3TC + ATV/r brain had to be excluded from analysis because of damage during preparation for imaging or image misregistration.

Volume analysis: An automated image registration approach implemented in the Pydpiper toolkit (Friedel et al., 2014) and using the Advanced Normalization Tools (ANTs) deformation algorithm (Avants et al., 2008) was used. The 53 MR images were registered together through a process of linear and non-linear steps to create an average image (Nieman et al., 2018). The registration yielded deformation fields for each brain, and the absolute and relative Jacobian determinants provided an estimate of the local volume changes at every voxel for comparison between the groups (Chung et al., 2001). Using the results of the linear alignment, multiple templates of a segmented anatomical atlas with 182 labeled structures (Dorr et al., 2008; Ullmann et al., 2013; Steadman et al., 2014) were created (the MAGeT procedure; Chakravarty et al., 2013). From the final voted segmentation, volume changes were calculated and expressed in absolute volumes (mm³). These volumes were then normalized to total brain volume.

Immunofluorescence studies were performed on paraffin-embedded fixed brain tissue (N = 10 for all treatment groups). Mice were anesthetized and perfused through the left ventricle with 30 mL PBS, followed by 30 mL of 4% PFA. The brains were then extracted, post-fixed in 4% PFA overnight at 4°C and then stored in a solution containing PBS and 0.01% sodium azide. Brains were processed and embedded in paraffin, and then coronally sectioned (6 um thick) and mounted on Superfrost Plus slides (20 slides series with 4 slices per slide). Antigen retrieval was performed by boiling slides in citrate buffer using a microwave (4 min at power 6 followed by a cool down and again at 3 min at power 6). Slides were then incubated in DAKO serum-free blocking solution (Agilent Technologies) for 45 min at room temperature, followed by incubation with primary antibodies suspended in PBS, either NeuN at 1:200 (Abcam; ab177487), or GFAP at 1:1000 (Abcam; ab4674) for 18–24 h at 4°C. Slides were washed in PBS for 9 min, and incubated with appropriate secondary antibodies conjugated to Alexa 488 or Alexa 647 (Abcam), and DAPI (4′,6 diamidino-2-phenylindole) for 1.5 h at room temperature. Prolong Diamond antifade mounting medium was used (ThermoFisher P36961). Slides were scanned with a Zeiss Axioscan 7 microscope slide scanner (ZEISS) at 20x objective, and images were analyzed. For counts, total number of cells were counted in every 6th section to ensure the same neurons were not counted in two sections, and the sections counted spanned the hippocampus (Bregma coordinates −1.35 to −2.6; Malberg et al., 2000) For neuronal counts, NeuN positive cells in the pyramidal layers of CA1 and dentate gyrus were manually counted on ImageJ software. For glial cells, GFAP positive cells co-labeled with DAPI were counted manually using Zen 3.6 Blue software (ZEISS) in the dendritic layers of CA1 and molecular layers of dentate gyrus. All data are reported as the mean ± SEM.

Brains were collected and dissected into specific regions and then snap frozen (N = 20–25 per treatment group). For this study, we isolated total RNA from the hippocampus using the MirVana kit (ThermoFisher AM1560) according to the manufacturer’s instructions. RNA quality and concentration were determined using the Nano-Drop1000 Spectrophotometer (Thermo Fisher Scientific). RNA was treated with DNase I to remove genomic DNA and reverse transcribed into cDNA using iScript^™ cDNA Synthesis Kit (Biorad 1,725,035) according to the manufacturer’s instructions. Target gene mRNA levels were assessed by quantitative PCR using SsoAdvanced Universal SYBR Green Supermix master mix (Biorad 1,725,270) and the CFX Opus RT-PCR System. The primer sequences for genes are shown in Table 1. Each reaction contained either 50 or 100 ng of cDNA, 1.24 μL of forward and reverse primers, and 3 μL of SYBR and was run in triplicate. The cycling conditions were as follows: 95°C – 3 min, 95°C – 10s, 60°C – 15 s, 72°C – 15 s, repeat 44 times, 65°C – 5 s, 95°C – 0.5°C/cycle. Gene expression was normalized to the geometric mean of 3 housekeeping genes (HPRT, TBP, and PPIA). Relative expression of target genes was obtained using the 2ΔΔCT method (Livak and Schmittgen, 2001).

