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Anhedonic behavior and γ-amino butyric acid during a sensitive period in female rats exposed to early adversity
Journal of Psychiatric Research 100 (2018) 8–15
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Anhedonic behavior and γ-amino butyric acid during a sensitive period in female rats exposed to early adversity
T
Jodi L. Lukkesa,b,1, Shirisha Medaa, Kevin J. Normana, Susan L. Andersena,b,∗ a b
Laboratory for Developmental Neuropharmacology, McLean Hospital, MA, 02478, United States Harvard Medical School, United States
A R T I C L E I N F O
A B S T R A C T
Keywords: Adolescence Amygdala Depression Helplessness Parvalbumin Prefrontal cortex sensitive period
Early life adversity increases depressive behavior that emerges during adolescence. Sensitive periods have been associated with fewer GABAergic interneurons, especially parvalbumin (PV), brain derived growth factor, and its receptor, TrkB. Here, maternal separation (MS) and social isolation (ISO) were used to establish a sensitive period for anhedonic depression using the learned helplessness (LH) paradigm. Female Sprague-Dawley rat pups underwent MS for 4-h/day or received typical care (CON) between postnatal days 2–20; for the ISO condition, separate cohorts were individually housed between days 20–40 or served as controls (CON2). Anhedonia was defined by dichotomizing subjects into two groups based on one standard deviation of the mean number of escapes for the CON group (< 14). This approach categorized 22% of CON subjects and 44% of MS subjects as anhedonic (p < 0.05), similar to the prevalence in maltreated human populations. Only 12.5% of ISO rats met criterion versus 28.5% in CON2 rats. Levels of PV and TrkB were reduced in the amygdala and prelimbic prefrontal cortex (PFC) in MS rats with < 14 escapes, but elevated in behaviorally resilient MS rats (> 13 escapes). The number of escapes in MS subjects significantly correlated with PV and TrkB levels (PFC: r = 0.93 and 0.91 and amygdala: r = 0.63 and 0.81, respectively; n = 9), but not in CON/ISO/CON2 subjects. Calretinin, but not calbindin, was elevated in the amygdala of MS subjects. These data suggest that low levels of PV and TrkB double the risk for anhedonia in females with an MS history compared to normal adolescent females.
1. Introduction Exposure to early life adversity (ELA) in the form of physical/sexual abuse, neglect, loss of a parent or caregiver or a natural disaster affects approximately 33% of the population. The lifetime prevalence rate of depression in an ELA-exposed population is 67% (Andersen, 2015; Andersen and Teicher, 2008; Teicher et al., 2009; Widom et al., 2007) compared to 20% in the general population. Elevated emotionality has been associated with increased amygdala activity in children younger than 11 years old with an ELA history (Malter Cohen et al., 2013; Marusak et al., 2015; Tottenham et al., 2011). More mature connectivity (e.g., resting state) between the amygdala and frontal cortex following ELA has also been described (Gee et al., 2013). In humans, elevated depressive/anhedonic behavior in adults and adolescents has been associated with less GABA in the PFC (Gabbay et al., 2012; Sanacora et al., 1999). However, the relationship of ELA, GABA, and depression during adolescence in animals on an individual basis is not known. During the preteen years, abused and non-abused children begin to ∗
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diverge in developmental trajectories (Widom et al., 2007). Depression emerges earlier than the general population, and coincides with increased prevalence of depression during a sensitive period of adolescent development (Andersen, 2015; Andersen and Teicher, 2008). A sensitive period is defined as a maturational stage when experience can exert maximal effect on development, but is not as necessary as it is during a critical period (Andersen, 2003; Greenough et al., 1987). Critical periods have been defined by changes in growth factors, such as brainderived growth factor (BDNF) and its receptor, tropomyosin receptor kinase B (TrkB), and the GABAergic interneuron that expresses the calcium-binding protein of parvalbumin (PV) (Huang et al., 1999; Morishita et al., 2015). While critical and sensitive periods are defined differently, sensitive periods of affective development may utilize the same underlying mechanisms. The sensitive period for the amygdala has been characterized as starting during the second week of postnatal (P) development in the rodent and ending at postnatal day (P)25 (Gogolla et al., 2009). Indeed, rats exposed to the ethologically relevant rodent model of maternal separation (MS) (Andersen, 2015; Lehmann and Feldon, 2000) show an increase in PV in the basolateral amygdala
Corresponding author. 115 Mill Street, Mailstop 333, Belmont, MA, 02478, United States. E-mail address: [email protected] (S.L. Andersen). Department of Psychiatry, Indiana University School of Medicine, Indianapolis, IN, United States.
https://doi.org/10.1016/j.jpsychires.2018.02.005 Received 4 November 2017; Received in revised form 22 December 2017; Accepted 8 February 2018 0022-3956/ © 2018 Elsevier Ltd. All rights reserved.
