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Published in final edited form as: Prog Neuropsychopharmacol Biol Psychiatry. 2019 Jul 10;95:109701. doi: 10.1016/j.pnpbp.2019.109701

Adolescent stress contributes to aberrant dopamine signaling in a heritable rodent model of susceptibility

Stephanie M Perez 1,*, Daniel J Lodge 1
PMCID: PMC6708463  NIHMSID: NIHMS1534901  PMID: 31299274

Abstract

Evidence suggests that both genetic and environmental factors contribute to the development of schizophrenia. Rodent models of the disorder have been developed that model either genetic or environment factors to recapitulate various aspects of the disease; however, the examination of gene by environment interactions requires a model of susceptibility. We have previously demonstrated that a proportion of the F2 generation of MAM-treated rats display a schizophrenia-like phenotype, defined as an increase in ventral tegmental area (VTA) dopamine neuron population activity. Here we use this model to examine the consequence of adolescent stress (AS), a known risk factor for psychiatric disease, on dopamine neuron activity in the VTA. Specifically, F2 MAM rats were exposed to predator odor, a stressor of high ethological relevance, intermittently over 10 days throughout the adolescent period and VTA dopamine neuron activity was evaluated in adulthood. Both saline and MAM F2 rats exposed to AS displayed significant increases in population activity; however, the proportion of F2 MAM rats exhibiting this increase was significantly greater (approximately 70 percent) compared to their respective controls. Given that we have previously demonstrated that the augmented dopamine neuron activity in rodent models of psychosis is directly attributable to aberrant activity in the ventral hippocampus (vHipp), we examined whether AS altered activity within the vHipp. Indeed, there was a positive correlation between dopamine neuron activity and hippocampal firing rates, such that those rats that displayed increases in population activity also had increases in the firing rates of vHipp putative pyramidal neurons. Taken together, these data further demonstrate a role for AS as a risk factor for psychosis, particularly in those with a heritable predisposition.

Keywords: Schizophrenia, MAM, F2 generation, Adolescent Stress, hippocampus

Introduction

Although there is significant evidence to support both genetic and environmental components in the development of schizophrenia, researchers have yet to pin point the exact etiology of this complex psychiatric disease (Tsuang and Faraone 1995, Brown and Derkits 2010, Gejman, Sanders et al. 2011, Greenwood, Swerdlow et al. 2013). No single gene has been identified as being a sole casual factor, instead, individuals with schizophrenia display genetic heterogeneity, and it is likely the cumulative interaction of environmental risk factors (i.e. psychosocial, biological, physical factors) combined with a genetic (or epigenetic) predisposition that leads to the development of the disease. In fact, one hypothesis of gene-environment interaction suggests that schizophrenia is a neurodevelopmental disease with a strong heritable component, whereby genes control embryonic neurodevelopment and are modified by the environment (Tsuang 2000, Howes, McDonald et al. 2004). Several neurodevelopmental rodent models of schizophrenia have been developed (i.e. maternal stress, maternal infection/immune challenge; for review see (Meyer and Feldon 2010)); however, there are few models available to study environmental factors on a heritable background.

The methylazoxymethanol acetate (MAM) developmental model of schizophrenia demonstrates both pathological and behavioral abnormalities consistent with those observed in individuals with schizophrenia (Flagstad, Mork et al. 2004, Moore, Jentsch et al. 2006, Lodge, Behrens et al. 2009). Studies have also shown that MAM-treated rats display aberrant activity in the ventral hippocampus (vHipp), which drives hyperfunction in ventral tegmental area (VTA) dopamine neurons (Lodge and Grace 2007), and is associated with psychosis in individuals with schizophrenia (Laruelle and Abi-Dargham 1999, Abi-Dargham 2004). Further, MAM-treated rats display epigenetic alterations that may be inherited by second filial (F2) and third filial (F3) generations (Perez, Aguilar et al. 2016, Neary, Perez et al. 2017, Aguilar, Giuffrida et al. 2018), suggesting an increased likelihood of developing a schizophrenia-like phenotype. Indeed, subpopulations of F2 and F3 generation rats (~40%) display increases in dopamine neuron activity and behavioral correlates of positive symptoms (Perez, Aguilar et al. 2016, Aguilar, Giuffrida et al. 2018). It is important to note that this is not a consequence of aberrant maternal care as offspring of F1 MAM-treated male and F1 saline-treated female rats display a similar phenotype, as do F2 MAM offspring that are cross-fostered by a control dam (Perez, Aguilar et al. 2016). In addition, we have recently reported that F2 MAM rats can model susceptibility and that adolescent cannabinoid exposure can increase the proportion of F2 MAM rats developing a schizophrenia-like phenotype (Aguilar, Giuffrida et al. 2018). Thus, F2 MAM rats represent a potentially novel model to examine environmental factors in rodents with a heritable predisposition.