Statistical analyses were performed using Prism (v8.2) and Stata (v13). All behavioral and expression data analyses were performed stratified by sex, and means with SEM were calculated. Differences in mean with 95% confidence intervals between the control group and the ART arms were calculated using mixed effects linear models with robust standard errors that included treatment as a categorical fixed effect, and litter as a random variable to account for litter effects. Neuronal and glial count data are shown as violin plots with individual data points displayed. Differences were assessed using Student’s t-test. The Pearson r test was used to assess correlations. MRI data analysis was performed using the R statistical software¹ and the RMINC package.² For the adult brain MR images, sex was included as a fixed effect. Multiple comparisons were controlled for using the false discovery rate (FDR; Genovese NeuroImage 2002, 15:870–878) and statistical significance was defined at an FDR threshold of 1%.

Some studies suggest altered motor skills and increased levels of anxiety in CHEU (Chase et al., 2000; Wedderburn et al., 2019b, 2022a,b; Yao et al., 2023). We first assessed locomotor activity, anxiety, and exploration using the open-field test. Both male and female TDF/FTC + ATV/r-exposed mice showed increased distance traveled (Figure 2A, difference in mean from control for male +12.8 m p = 0.01, for female +8.1 m, p = 0.04) compared to control and ABC/3TC + ATV/r-exposed mice. Both male and female TDF/FTC + ATV/r exposed mice also showed increased mobile time compared to control and ABC/3TC + ATV/r, although this only reached significance for the males (Figure 2B, difference in mean from control for male TDF/FTC + ATV/r + 66.5 s, p = 0.01, for female +41.4 s, p = 0.07). When we adjusted for weight at postnatal day 3, the increase in mobile time seen in the TDF/FTC + ATV/r-exposed females did reach significance (Supplementary Table S2). No difference was observed in distance traveled between ABC/3TC + ATV/r-exposed mice and controls for either sex, although female ABC/3TC + ATV/r-exposed mice had a lower mobile time compared to controls that did not reach significance (Figure 2A). Time spent in the center and time spent by the wall (thigmotaxis zone) were similar between groups (Table 2; Supplementary Table S1). These data suggest an increased locomotor activity and exploratory drive with TDF/FTC + ATV/r, but not with ABC/3TC + ATV/r exposure.

We also assessed rearing time in the open field test, another indicator of locomotor activity, exploration, and anxiety. Increased rearing was observed in both male and female TDF/FTC + ATV/r-exposed mice compared to the other groups, again suggesting increased activity and reduced anxiety (Figure 2C, difference in mean from control for male TDF/FTC + ATV/r + 80.6 s, p < 0.001, for female +70.2 s, p < 0.001). Rearing time did not differ significantly between the ABC/3TC + ATV/r-exposed mice and controls.

Next, we assessed grooming in the open field. Grooming in rodents is a natural response to displacement which is often exhibited upon exposure to a novel environment. In a stressful environment with a high anxiety level, rodents tend to groom longer than usual (Estanislau, 2012; Kalueff et al., 2016; Liu et al., 2021). Both male and female ABC/3TC + ATV/r-exposed mice spent more time grooming compared to controls and TDF/FTC + ATV/r-exposed mice, although this only reached significance for the female mice (Figure 2D, difference in mean from control for male ABC/3TC + ATV/r + 16.6 s, p = 0.1, for female +32.6 s, p < 0.001), suggesting greater anxiety with ABC/3TC + ATV/r exposure. Supporting a phenotype of reduced anxiety, TDF/FTC + ATV/r-exposed mice had the lowest grooming times (Figure 2D, difference in mean from control for male TDF/FTC + ATV/r − 14.5 s, p = 0.04, for female −10.7 s, p = 0.02). The significant difference in the TDF/FTC + ATV/−exposed males was lost when we adjusted for weight at postnatal day 3 (Supplementary Table S2). As expected, we observed a strong correlation between rearing time and distance traveled (r = 0.57, p < 0.0001; Figure 2E). In support of increased grooming being an anxiety-related behavior, we observed a negative correlation between grooming time and distance traveled (r = −0.44, p < 0.0001; Figure 2F).

Next, we used the zero maze and light/dark box, two tests of exploratory and avoidance-related behaviors that are more sensitive to anxiety-related behavior (Hånell and Marklund, 2014; Thompson et al., 2018; La-Vu et al., 2020). Mice exposed to TDF/FTC + ATV/r spent more time in the light chamber of the light/dark chamber test, although this reached significance compared to controls only in the female mice (Figure 2G, difference in mean from control for male TDF/FTC + ATV/r + 20.8 s, p = 0.18, for female +33.0 s, p = 0.002). As predicted, male and female mice exposed to TDF/FTC + ATV/r spent more time in the open area of the zero maze compared to controls although this only reached significance for male mice (Figure 2H, difference in mean from control for male TDF/FTC + ATV/r + 21.1 s, p < 0.001, for female +11.9 s, p = 0.11). Male mice exposed to ABC/3TC + ATV/r also spent significantly more time in the open area compared to controls (mean difference from control for male ABC/3TC + ATV/r + 12.9 s, p = 0.036), although significance was lost when we adjusted for weight at postnatal day 3 (Supplementary Table S2).