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day 16 of gestation. The day of birth was designated as P0. One day after birth, litters were culled to 10 pups (5 males and 5 females), and randomly assigned to either a MS or animal facility reared control group (CON). Only one pup per litter was assigned to a single condition. We acknowledge that the stress of shipping may have influenced our findings (Ogawa et al., 2007), but was minimized by distributing subjects both CON and MS rats equally across all conditions. Maternal separation: Pups in the MS group (n = 9) were isolated from their peers and mother for 4 h per day between P2 and P20, and kept at a thermo neutral temperature, following our methodology (Andersen, 2015; Brenhouse and Andersen, 2011; Leussis et al., 2012) and others (Plotsky and Meaney, 1993). Pups in the CON (n = 18) group were not disturbed after day two except for routine weekly cage changes, during which pups were weighed. A small, but significant, reduction in weight occurs in MS animals relative to CON during early development (Freund et al., 2013). Rats were housed with food and water available ad libitum in constant temperature and humidity conditions on a 12-h light/dark cycle (light period 07:00–19:00). Rats were weaned on P21, and group-housed in same-sex caging in male- or female-only vivarium until behavioral testing. Only females were used in this study. Social isolation: Rats were born and housed at McLean, as described above. Between P21-40, subjects were individually housed within the vivarium (ISO; n = 7); only females were used and were housed in a female-only room. No other manipulation occurred during this time. The controls (CON2; n = 7) were group housed (3/cage) under the same conditions. The experimental timeline is outlined in Fig. 1A. These experiments were conducted in accordance with the 2011 Guide for the Care and Use of Laboratory Animals (Committee for the Update of the Guide for the Care and Use of Laboratory Animals, 2011), and were approved by the Institutional Animal Care and Use Committee at McLean Hospital. Learned helplessness: In an initial cohort, CON (n = 9) and MS females (n = 9) were tested for depressive-like behavior on P40 and P41. Our previous studies have shown that females exposed to MS exhibited increased escape latency in the no shock (NS) condition (Leussis et al., 2012; Maier and Watkins, 2005; Pryce et al., 2011), which is associated with motivational deficits (Pryce et al., 2011). Females were gently restrained in the testing apparatus on Day 1 while a rat underwent the escapable shock (ES) condition of LH in their presence. This exposure affects escape behavior relative to rats that were not exposed to an ES rat on Day 1 (Lukkes et al., 2017). On Day 2, rats were individually tested in the shuttle box for 30 trials. Subjects could terminate a one mA foot-shock by shuttling to the other side for trials 1–5, or by shuttling to the other side and back again for trials 6–30. This response was cued by a tone that preceded the shock by 2 s. The shock remained on for 30 s, or until terminated by escape. The number of escapes was measured for trials 6–30. A second cohort of n = 9 females was added to the CON condition to increase our sample size for behavior only to increase statistical power to determine the prevalence of anhedonia in a typical population. These subjects did not differ from the first CON cohort on any measure. Western Immunoblots: Ninety minutes following the onset of behavioral testing on Day 2, animals were decapitated and tissue was regionally dissected in the amygdala and PFC and stored at −80 °C until processed. Tissue was homogenized in 1% sodium dodecyl sulfate (SDS) solution containing a protease inhibitor cocktail (Pierce, Rockford, IL). Protein concentration was determined by the Bradford method (BioRad Laboratories, Hercules, CA; Bradford, 1976). Proteins from the amygdala and PFC were analyzed for PV, CB, CR, and TrkB with each condition represented on a single blot for the CON/MS cohort of subjects. This approach minimized variability for the correlational analyses. The second cohort CON2/ISO was run on a single blot together for PV and TrkB. Eighty μg of protein was mixed in 6× SDS, centrifuged, and boiled for 3 min prior to separation by 15% SDS-PAGE. Proteins were then transferred to a nitrocellulose membrane (Bio-Rad
(BLA) by P35 (Giachino et al., 2007), consistent with an end to the sensitive period. Defining the sensitive period in the PFC has not received as much attention as the amygdala. Levels of PFC PV are low during juvenility and rise during adolescence into adulthood in normal rats (Brenhouse and Andersen, 2011; Caballero et al., 2014; Leussis et al., 2012). These PV data suggest a closing of the PFC sensitive period during adolescence. An early and protracted sensitive period in individuals with a stress history (Andersen and Teicher, 2008) implies that the brain remains highly sensitive to environmental stimuli, and thus, vulnerable during adolescence and even into adulthood. To better determine whether a sensitive period is present for anhedonia, the current study compared MS to the social isolation (ISO (Lukkes et al., 2009a);) as each of these stressor occur at different developmental stages. Comparison of these two 20-day manipulations may shed light on the role of sensitive periods in depression. Depressive and GABA changes are found in MS rats. MS increases learned helplessness (LH) and impaired working memory in adolescent rats (Brenhouse and Andersen, 2011; Leussis et al., 2012; Matthews and Robbins, 2003). GABA-A receptors are reduced in the amygdala of rats with a history of MS (Caldji et al., 2003), which is likely to lower GABAergic activity. Lower GABA levels or GABAergic activity in the amygdala is associated with increased anxiety and depressive-like behavior (Caldji et al., 1998; Raineki et al., 2012; Ritov et al., 2016). Finally, human post-mortem studies found low amygdala BDNF and GABA mRNA in depressed women (Guilloux et al., 2012). Reduced GABA activity in the amygdala during development can affect cortical development (Berretta and Benes, 2006). Together, these findings support a role for amygdala GABA in affective dysfunction, but has neither established the relationship between the loss of GABA in the amygdala as a direct consequence of MS nor its influence on behavior. Significant decreases in PFC PV-immunoreactive interneurons (counted by stereology) or PV expression (Western immunoblot) have been described during adolescence following MS between P2-20 days of age (Brenhouse and Andersen, 2011; Leussis et al., 2012). Sex differences in PV