Symptoms of schizophrenia commonly arise in late adolescence to early adulthood (Lieberman 2006), because of the timing of disease onset, adolescence presents itself as a critical period for schizophrenia (McGorry 2011, Gomes and Grace 2017). Environmental stressors, such as drug use and social stressors, can precipitate schizophrenia in adolescents with a genetic vulnerability (Di Maggio, Martinez et al. 2001, Miller, Lawrie et al. 2001, Read and Ross 2003). Similarly, F2 MAM rats exposed to synthetic cannabinoids are more likely to develop schizophrenia-like pathologies (Aguilar, Giuffrida et al. 2018). Here we examine a psychogenic stressor that can be modeled in rats by exposure to the odor of natural predators [i.e. fox and coyote (Dielenberg, Hunt et al. 2001, Dielenberg and McGregor 2001, Apfelbach, Blanchard et al. 2005)]. A major strength of this approach is the ecological relevance, whereby, the life or death circumstances associated with a predator can be chronically administered to produce an enduring model that recapitulates some aspects associated with psychological stress. Indeed, the exposure to predator odors has been previously reported to produce robust behavioral alterations in rodents indicative of extreme manifestations of anxiety and abnormal HPA axis regulation (Dielenberg, Hunt et al. 2001, Dielenberg and McGregor 2001, Apfelbach, Blanchard et al. 2005). Although there is a possibility that rats may habituate to the stressor, it has been demonstrated that even a single exposure to predator stress induces enduring behavioral and physiological abnormalities that can persist for up to three months after exposure (Cohen, Matar et al. 2006, Deslauriers, Toth et al. 2018). While the immediate effects of predator stress are not always persistent, and more accurately reflect acute effects associated with trauma, in the context of a susceptible model (such as the F2 MAM rat), we posit that exposure to predator odor will induce enduring alterations. Here, we investigate environmental interactions with a heritable predisposition in F2 MAM rats and posit that F2 generation MAM rats exposed to adolescent stress (AS) will have an increased likelihood of developing a schizophrenia-like hyperdopaminergic phenotype in adulthood.

Material and Methods

All experiments were performed in accordance with the guidelines outlined in the United States Public Health Service Guide for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee of UT Health San Antonio.

Animals

MAM treatments were performed as previously described (Moore, Jentsch et al. 2006, Lodge 2013). In brief, timed pregnant female rats were obtained from Envigo on gestational day (GD) 16 and injected with methylazoxymethanol acetate (MAM diluted in saline; 22mg/kg, i.p.) or saline (1mL/kg, i.p.) on GD17. Male first filial (F1) generation pups were weaned on postnatal (PN) day 21 and housed in groups of 2 to 3 until adulthood (> PN 60). Second filial (F2) generation rats were obtained by crossing F1 saline-treated (♂) x F1 saline-treated (♀) for F2 saline rats or F1 saline-treated (♂) x F1 MAM-treated (♀) for F2 MAM rats. Previous studies have demonstrated that mating female F1 MAM treated rats with a control male results in ~40% of the rats developing a hyperdopaminergic phenotype versus ~60% in offspring from a F1 MAM-treated father (Perez, Aguilar et al. 2016). As our a priori hypothesis was that AS would increase the proportion of rats developing aberrant dopamine signaling, we used offspring from female F1 MAM-treated rats, since they started at a lower baseline. All experiments were performed on multiple litters of male F2 MAM and F2 saline rats.

Stress Paradigm

Starting on PN day 42, F2 rats were exposed to AS (predator urine) for two hours each day for a total of 10 days. Rats were singly contained in a novel cage, where two predator urines (5 mL; fox and coyote) were applied to different sponges (reapplied twice weekly) and suspended from opposing ends of the cage. Control rats were not exposed to any predator urine but were taken to the same procedure room for two hours a day for the same period of 10 days.