Collectively our data suggest increased mobility and exploratory behavior, as well as indications of reduced anxiety or increased risk-taking behavior, in mice exposed to TDF/FTC + ATV/r.

Deficits in cognitive abilities have been reported in CHEU as compared to unexposed children, including learning deficits and mild cognitive impairment (Wedderburn et al., 2019a, 2022b; Yao et al., 2023). We first assessed hippocampal-dependent working memory, requiring neural circuits associated with the prefrontal cortex and medial temporal lobe (Frank et al., 2004; Chao et al., 2022; Voitov and Mrsic-Flogel, 2022). The novel object recognition test is a test of non-spatial, episodic memory that is independent of neuromotor deficits and emotional cues. A “memory index” was calculated as (T_N-T_F)/(T_N + T_F), wherein “T_N” represents time exploring a novel object and “T_F” the duration of familiar object exploration. A positive memory index indicates preference for the novel object, which is normal behavior for mice. As expected, the control mice spent statistically significantly more time exploring the novel object rather than the familiar object, with a mean memory index with SEM of 0.22 ± 0.03 for males and 0.19 ± 0.03 for females (Table 2). The time spent exploring the novel object, as well as memory index, were lower in ABC/3TC + ATV/r treatment group compared to controls for both males and females (Figures 3A,B), with the memory index being close to zero indicating failure to differentiate between the novel and familiar object (memory index with SEM for ABC/3TC + ATV/r: 0.036 ± 0.06 for male, −0.0003 ± 0.04 for female; Table 2). While time spent exploring the novel object was similar between the TDF/FTC + ATV/r group and control, total exploration time was significantly higher in mice exposed to TDF/FTC + ATV/r, which supports the open field results of hyperactivity (Figure 3C, difference in mean from control for male TDF/FTC + ATV/r + 55.7 s, p = 0.001, for female +86.7 s, p < 0.001). This also resulted in a lower memory index for both male and female mice exposed to TDF/FTC + ATV/r as compared to controls (Figure 3B), with the memory index being 0.11 ± 0.03 for males and 0.089 ± 0.03 for female mice (Table 2). These findings suggest an impairment in working/reference memory encoding in mice exposed to both regimens in utero.

We next investigated social memory in our mouse model using 3-chambered social approach test. Mice exposed to TDF/FTC + ATV/r showed reduced performance (lower sociability index) in the first task of the test, although this only reached significance for the female mice (Figure 3D, difference in mean from control for male TDF/FTC + ATV/r − 0.093, p = 0.09, for female −0.10, p = 0.008). ABC/3TC + ATV/r exposed females showed reduced social novelty (lower social novelty index) in the second task (Figure 3E, difference in mean from control for male ABC/3TC + ATV/r − 0.037, p = 0.56, for female −0.18, p = 0.005). Females exposed to TDF/FTC + ATV/r also had a lower social novelty index compared to controls although this did not reach significance (p = 0.08). Our data suggest that female mice may be more susceptible to sociability deficits due to ART exposure and that NRTI backbone can also influence responses.

With deficits observed in learning and memory functions of the hippocampus, we next investigated the effect of in utero ART exposure on the macroscopic morphology of the brain using magnetic resonance imaging (MRI). At a false discovery rate of 1%, we observed statistically significant reductions in total brain volume in the ABC/3TC + ATV/r group compared to controls. The decrease in total brain volume was not uniform across the brain, with significant brain volume decreases observed primarily in the cortex and thalamus (Figure 4A, center panel). When normalized to account for total brain volume, we also observed significant relative volume differences in brain regions of ABC/3TC + ATV/r exposed mice as compared to controls (Figure 4A, right panel). Of interest areas associated with memory, including the hippocampus, were significantly smaller in the ABC/3TC + ATV/r exposed mice vs. control. No differences were observed in TDF/FTC + ATV/r treatment group compared to controls (Figure 4A, left panel).