loss following MS occur earlier in females than males (Holland et al., 2014), but PV loss secondary to MS has been found in both sexes in adolescence (Leussis et al., 2012). Notably, one study of MS failed to find changes in PV in octus degus, a precocial rodent species (Seidel et al., 2008). Relatively little data is available on GABA in the cortex of ISO rats. Cortical chandelier cells (some expressing PV) decreased in males that underwent ISO (P28-84) (Bloomfield et al., 2008). However, magnetic resonance spectroscopy reported no differences in GABA levels between controls and ISO rats (Napolitano et al., 2014). The current study examined whether a sensitive period of ELA exists for anhedonic depression by comparing behavior, PV, and TrkB in MS and ISO rats during adolescence. As the prevalence of anhedonia was affected by MS, and by ISO, changes in other GABAergic interneurons that express calbindin (CB) and calretinin (CR) were only investigated in MS subjects. There is less evidence supporting their involvement in mood disorders (Sibille et al., 2011), even though the expression of CB changes during adolescence, but CR expression remains steady (Caballero et al., 2014). BDNF levels in cortical cell culture differentially modulated CB and CR (Fiumelli et al., 2000), raising the possibility that MS exposure will effect their expression. To better understand sensitive period-related changes, the inter-relationships between these GABAergic markers, TrkB, and depressive-like behavior were examined in the amygdala and the PFC across two different manipulations. 2. Methods and materials 2.1. Subjects Pregnant female multiparous Sprague-Dawley rats (250–275 g) were obtained from Charles River Laboratories (Wilmington, MA) on 9
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markers (PV, CR, CB, and TrkB) and the number of escapes in the CON/ MS group. All correlation data were subject to bootstrap analysis with 10,000 random resampling analyses; data were considered significant following this analysis if they met the adjusted P values (P < 0.05). The relationships between CON versus MS were then statistically compared with Fisher's z transformation followed by Bonferroni correction to determine if they were significantly different. Post-hoc analyses were adjusted with Bonferroni correction. Significance was set at P < 0.05. There were too few depressive subjects in the CON2 or ISO groups to perform correlational analyses on these data. 3. Results Dichotomized data analysis: Prior to dichotomization, rats in the MS group had an average number of 16 ± 2.9 escapes and CONs had 19.3 ± 1.9, escapes, which was not statistically different (P > 0.2). Isolation rats had an average of 19.6 ± 3.4 versus CON2 rats with 20.3 ± 3.6, and were not different (P > 0.5). Helplessness data were dichotomized based on one standard deviation from the mean (Ritov et al., 2016). As the average of the CON group was 20 ± 7 (1 SD), we used < 14 escapes as our cut-off to define anhedonia. Subjects with < 14 escapes were considered anhedonic and > 13 escapes considered non-anhedonic (Fig. 1B). Significant differences were found between the number of depressive versus non-depressive subjects overall (Mann-Whitney U, P < 0.001). The percentage of subjects who met criteria within each group are shown in Fig. 1C. Those meeting criterion were n = 4 of 18 and 4 of 9, for CON and MS respectively. The ISO group had a different profile. An n = 2 of 7 and 1 of 7 for CON2 and ISO, respectively, were anhedonic. Protein levels of PV and TrkB were dichotomized based on the number of escapes described above (Fig. 2A and B). In the amygdala, escape number significantly identified different levels of protein expression for both PV and TrkB in MS subjects (Kruskal-Wallis: P < 0.05 for both), but scores did not distinguish the data further between CON and MS within each dichotomized group (Fig. 2A). In the PFC, both PV and TrkB were significantly different in MS subjects between non-depressive and depressive groups; TrkB differed by escape number between CON and MS subjects in > 13 escapes (Kruskal-Wallis: P < 0.01). Individual comparisons were determined with a Dunn's test, which revealed that TrkB was significantly higher in MS subjects relative to the CON subjects in the non-depressive like group (P < 0.02). In CON2 and ISO animals, PV was significantly reduced in the < 14 depressive group in amygdala only. Correlational analysis: Since we found overall changes in PV expression levels in the amygdala, we investigated GABA's interactions with depressive behavior further by including other markers of GABA expression, such as CB and CR for the CON/MS groups. As the CON2 and ISO data are highly polarized between low and high escape values, this analysis is invalid. Dichotomizing the data minimizes individual differences that may offer additional insight into how GABA markers (PV, CB, and CR) and TrkB influence escape behavior. In Fig. 3A (top), Pearson's correlation with bootstrap adjustment indicated that MS subjects show a positive relationship with the number of escapes and PV expression in the amygdala (r = 0.63, P < 0.05), whereas the relationship was weaker in CON subjects, r = 0.36, P > 0.05. In the PFC, the correlation between escape number and PV was r = 0.14 (NS) in CON subjects. However, the strong relationship (r = 0.93, P < 0.001) in MS subjects indicates that fewer escapes were highly correlated with lower PV expression in this region. The correlations between the number of escapes and TrkB were similar to PV relationships. In the amygdala, higher levels of TrkB were associated with a less depressive phenotype (increased escapes); this relationship is significant in the MS group (r = 0.81; P < 0.01), but not the CON group (r = 0.41; P > 0.1). TrkB, however, was very distinctively associated with depressive scores in the MS group only (r = 0.91, P > 0.01) with no relationship in the CON group (r = −0.05; P > 0.6) in the PFC. Fig. 3
Fig. 1. Experimental design and prevalence of anhedonia. A) Timeline of treatments; LH: learned helplessness; P: postnatal day; maternal separation (MS), Control (CON) and social isolation (ISO) and CON2, the control group for ISO; B) Subjects were dichotomized based on one standard deviation from the number of escapes in the CON or CON2 group, for MS and ISO, respectively with < 14 suggesting anhedonia. Means ± SE presented, with the n for each group included; **P < 0.01. C) A pie chart represents the percentage of rats with a non-anhedonic phenotype and an anhedonic phenotype within the (left) CON group (n = 18) and the MS group (n = 9) and the (right) CON2 (n = 7) and ISO group (n = 7).