In Vivo Extracellular Recordings

F2 MAM and saline rats (250-400 g), a minimum of two weeks post-AS, were anesthetized with 8 % chloral hydrate (400mg/kg, i.p.). Chloral hydrate is the preferred anesthetic for dopamine recordings, as this anesthetic does not significantly depress dopamine neuron activity (Hyland, Reynolds et al. 2002). Rats were then placed in a stereotaxic apparatus and a core body temperature of 37°C was maintained with a thermostatically controlled heating pad. Anesthesia was supplemented as required to maintain suppression of limb compression withdrawal reflex. Extracellular glass electrodes (impedance 6-10 MΩ) were lowered into the VTA (A/P −5.3 and M/L ±0.6mm from bregma; D/V −6.5 to −9.0 mm ventral of the brain surface) using a hydraulic micro-positioner (Model 640; Kopf Instruments; Tujunga, CA, USA). Spontaneously active dopamine neurons were identified using previously established electrophysiological criteria (Grace and Bunney 1983, Ungless and Grace 2012) with open filter settings (low frequency cut off: 30 Hz; high frequency cut off: 30 KHz). Spontaneously active dopamine neurons were recorded (for approximately 3 to 5 minutes) while making multiple ventral passes (typically 6-9), separated by 200 μm, in a predetermined pattern to sample various regions of the VTA. Three parameters of dopamine neuron activity were measured: (1) population activity, defined as the number of dopamine firing spontaneously (Lodge and Grace 2011) (2) basal firing rate and (3) the proportion of action potentials occurring in bursts.

To record putative pyramidal neurons in the vHipp, extracellular glass microelectrodes (impedance 6-14 MΩ) were lowered into the vHipp (A/P: −5.0 and M/L: ±4.5mm from bregma; D/V: −4.0 to −8.0 mm ventral of the brain surface) with a hydraulic micro-positioner. Putative pyramidal neurons were identified as reported previously (van der Meer and Redish 2011, Perez and Lodge 2013, Shah and Lodge 2013, Boley, Perez et al. 2014) and defined as those neurons with a firing frequency less than 2 Hz. Spontaneously active putative pyramidal neurons were isolated by making vertical passes (typically 4-6 separated by 200 μm) and recorded for approximately 3-5 minutes.

Histology

At the cessation of all experiments, rats were rapidly decapitated; brains were removed and fixed for a minimum of 24 hours (4% formaldehyde in saline) then cryoprotected (10% w/v sucrose in phosphate-buffered saline) until saturated. Brains were sectioned (25 μm coronal sections) on a cryostat (Leica; Buffalo Grove, IL, USA). Sections containing electrode placements were mounted onto gelatin-chrom alum-coated slides and stained with neutral red (0.1%) and thionin acetate (0.01%) for histological verification of electrode tracks with in the VTA and vHipp (Paxinos and Watson 1998).

Analysis

Electrophysiological analysis of dopamine neuron activity was performed with commercially available computer software (LabChart version 7.1; ADInstruments, Chalgrove, Oxfordshire, UK) and analyzed using Prism software (GraphPad Software; San Diego, CA, USA). Data are represented as the mean ± s.e.m. unless otherwise stated, with n values representing the number of rats per experimental group or the number of neurons per group where indicated. Electrophysiological data were primarily analyzed by two-way analysis of variance (ANOVA; prenatal treatment and stress as factors), followed by a Holm-Sidak post hoc test, with significance determined at P< 0.05. For data segregated by dopamine activity (less than or greater than 1.5 cells per track), a Kruskal-Wallis one-way ANOVA on ranks was performed (as data failed tests for normality and/or equal variance), followed by an all pairwise multiple comparison procedure (Dunn’s method). Data comparing proportions were analyzed by Marascuilo’s Procedure for Comparing Multiple Proportions.

Materials

MAM was purchased from Midwest Research Institute (Kansas City, MO, USA). Fox and coyote urine were obtained from the Predator Pee store (www.predatorpeestore.com). Chloral hydrate was purchased from Sigma-Aldrich (St. Louis, MO, USA). All other chemicals and reagents were of either analytical or laboratory grade and purchased from various suppliers.