When further dissected into regions, ABC/3TC + ATV/r exposed mice showed significantly lower gray matter volume as compared to controls and TDF/FTC + ATV/r exposed mice (Figure 4B, difference in mean from control -17 mm³, p < 0.0001). The volume of the CA1 region of the hippocampus was also lower in ABC/3TC + ATV/r exposed mice as compared to controls (Figure 4C, difference in mean from control −0.0004mm³, p < 0.0001). Since there were no significant sex-by-group interactions, for these analyses we combined males and females. We observed a strong positive correlation between CA1 volume and memory index in ABC/3TC + ATV/r exposed mice, but not in the control or TDF/FTC + ATV/r exposed mice (Figures 4D–F).

Supporting the MRI findings of smaller hippocampal volume, immunostaining of paraffin-embedded fixed sections of the brain with the neuronal marker NeuN, showed a decreased count of pyramidal neurons in the CA1 and dentate gyrus granule cells of ABC/3TC + ATV/r exposed mice compared to controls (Figures 5A–C). We also investigated changes in the number of glial cells using glial fibrillary acidic protein (GFAP) as a marker, but observed no differences between treatment groups (Figures 5D,E). These results support an association between brain volumetric reductions, reduced neuronal counts, and memory deficit in mice exposed to ABC/3TC + ATV/r. In contrast TDF/FTC + ATV/r exposure was not associated with macroscopic brain volumetric differences.

We next investigated if there are molecular changes in the brain that could underly the observed behavior phenotypes in our model. Experience-dependent modification of neural circuits occurs through changes in synaptic transmission and synaptic plasticity (Malenka, 2003; Nicoll, 2003; Citri and Malenka, 2008; Südhof and Malenka, 2008). Two glutamate neurotransmitter receptors that drive synaptic plasticity in the hippocampus of the mammalian brain are amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) and N-methyl-D-aspartate receptor (NMDAR; Lüscher and Malenka, 2012; Penn et al., 2017). Further, postsynaptic density protein-95 (PSD95) is a major regulator of synaptic maturation by interacting, stabilizing, and trafficking NMDARs and AMPARs to the postsynaptic membrane in the neurons (Kornau et al., 1995; Schnell et al., 2002). In utero exposure to ART led to deficits in hippocampal-dependent working memory in our mouse model and increased exploration. Thus, we investigated changes in gene expression of major neurotransmitter receptors affecting memory encoding in the hippocampus. We identified lower mRNA expression of AMPAR subunits GluA1 and GluA2 (Figures 6A,B; Supplementary Table S1) in TDF/FTC + ATV/r exposed females and males, respectively. In contrast, we observed lower mRNA expression of the NMDAR subunits GluN2A and GluN2B in ABC/3TC + ATV/r exposed females (Figures 6C,D; Supplementary Table S1). Lower mRNA expression levels of the postsynaptic scaffolding protein PSD95 were also observed in ABC/3TC + ATV/r exposed females compared to controls (Figure 6E; Supplementary Table S1).

Next, we assessed potential neurotrophic factors that are associated with modulation of neurotransmitter receptors. Several studies have reported the role of neurotrophic signaling of brain-derived neurotrophic factor (BDNF) and its downstream modulatory effect on the expression of AMPARs (Martinez-Turrillas et al., 2002; Martínez-Turrillas et al., 2005; Jourdi et al., 2009; Autry and Monteggia, 2012; Keifer, 2022; Gliwińska et al., 2023). Not only does BDNF signaling promote the formation of new synapses and exert an effect on the pre-synapse, but it also plays a role in promoting synaptic plasticity in the post-synapse (Jourdi et al., 2009; Autry and Monteggia, 2012; Diering and Huganir, 2018; Park, 2018). Given the role of BDNF signaling in the brain, we assessed the expression of BDNF and its receptors, tyrosine kinase B (TrkB) full-length and TrkB truncated in the hippocampus. We observed statistically significantly higher mRNA levels of BDNF in ABC/3TC + ATV/r exposed males (Figure 6F; Supplementary Table S1). Further, we observed lower mRNA levels of the BDNF receptors TrkB full-length (both males and females) and TrkB truncated (males only) in TDF/FTC + ATV/r exposed mice compared to controls (Figures 6G,H; Supplementary Table S1). In contrast, we found upregulation of the TrkB truncated in ABC/3TC + ATV/r exposed females (Figure 6H; Supplementary Table S1). Thus, our findings suggest that in utero exposure to ART regimes can lead to an alteration in the receptors associated with synaptic plasticity and neuromodulator proteins in the hippocampal region of the brain.