Laboratories), blocked with Odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE) in PBS for 60 min at room temperature, and incubated with primary polyclonal antibodies to PV (14–17 kDa; 1:10,000; Thermo Scientific, Rockford, IL), CB (28 kDa; 1:3000; Sigma), CR (31 kDa; 1:1000; Millipore), or TrkB (140 kDa; 1:1000; Cell Signaling Technology) in Odyssey blocking buffer (LI-COR Biosciences) in PBS containing 0.1–0.2% Tween (PBS-T) overnight at 4 °C. The membranes were rinsed in PBS-T and incubated for 1 h at room temperature in IRDye 800-conjugated anti-rabbit (LI-COR Biosciences) or anti-rat (LI-COR Biosciences) in 1:20,000 Odyssey blocking buffer in 0.1% PBST. Control for protein loading was achieved with β-tubulin (55 kDa; anti-mouse; 1:10,000, Covance Laboratories, Dedham, MA) visualized with a LI-COR secondary in PBS-T. Proteins were detected using the Odyssey infrared imaging system (excitation/emission filters at 780 nm/820 nm range). All protein values were normalized to β-tubulin expression. Statistical Analyses: Following dichotomization into depressive versus non-depressive groups based on one standard deviation from the CON mean, categorical data for helplessness were analyzed with the non-parametric analysis of Mann-Whitney U. Dichotomized Western data were analyzed with a Kruskal-Wallis for non-parametric data followed by a Dunn's test when significant overall differences were found (SPSS v. 22.0 Chicago, IL). Correlational analyses (Pearson's r) were also used to examine individual relationships between biochemical
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Fig. 3. A) Correlations between PV (top) and TrkB (middle) expression and depressivelike behavior in the (left) amygdala and the (right) prefrontal cortex (PFC) in the CON and MS groups. Too few depressive subjects (< 14) in the CON2/ISO group prevented a meaningful analysis. Pearson's r values are presented; with Fisher's Z transformation showing significance * P < 0.05, **P < 0.001. B) Representative Western immunoblots of tubulin (a loading control), PV, and TrkB in the amygdala (left) and the PFC (right).
between the regions. The correlation between the amygdala/PFC in CON subjects was not significant for PV or TrkB (r = −0.29 and 0.24, respectively, P > 0.1), but they were for MS subjects (PV and TrkB: r = 0.62 and 0.89, respectively, P > 0.05). The relationships between the number of escapes and CB and CR were also investigated. Both CB and CR levels in the amygdala demonstrated significant correlations with the number of escapes in the MS subjects (Fig. 4A; r = 0.64 and −0.64; P < 0.05), but not in CON subjects (P > 0.1 based on Fisher's z transformation). When the data were dichotomized, group differences between MS and CON females were observed for CR in the non-depressive condition for both regions (Fig. 4B), but not for CB (not shown). Significant differences were observed for CR in the amygdala (Kruskal-Wallis: P < 0.02), with Dunn's test determining that group differences were present in both non-depressive and depressive groups (P < 0.02). However, while significant differences were observed in the PFC between depressive groups (Kruskal-Wallis: P < 0.02), these differences in PFC CR were mainly driven by the non-depressive group (Dunn's: P < 0.01).
Fig. 2. Parvalbumin (PV) and TrkB protein levels for the A) amygdala and B) prefrontal cortex; (left) CON and MS subjects and (right) CON2 and ISO subjects dichotomized based on depression scores that were based on one standard deviation from CON and CON2 means. Western blot data were then normalized to the > 13 CON and CON2 values within each period. Means ± SE presented, with * P < 0.05; **P < 0.01.
B shows representative immunoblots of TrkB and PV. Fisher's z transformation was used to determine whether these correlations differed from each other by taking into account multiple comparisons. The MS and CON group correlations were significantly different for PV in the amygdala (z = −0.64, P < 0.05), but not for TrkB. In the PFC, the PV correlations between groups were different (z = −3.1, P < 0.001) as were the group comparisons for TrkB (z = −3.3, P < 0.001). Inter-correlations between the PFC and amygdala of individual subjects overall was investigated to determine any relationships
4. Discussion Individual changes in the GABA markers as they relate to helplessness in the amygdala and the PFC have not been examined previously in MS and ISO females. The strong correlation between escape number and PV following MS or in CON subjects within the PFC 11
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why ISO alone in weaned rats was insufficient to produce anhedonia. Exposure to ISO between P20-40 did not alter the prevalence of anhedonia, although we have recently observed an increase in helplessness in ISO females (Lukkes and Andersen, unpublished observation). It is also possible that the testing of subjects two days after isolation was insufficient to allow the stress effects to incubate. Whether the effects of MS and ISO produce similar levels of stress is unclear. When comparisons between MS and ISO are made directly within the same laboratory, the effects of MS versus ISO are either different (Biggio et al., 2014) or the same (Vargas et al., 2016). In a study by Biggio et al. (2014), ISO starting at P51 in male rats produced a significantly greater decrease in baseline levels of plasma corticosterone relative to MS subjects. In contrast, ISO that is initiated at P21, like our study, found plasma corticosterone levels were the same across these two conditions and depression in MS, but not ISO, animals (Vargas et al., 2016). These data suggest that MS has a greater effect on depressive effects than ISO when ISO is initiated at the time of weaning. While ELA is associated with a delay in depression, maltreatment during adolescence typically results in depression during adolescence (Teicher et al., 2009). A possible protective effect of ISO is suggested by a decrease in the prevalence of anhedonia from 44% in MS rats to 22–28% in controls to 12% in ISO rats. Two differences of significance exist between these groups. First, PV levels are elevated in the MS rats that exhibited > 13 escapes relative to those subjects that were depressive-like (< 14). The > 13 group could represent either rats that were resilient to MS, exhibited a different abnormal behavior associated with elevated PV levels, or both. Second, PV and TrkB levels were significantly lower in the PFC of MS, but not ISO, subjects who met the criterion of < 14 escapes for depressive behavior. Reduced PV and TrkB that are found in this < 14 group are consistent with the correlations in Fig. 3, where low PV and TrkB were strongly associated with depressive-like