Results

VTA Dopamine Neuron Extracellular Recordings

A consistent observation in F1 MAM rats (both male and female) is elevated VTA dopamine neuron population activity when compared to F1 saline control rats (Lodge and Grace 2007, Perez and Lodge 2013, Perez, Shah et al. 2013, Perez, Chen et al. 2014). Additionally, a proportion (typically ~40%) of F2 MAM rats also display an augmented dopamine system function (Perez, Aguilar et al. 2016, Aguilar, Giuffrida et al. 2018). To investigate whether F2 MAM rats exposed to AS will have an increased susceptibility to developing aberrant dopamine neuron activity, we recorded spontaneously active dopamine neurons from F2 MAM and F2 saline rats exposed to predator urine during adolescence (Figure 1A). Indeed, F2 MAM controls (1.32 ± 0.11 cells per track) displayed a population activity that is significantly higher than F2 saline control rats (0.99 ± 0.04 cells per track; two-way ANOVA; FMAM(1,70)= 13.07; FStress(1,70)= 7.74; FInteraction(1,70)= 0.26; Holm-Sidak; P= 0.042; t= 2.07). Additionally, F2 MAM rats exposed to AS displayed a significantly higher population activity (1.66 ± 0.14 cells per track) when compared to F2 saline rats exposed to the same AS (1.23 ± 0.11 cells per track; Holm-Sidak; P= 0.003; t= 3.12) and F2 MAM control rats (Holm-Sidak; P= 0.017; t= 2.45). No significant differences were observed in the firing rates (Figure 1B) or percent bursting (Figure 1C) between any of the groups.

Figure 1.

Figure 1.

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Offspring born from methylazoxymethanol acetate (MAM)-treated parents exhibit enhanced ventral tegmental area (VTA) dopamine neuron activity. Similarly, F2 MAM rats exposed to adolescent stress (AS) also display aberrant dopamine neuron population activity; however, this increase in population activity is significantly higher in those F2 MAM rats not exposed to AS (A). No differences were observed in the firing rate (B) and percent bursting (C) of dopamine neurons between any of the groups. F2 saline control: n= 14 rats, 80 neurons; F2 MAM control: n= 17 rats, 125 neurons; F2 saline AS: n= 18 rats, 132 neurons; F2 MAM AS: n= 22 rats, 204 neurons. *represents significant difference from respective F2 saline P< 0.05. #denotes significance from F2 MAM control P= 0.017.

Previous studies performed in F2 MAM rats revealed a clear bimodal distribution of dopamine neuron activity (Perez, Aguilar et al. 2016, Aguilar, Giuffrida et al. 2018). In this study, further analysis of VTA dopamine neuron activity again revealed a bimodal distribution with an R2 of 0.9420 (Figure 2A). Thus, we identified two distinct groups of offspring: those that inherited (≥1.5 cells per track) and those that did not inherit the (<1.5 cells per track) the schizophrenia-like phenotype (elevated VTA dopamine neuron population activity). The percent of F2 rats that display ≥1.5 cells per track was increased in both F2 saline and F2 MAM rats when exposed to AS (Figure 2B; Chi Square: 17.30; df= 3; α= 0.0006). To evaluate if F2 generation rats with ≥1.5 cells per track were significantly higher than their respective <1.5 cells per track groups we performed a Kruskal-Wallis one-way ANOVA on Ranks followed by an all pairwise multiple comparison using the Dunn’s Methods (H= 51.92 with 6 degrees of freedom; P< 0.001; Figure 2C). Significance was observed between F2 saline AS <1.5 cells per track and ≥1.5 cells per track (Q= 3.36; P< 0.05), and F2 MAM AS <1.5 cells per track and ≥1.5 cells per track (Q= 4.11; P< 0.05).

Figure 2.

Figure 2.

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A subset of F2 generation rats displays an augmented dopamine neuron population activity. Further examination of the data revealed a bimodal distribution, with an R2 of 0.94, such that rats displayed a population activity of either <1.5 or ≥1.5 cells per track (A). The percentage of F2 generation rats exhibiting a population activity ≥1.5 cells per track is presented in panel (B) and the population activities for each subset is depicted in (C;F2 saline control <1.5: n= 14 rats; F2 MAM control <1.5: n= 11 rats; F2 MAM control ≥1.5: n= 6 rats; F2 saline AS <1.5: n= 12 rats; F2 saline AS ≥1.5: n= 6 rats; F2 MAM AS <1.5: n= 7 rats; F2 MAM AS ≥1.5: n= 15 rats; *represents significant difference from respective <1.5 cells per track population activity; P< 0.05.