behavior. The preclinical data of low PV in the amygdala of MS subjects are consistent with observations of increased emotionality and blood flow responses to emotional stimuli in children with an ELA history. Independent of condition, rats with fewer escapes had low levels of PV in the amygdala. These results are similar to reports of reduced forced swim immobility following muscimol in the amygdala (Raineki et al., 2012), but forced swim changes in ISO animals have not been found (Hall et al., 1998), (Raz, 2013), but see (Gilles and Polston, 2017). A decrease in amygdala PV was observed in a subset of CON2 and ISO subjects, consistent with reports of less c-fos activity in amygdalar GABAergic interneurons following ISO (Lukkes et al., 2012) and greater anxiety (Lukkes et al., 2009b). Finally, low BDNF in the amygdala has been found in rats that receive less maternal care (Macri et al., 2010). Low PV levels in the PFC of CON or MS females with few escapes are common to reports of low GABA in humans with depressive symptoms (Gabbay et al., 2012; Klempan et al., 2009; Sanacora et al., 2000). Surprisingly, the results of this study show that low PV in the PFC can occur independently of an ELA history. ELA may further decrease PFC PV, doubling the prevalence of anhedonia. The strong amygdala/PFC correlation in MS, but not in CON, CON2 or ISO subjects, suggests that PV loss in both regions simultaneously is needed to increase depression risk (Fig. 5). Alternatively, the shock in the LH paradigm could produce the observed changes in PV and TrkB. Our previous observations of PV loss in MS animals that were not exposed to the LH paradigm argue against this possibility (Brenhouse and Andersen, 2011; Leussis et al., 2012). Inflammation may also enhance PV vulnerability in MS animals (Brenhouse and Andersen, 2011; Canetta et al., 2016; Dugan et al., 2009; Hennessy et al., 2011; Wieck et al., 2013). The dichotomization of behavioral data into anhedonia and nonanhedonia resulted in a distribution of 22–28% of the subset of CON/ CON2 rats versus 44% of the MS rats. The lifetime prevalence rate of depression is 20% in the general population (Kessler et al., 1994) and 67% in an ELA-exposed population (Andersen, 2015; Teicher et al., 2009). While we categorized groups by one standard deviation of the
Fig. 4. A) Correlations between calbindin (top) and calretinin (middle) expression and depressive-like behavior in the amygdala and the prefrontal cortex (PFC). Pearson's r values are presented; *P < 0.05. B) Calretinin data from the amygdala (left) and PFC (right) from CON/MS subjects dichotomized for anhedonia scores. Means ± SE presented, with * P < 0.05; **P < 0.01; #P < 0.06.
supports the importance of GABAergic expression in depressive/anhedonic behavior, similar to reports in humans (Gabbay et al., 2012; Klempan et al., 2009; Sanacora et al., 1999). Changes in amygdala PV also correlated with escape behavior in MS subjects, although not as strongly as in the PFC. Significant correlations between the number of escapes and PV and TrkB in the PFC suggest that depressive-like behaviors following MS exposure are associated with a sensitive period. Exposure to the ISO stressor during a later developmental period (Lukkes et al., 2009b) failed to change anhedonia with this paradigm. Clinically, early life maltreatment accelerates the onset of depression (Teicher et al., 2009; Widom et al., 2007). We hypothesized that exposure to MS would facilitate depressive behavior by keeping the sensitive period open. Low PV/TrkB levels in both amygdala and PFC could be interpreted as an open sensitive period for emotionality and its cognitive control, where the environment would have greater influences on mood. This hypothesis requires additional testing, but a proposed model of how ELA alters sensitive periods is presented in Fig. 5. We include inflammation as an additional factor whereby exposure to MS during a sensitive period of development may render subjects more vulnerable to later insult. Inflammation is elevated in some animals with a MS (Brenhouse and Andersen, 2011; Lukkes et al., 2017; Wieck et al., 2013) or ISO history (Ko and Liu, 2016), although we did not observe changed in interleukin-6 (unpublished). As the dam may mediate the inflammation (Hennessy et al., 2013), this may explain 12
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Fig. 5. Proposed model of sensitive period changes following early life adversity that leads to an anhedonic depression. Evidence suggests that the sensitive period for anxiety as mediated by the amygdala opens during the juvenile period. As PV and TrkB levels naturally rise and close the amygdala sensitive period, the sensitive period in the prefrontal cortex (PFC) opens to environmental stimuli during adolescence. If allowed to develop normally, PV and TrkB levels rise and close the window at each stage. However, if inflammation or other mediating factors are present and PV and TrkB levels remain low, the sensitive period is extended and vulnerability to outside events remains. The result is anxiety and depression, two common observations following exposure to early life adversity. In contrast, animals exposed to social isolation demonstrate only decreased PV levels in the amygdala, with no changes in the PFC, and relatively little anhedonic depression.
from the number of immunoreactive cells (Caballero et al., 2014), although our studies in MS males demonstrated a 38% decrease in PVimmunoreactive neurons (Brenhouse and Andersen, 2011) and a 42 ± 8% decrease with Westerns (Leussis et al., 2012). Second, the design of our blots minimized variability by running groups of subjects on a single gel with CON/MS and CON2/ISO run simultaneously. Protein values were normalized to tubulin levels to further minimize variance. Third, we included different cohorts of animals as a replication sample. These additional subjects demonstrated reliability in the findings. For example, we observed a correlation of r = 0.97 in the first MS group (n = 6) and a correlation of r = 0.93 in the second cohort (n = 6 + 3 additional). The results of this study demonstrate the importance of individual differences in PV and TrkB levels in the expression of anhedonia. By comparing the results from MS subjects to ISO subjects, we revealed a sensitive period for anhedonia as indicated by a 1) doubled prevalence following MS, but not ISO; and 2) reductions in PV in both the amygdala and the PFC that was absent in ISO animals. Notably, CON and CON2 and ISO subjects with atypical escapes had lower PV levels in the amygdala. These data further suggest that ELA exposure may maintain heightened environmental sensitivity and advocate for earlier interventions in this vulnerable population.