Putative Pyramidal Neuron Extracellular Recordings

Augmented VTA dopamine neuron population activity and corresponding hyperactivity in the vHipp has been observed in MAM-treated rats (Lodge and Grace 2007, Perez and Lodge 2013). To examine whether this vHipp hyperactivity is also observed in F2 MAM rats, and if it is affected by AS, we recorded the firing rates of spontaneously active putative pyramidal neurons. The average firing rate of putative pyramidal neurons of the vHipp in control F2 MAM rats was significantly higher than F2 saline rats (Figure 3A; F2 saline control: 0.63Hz ± 0.010; F2 MAM control: 0.91Hz ± 0.08; two way-ANOVA; Holm-Sidak post-hoc; P= 0.022; t= 2.31). No differences were observed between the AS groups. Further analysis was performed on F2 rats as previously mentioned, based on dopamine neuron population activity (<1.5 or ≥1.5 cells per track; Figure 3B; Kruskal-Wallis one-way ANOVA on ranks; H= 17.09 with 6 degrees of freedom; P= 0.009). F2 MAM rats with ≥1.5 cell per track exhibited a vHipp frequency that was significantly higher than F2 MAM rats exposed to AS with <1.5 cell per track (Dunn’s method; Q= 3.24; P< 0.05). No significant differences were observed in the F2 saline rats exposed to AS. Additionally, a significant correlation exists between the putative pyramidal neurons of the vHipp (Frequency in Hz) and VTA dopamine neuron population activity (n= 38 rats; R2= 0.1615; P= 0.0124), such that those rats that displayed an increase in population activity also had increase in the firing rate of vHipp putative pyramidal neurons (Figure 3C). Interestingly, when we compared vHipp firing rates from rats that inherited the schizophrenia-like phenotype (population activity ≥1.5 cells per track) to those that did not inherit the phenotype (population activity <1.5 cells per track), we found that those with the phenotype displayed a significantly higher vHipp activity (Kruskal-Wallis one-way ANOVA on ranks; H= 8.18 with 1 degrees of freedom; P= 0.004; Figure 3D).

Figure 3.

Figure 3.

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F2 MAM control rats exhibit an average ventral hippocampal (vHipp) firing frequency that is significantly higher than F2 saline control rats (A; *P= 0.022; F2 saline control: n= 36 neurons; F2 MAM control: n= 48 neurons; F2 saline AS: n= 53 neurons; F2 MAM AS: n= 88 neurons). F2 generation rats were separated based on dopamine neuron population activity (<1.5 or ≥1.5 cells per track) and vHipp firing frequency was presented (B; *represents significant difference from F2 saline control <1.5 cells per track population activity; P< 0.05; F2 saline control <1.5: n= 36 neurons; F2 MAM control <1.5: n= 36 neurons; F2 MAM control ≥1.5: n= 12 neurons; F2 saline AS <1.5: n= 40 neurons; F2 saline AS ≥1.5: n= 13 neurons; F2 MAM AS <1.5: n= 17 neurons; F2 MAM AS ≥1.5: n= 71 neurons). There is a correlation between vHipp frequency and the population activity of ventral tegmental area (VTA) dopamine neuron population activity (C; R2= 0.1615; P= 0.0124) such that increases in vHipp putative pyramidal neuron firing frequency correlates with increases in dopamine neuron cells per track. F2 generation rats that display the schizophrenia-like phenotype (≥1.5 cells per track; n= 129 neurons) display vHipp frequencies that are significantly higher than those that do not display the phenotype (<1.5cells per track, D; n= 96 neurons; *P< 0.05).