CON mean (Ritov et al., 2016), others have a priori defined a few missed escapes as ‘depressive’ (Shirayama et al., 2002) or did not dichotomize (Leussis et al., 2012). Regardless, the doubling of anhedonia prevalence is similar to clinical prevalence levels, and thus may further aid in determining common causes of depression where ELA plays a permissive role. Increased levels of PV and TrkB in MS relative to CON females may signal resilience, or possibly, another outcome of ELA such as an externalizing disorder (D'Andrea et al., 2012). In depression-resilient humans, greater PFC activity and less amygdala activity facilitate adapting to stress quickly (Gee et al., 2013). As changes in GABA inversely correlate with blood oxygenated levels in the brain (Northoff et al., 2007), the observation that elevated PFC PV levels were associated with more escapes is consistent with its role in adapting to stress. Calretinin levels were increased in the amygdala, but not PFC, of MS subjects with greatest increases in the anhedonia phenotype. A positive relationship was observed between TrkB levels and CB expression (not shown), consistent with earlier findings in cell culture (Fiumelli et al., 2000). However, non-significant relationships were observed for CR neurons and TrkB in both regions, where BDNF is reported to decrease CR expression (Fiumelli et al., 2000). We readily acknowledge caveats to our methodological/analytical approaches. Proteins were quantified with Westerns, without quantifying the number of neurons. Functional protein expression may differ 13
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Conflicts of interest
Gilles, Y.D., Polston, E.K., 2017. Effects of social deprivation on social and depressive-like behaviors and the numbers of oxytocin expressing neurons in rats. Behav. Brain Res. 328, 28–38. Gogolla, N., Caroni, P., Luthi, A., Herry, C., 2009. Perineuronal nets protect fear memories from erasure. Science 325 (5945), 1258–1261. Greenough, W.T., Black, J.E., Wallace, C.S., 1987. Experience and brain development. Child Dev. 58 (3), 539–559. Guilloux, J.P., Douillard-Guilloux, G., Kota, R., Wang, X., Gardier, A.M., Martinowich, K., Tseng, G.C., Lewis, D.A., Sibille, E., 2012. Molecular evidence for BDNF- and GABArelated dysfunctions in the amygdala of female subjects with major depression. Mol. Psychiatr. 17 (11), 1130–1142. Hall, F.S., Huang, S., Fong, G.F., Pert, A., 1998. The effects of social isolation on the forced swim test in Fawn hooded and Wistar rats. J. Neurosci. Meth. 79 (1), 47–51. Hennessy, M.B., Jacobs, S., Schiml, P.A., Hawk, K., Stafford, N., Deak, T., 2013. Maternal inhibition of infant behavioral response following isolation in novel surroundings and inflammatory challenge. Dev. Psychobiol. 55 (4), 395–403. Hennessy, M.B., Paik, K.D., Caraway, J.D., Schiml, P.A., Deak, T., 2011. Proinflammatory activity and the sensitization of depressive-like behavior during maternal separation. Behav. Neurosci. 125 (3), 426–433. Holland, F.H., Ganguly, P., Potter, D.N., Chartoff, E.H., Brenhouse, H.C., 2014. Early life stress disrupts social behavior and prefrontal cortex parvalbumin interneurons at an earlier time-point in females than in males. Neurosci. Lett. 566, 131–136. Huang, Z.J., Kirkwood, A., Pizzorusso, T., Porciatti, V., Morales, B., Bear, M.F., Maffei, L., Tonegawa, S., 1999. BDNF regulates the maturation of inhibition and the critical period of plasticity in mouse visual cortex. Cell 98 (6), 739–755. Kessler, R.C., McGonagle, K.A., Zhao, S., Nelson, C.B., Hughes, M., Eshleman, S., Wittchen, H.U., Kendler, K.S., 1994. Lifetime and 12-month prevalence of DSM-III-r psychiatric disorders in the United States. Results from the National comorbidity survey. Arch. Gen. Psychiatr. 51 (1), 8–19. Klempan, T.A., Sequeira, A., Canetti, L., Lalovic, A., Ernst, C., ffrench-Mullen, J., Turecki, G., 2009. Altered expression of genes involved in ATP biosynthesis and GABAergic neurotransmission in the ventral prefrontal cortex of suicides with and without major depression. Mol. Psychiatr. 14 (2), 175–189. Ko, C.Y., Liu, Y.P., 2016. Disruptions of sensorimotor gating, cytokines, glycemia, monoamines, and genes in both sexes of rats reared in social isolation can be ameliorated by oral chronic quetiapine administration. Brain Behav. Immun. 51, 119–130. Lehmann, J., Feldon, J., 2000. Long-term biobehavioral effects of maternal separation in the rat: consistent or confusing? Rev. Neurosci. 11 (4), 383–408. Leussis, M.P., Freund, N., Brenhouse, H.C., Thompson, B.S., Andersen, S.L., 2012. Depressive-like behavior in adolescents after maternal separation: sex differences, controllability, and GABA. Dev. Neurosci. 34 (2–3), 210–217. Lukkes, J.L., Burke, A.R., Zelin, N.S., Hale, M.W., Lowry, C.A., 2012. Post-weaning social isolation attenuates c-Fos expression in GABAergic interneurons in the basolateral amygdala of adult female rats. Physiol. Behav. 107 (5), 719–725. Lukkes, J.L., Meda, S., Thompson, B.S., Freund, N., Andersen, S.L., 2017. Early life stress and later peer distress on depressive behavior in adolescent female rats: effects of a novel intervention on GABA and D2 receptors. Behav. Brain Res. 330, 37–45. Lukkes, J.L., Mokin, M.V., Scholl, J.L., Forster, G.L., 2009a. Adult rats exposed to earlylife social isolation exhibit increased anxiety and conditioned fear behavior, and altered hormonal stress responses. Horm. Behav. 55 (1), 248–256. Lukkes, J.L., Watt, M.J., Lowry, C.A., Forster, G.L., 2009b. Consequences of post-weaning social isolation on anxiety behavior and related neural circuits in rodents. Front. Behav. Neurosci. 3, 18. Macri, S., Laviola, G., Leussis, M.P., Andersen, S.L., 2010. Abnormal behavioral and neurotrophic development in the younger sibling receiving less maternal care in a communal nursing paradigm in rats. Psychoneuroendocrinology 35, 392–402. Maier, S.F., Watkins, L.R., 2005. Stressor controllability and learned helplessness: the roles of the dorsal raphe nucleus, serotonin, and corticotropin-releasing factor. Neurosci. Biobehav. Rev. 29 (4–5), 829–841. Malter Cohen, M., Jing, D., Yang, R.R., Tottenham, N., Lee, F.S., Casey, B.J., 2013. Earlylife stress has persistent effects on amygdala function and development in mice and humans. Proc. Natl. Acad. Sci. U.S.A. 110 (45), 18274–18278. Marusak, H.A., Martin, K.R., Etkin, A., Thomason, M.E., 2015. Childhood trauma exposure disrupts the automatic regulation of emotional processing. Neuropsychopharmacology 40 (5), 1250–1258. Matthews, K., Robbins, T.W., 2003. Early experience as a determinant of adult behavioural responses to reward: the effects of repeated maternal separation in the rat. Neurosci. Biobehav. Rev. 27 (1–2), 45–55. Morishita, H., Cabungcal, J.H., Chen, Y., Do, K.Q., Hensch, T.K., 2015. Prolonged period of cortical plasticity upon redox dysregulation in fast-spiking interneurons. Biol. Psychiatr. 78 (6), 396–402. Napolitano, A., Shah, K., Schubert, M.I., Porkess, V., Fone, K.C., Auer, D.P., 2014. In vivo neurometabolic profiling to characterize the effects of social isolation and ketamineinduced NMDA antagonism: a rodent study at 7.0 T. Schizophr. Bull. 40 (3), 566–574. Northoff, G., Walter, M., Schulte, R.F., Beck, J., Dydak, U., Henning, A., Boeker, H., Grimm, S., Boesiger, P., 2007. GABA concentrations in the human anterior cingulate cortex predict negative BOLD responses in fMRI. Nat. Neurosci. 10 (12), 1515–1517. Ogawa, T., Kuwagata, M., Hori, Y., Shioda, S., 2007. Valproate-induced developmental neurotoxicity is affected by maternal conditions including shipping stress and environmental change during early pregnancy. Toxicol. Lett. 174 (1–3), 18–24. Plotsky, P.M., Meaney, M.J., 1993. Early, postnatal experience alters hypothalamic corticotropin-releasing factor (CRF) mRNA, median eminence CRF content and stressinduced release in adult rats. Brain research. Mol. Brain Res. 18 (3), 195–200. Pryce, C.R., Azzinnari, D., Spinelli, S., Seifritz, E., Tegethoff, M., Meinlschmidt, G., 2011. Helplessness: a systematic translational review of theory and evidence for its
None. Funding The National Institute on Drug Abuse (DA-015403 and DA-026485 [SLA]), the Simches Family (SLA), and a 2013 NARSAD Young Investigator Award from the Brain and Behavior Research Foundation (JLL) supported the project. Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx. doi.org/10.1016/j.jpsychires.2018.02.005. References Andersen, S.L., 2003. Trajectories of brain development: point of vulnerability or window of opportunity? Neurosci. Biobehav. Rev. 27 (1–2), 3–18. Andersen, S.L., 2015. Exposure to early adversity: points of cross-species translation that can lead to improved understanding of depression. Dev. Psychopathol. 27 (2), 477–491. Andersen, S.L., Teicher, M.H., 2008. Stress, sensitive periods and maturational events in adolescent depression. Trends Neurosci. 31 (4), 183–191. Berretta, S., Benes, F.M., 2006. A rat model for neural circuitry abnormalities in schizophrenia. Nat. Protoc. 1 (2), 833–839. Biggio, F., Pisu, M.G., Garau, A., Boero, G., Locci, V., Mostallino, M.C., Olla, P., Utzeri, C., Serra, M., 2014. Maternal separation attenuates the effect of adolescent social isolation on HPA axis responsiveness in adult rats. European Neuropsychopharmacology. J. Eur. Coll. Neuropsychopharmacol. 24 (7), 1152–1161. Bloomfield, C., French, S.J., Jones, D.N., Reavill, C., Southam, E., Cilia, J., Totterdell, S., 2008. Chandelier cartridges in the prefrontal cortex are reduced in isolation reared rats. Synapse 62 (8), 628–631. Bradford, M., 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 7, 248–254. Brenhouse, H.C., Andersen, S.L., 2011. Nonsteroidal anti-inflammatory treatment prevents delayed effects of early life stress in rats. Biol. Psychiatr. 70 (5), 434–440. Caballero, A., Flores-Barrera, E., Cass, D.K., Tseng, K.Y., 2014. Differential regulation of parvalbumin and calretinin interneurons in the prefrontal cortex during adolescence. Brain Struct. Funct. 