Discussion

Research involving families of patients with schizophrenia, including fraternal and identical twin studies, support a familial or genetic component to schizophrenia (Tsuang 1991, Kendler, McGuire et al. 1993, Cardno, Marshall et al. 1999, Tsuang 2000). Although a single gene has not been implicated as the sole contributing factor to the development of the disease, at least 20 genes have been linked to schizophrenia (Bergen and Petryshen 2012). Further, possessing a mutation or disruption in one of the candidate genes does not guarantee an individual will develop schizophrenia, but instead, they may have a higher risk (Tsuang, Gilbertson et al. 1991, Tsuang 2000). Twin studies provide evidence in support of a role for environmental factors, as monozygotic twins (with identical genetic makeup) who are exposed to different environments may cause schizophrenia to develop in one twin while rendering the other twin unaffected (Cardno and Gottesman 2000, Selemon and Zecevic 2015). Thus, when exposed to various prenatal and perinatal factors, schizophrenia may be triggered in susceptible individuals (Weinberger 1987, Sullivan, Kendler et al. 2003).

The typical age of onset of schizophrenia is generally in late adolescence, which also reflects a critical time in brain development, and thus presents itself as a vulnerable period (Hafner, Maurer et al. 1994, Gogtay, Vyas et al. 2011, Selemon and Zecevic 2015). Causative factors associated with the onset of psychosis include social and psychological factors (i.e. ethnicity, urban versus rural upbringing, childhood trauma, social isolation), as well as, cannabis or substance abuse (Marcelis, Takei et al. 1999, Hall and Degenhardt 2000, Boydell, van Os et al. 2001, Miller, Lawrie et al. 2001, van Os, Bak et al. 2002). Pre-clinical studies support these data, as changes in dopamine system function, a phenotype commonly observed in schizophrenia, has been associated with social isolation in rodents (Hall, Wilkinson et al. 1998, Hall, Wilkinson et al. 1999). Schizophrenia patients display an augmented dopamine system function (Laruelle and Abi-Dargham 1999, Abi-Dargham 2004), which can be assessed in rodent models of the disease by directly recording spontaneous dopamine neuron activity in the VTA. Positive symptoms of the disease are typically associated with aberrant dopamine system function (Schobel, Lewandowski et al. 2009); however, no observable pathology has been identified within the dopamine system, indicating that it is actually the regulation of this system that is dysfunctional (Lodge and Grace 2007). Individuals with schizophrenia and rodent models of the disease display hippocampal hyperactivity, which has been shown to drive aberrant dopamine system function (Heckers, Rauch et al. 1998, Floresco, Todd et al. 2001, Lodge and Grace 2007, Malaspina, Schobel et al. 2008, Schobel, Lewandowski et al. 2009).

An array of phenotypes that are commonly observed in individuals with schizophrenia can be modeled in rodents who receive the mitotoxin, MAM, during gestation for review see (Lodge and Grace 2009)). The mechanism of action of MAM has not been conclusively demonstrated, thus, we do not know exactly how the schizophrenia-like phenotype (increased dopamine neuron population activity) is produced. Alterations associated with cellular development, morphology, signaling, as well as, bidirectional changes in the DNA methylation of promoters was observed in the brain tissue of these rats, indicating epigenetic modifications (Perez, Aguilar et al. 2016). A schizophrenia-like phenotype has been observed in F1 MAM rats, and epigenetic modifications are inherited by the F2 and F3 generations (Perez, Aguilar et al. 2016, Aguilar, Giuffrida et al. 2018). Specifically, subpopulations of F2 and F3 MAM rats display increases in dopamine neuron population activity (Perez, Aguilar et al. 2016). Because F2 MAM rats possess epigenetic alterations, as a consequence of MAM administration (Perez, Aguilar et al. 2016), and a heritable predisposition to the development of aberrant dopamine system function, (Aguilar, Giuffrida et al. 2018), we hypothesized that the number of rats that exhibit a schizophrenia-like dopaminergic phenotype would increase in rats exposed to an adolescent stressor (predator urine). Indeed, Grace and colleagues (Gomes and Grace 2017, Gomes and Grace 2017) have demonstrated that decreasing stress during adolescence can reverse behavioral deficits in the F1 MAM model. Further, we have previously shown that the proportion of rats that exhibit this schizophrenia-like phenotype can be increased when exposed to cannabis during adolescence (Aguilar, Giuffrida et al. 2018). Similarly, in this study we show the same increase in VTA population activity in F2 MAM control rats, and in F2 MAM rats exposed to AS (Figure 1A). This increase in dopamine neuron activity is consistent with imaging studies in schizophrenia patients who display an augmented dopamine system function (Laruelle and Abi-Dargham 1999, Abi-Dargham 2004). It should be noted that F2 MAM rats do not follow a standard pattern of Mendelian inheritance, rather they demonstrate a pattern of susceptibility to inherit the schizophrenia-like pathology, and the likelihood of developing this can be increased by exposing the rats to an environmental insult. Indeed, consistent with previous studies only ~40% of F2 MAM rats display an altered dopamine neuron phenotype (Figure 2B) (Perez, Aguilar et al. 2016, Aguilar, Giuffrida et al. 2018). It is important to note that additional behavioral studies are needed to determine the consequences of this increased dopamine neuron activity. Further, we show that ~40% of F2 saline rats exposed to AS display aberrant dopamine neuron activity, thus, it is possible that the effects we observed were additive in nature, and not due to a synergistic relationship between heritable predisposition and AS. Exposing control rats to a subthreshold stressor would likely answer this question; however, a different stressor would have to be chosen, as even a single exposure to a psychological stressor, such as the predator urine chosen can induce robust and lasting effects (Cohen, Matar et al. 2006, Deslauriers, Toth et al. 2018). In humans, aberrant dopamine signaling has been linked to psychosis (Schobel, Lewandowski et al. 2009), as well as, alterations in sensorimotor gating (Abi-Dargham 2004, Swerdlow, Light et al. 2014, Kesby, Eyles et al. 2018). Whether similar behavioral alterations are present following AS in this model remains to be established. Nonetheless, these data confirm our previous work demonstrating that a heritable predisposition in the F2 MAM rat can be exacerbated by AS. This provides a novel, heritable model of susceptibility, whereby exposure to AS (predator urine) or cannabinoids (Aguilar, Giuffrida et al. 2018), increases the proportion of F2 MAM rats developing a hyperdopaminergic phenotype to about 70% percent.

As mentioned previously, aberrant hippocampal activity has been shown to drive dysfunction in the dopamine system (Lodge and Grace 2007). We observed an increase in the average firing rate of putative pyramidal neurons of the vHipp, specifically in F2 MAM rats (Figure 3A). However, separating F2 rats based on the bimodal distribution of dopamine neuron population activity, F2 rats that had ≥1.5 cells per track also displayed higher vHipp firing rates (Figure 3B). Furthermore, the firing frequency of vHipp neurons is correlated with the population activity of VTA dopamine neurons, such that more activity in the vHipp leads to a subsequent increase in dopamine neuron activity (Figure 3C), which is consistent with human imaging data demonstrating hyperactivity of hippocampal subfields that is correlated with psychosis in schizophrenia patients (Schobel, Lewandowski et al. 2009). It is interesting to note that the magnitude of this increase was attenuated in those F2 MAM rats exposed to AS. This is consistent with alterations in hippocampal activity observed in patients with PTSD (Bremner, Vythilingam et al. 2003, Francati, Vermetten et al. 2007), as well as, rodent models of predator-stress (Mesches, Fleshner et al. 1999, Borghans and Homberg 2015, Deslauriers, Toth et al. 2018). Additional studies are required to clarify the physiological and/or circuit level hippocampal alterations induced by AS in this model with a heritable predisposition.

Taken together, these data provide evidence that F2 MAM rats, which possess a heritable predisposition to inherit a schizophrenia-like pathology, are susceptible to AS. Additionally, adolescence is critical period in the development of schizophrenia (Gomes and Grace 2017) and models such as the F2 MAM model of heritable susceptibility are essential to mechanistically evaluate how genes interact with environmental alterations at different time points of development and in adulthood.

Highlights.

  • Adolescent stress increases the number of rats with a schizophrenia-like phenotype

  • The number of spontaneously activity dopamine neurons is specifically altered

  • A positive correlation exists for aberrant dopamine neuron and hippocampal activity

  • Adolescent stress may be a risk for psychosis in those with a genetic predisposition

Acknowledgements

We thank Angela Boley and David Aguilar for valuable assistance with the stress paradigm.

Funding and Disclosure

This work was supported by an RO1 (MH090067) from the NIMH (DJL) and a San Antonio Life Science Institute (SALSI) Postdoctoral Scholar Fellowship (SMP).

Footnotes

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