219 (1), 395–406. Caldji, C., Diorio, J., Meaney, M.J., 2003. Variations in maternal care alter GABA(A) receptor subunit expression in brain regions associated with fear. Neuropsychopharmacology 28 (11), 1950–1959. Caldji, C., Tannenbaum, B., Sharma, S., Francis, D., Plotsky, P.M., Meaney, M.J., 1998. Maternal care during infancy regulates the development of neural systems mediating the expression of fearfulness in the rat. Proc. Natl. Acad. Sci. U.S.A. 95 (9), 5335–5340. Canetta, S., Bolkan, S., Padilla-Coreano, N., Song, L.J., Sahn, R., Harrison, N.L., Gordon, J.A., Brown, A., Kellendonk, C., 2016. Maternal immune activation leads to selective functional deficits in offspring parvalbumin interneurons. Mol. Psychiatr. 21 (7), 956–968. Committee for the Update of the Guide for the Care and Use of Laboratory Animals, 2011. N.R.C. Guide for the Care and Use of Laboratory Animals Eighth Ed. The National Academies. (Washington, D.C). D'Andrea, W., Ford, J., Stolbach, B., Spinazzola, J., van der Kolk, B.A., 2012. Understanding interpersonal trauma in children: why we need a developmentally appropriate trauma diagnosis. Am. J. Orthopsychiatry 82 (2), 187–200. Dugan, L.L., Ali, S.S., Shekhtman, G., Roberts, A.J., Lucero, J., Quick, K.L., Behrens, M.M., 2009. IL-6 mediated degeneration of forebrain GABAergic interneurons and cognitive impairment in aged mice through activation of neuronal NADPH oxidase. PLoS One 4 (5), e5518. Fiumelli, H., Kiraly, M., Ambrus, A., Magistretti, P.J., Martin, J.L., 2000. Opposite regulation of calbindin and calretinin expression by brain-derived neurotrophic factor in cortical neurons. J. Neurochem. 74 (5), 1870–1877. Freund, N., Thompson, B.S., Denormandie, J., Vaccarro, K., Andersen, S.L., 2013. Windows of vulnerability: maternal separation, age, and fluoxetine on adolescent depressive-like behavior in rats. Neuroscience 249, 88–97. Gabbay, V., Mao, X., Klein, R.G., Ely, B.A., Babb, J.S., Panzer, A.M., Alonso, C.M., Shungu, D.C., 2012. Anterior cingulate cortex gamma-aminobutyric acid in depressed adolescents: relationship to anhedonia. Arch. Gen. Psychiatr. 69 (2), 139–149. Gee, D.G., Gabard-Durnam, L.J., Flannery, J., Goff, B., Humphreys, K.L., Telzer, E.H., Hare, T.A., Bookheimer, S.Y., Tottenham, N., 2013. Early developmental emergence of human amygdala-prefrontal connectivity after maternal deprivation. Proc. Natl. Acad. Sci. U.S.A. 110 (39), 15638–15643. Giachino, C., Canalia, N., Capone, F., Fasolo, A., Alleva, E., Riva, M.A., Cirulli, F., Peretto, P., 2007. Maternal deprivation and early handling affect density of calcium binding protein-containing neurons in selected brain regions and emotional behavior in periadolescent rats. Neuroscience 145 (2), 568–578.
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Journal of Psychiatric Research 100 (2018) 8–15
J.L. Lukkes et al.
Shirayama, Y., Chen, A.C., Nakagawa, S., Russell, D.S., Duman, R.S., 2002. Brain-derived neurotrophic factor produces antidepressant effects in behavioral models of depression. J. Neurosci. 22 (8), 3251–3261. Sibille, E., Morris, H.M., Kota, R.S., Lewis, D.A., 2011. GABA-related transcripts in the dorsolateral prefrontal cortex in mood disorders. Int. J. Neuropsychopharmacol. 14 (6), 721–734. Teicher, M.H., Samson, J.A., Polcari, A., Andersen, S.L., 2009. Length of time between onset of childhood sexual abuse and emergence of depression in a young adult sample: a retrospective clinical report. J. Clin. Psychiatr. 70 (5), 684–691. Tottenham, N., Hare, T.A., Millner, A., Gilhooly, T., Zevin, J.D., Casey, B.J., 2011. Elevated amygdala response to faces following early deprivation. Dev. Sci. 14 (2), 190–204. Vargas, J., Junco, M., Gomez, C., Lajud, N., 2016. Early life stress increases metabolic risk, HPA Axis Reactivity, and depressive-like behavior when combined with postweaning social isolation in rats. PLoS One 11 (9) e0162665. Widom, C.S., DuMont, K., Czaja, S.J., 2007. A prospective investigation of major depressive disorder and comorbidity in abused and neglected children grown up. Arch. Gen. Psychiatr. 64 (1), 49–56. Wieck, A., Andersen, S.L., Brenhouse, H.C., 2013. Evidence for a neuroinflammatory mechanism in delayed effects of early life adversity in rats: relationship to cortical NMDA receptor expression. Brain Behav. Immun. 28, 218–226.
relevance to understanding and treating depression. Pharmacol. Therapeut. 132 (3), 242–267. Raineki, C., Cortes, M.R., Belnoue, L., Sullivan, R.M., 2012. Effects of early-life abuse differ across development: infant social behavior deficits are followed by adolescent depressive-like behaviors mediated by the amygdala. J. Neurosci. 32 (22), 7758–7765. Raz, S., 2013. Ameliorative effects of brief daily periods of social interaction on isolationinduced behavioral and hormonal alterations. Physiol. Behav. 116–117, 13–22. Ritov, G., Boltyansky, B., Richter-Levin, G., 2016. A novel approach to PTSD modeling in rats reveals alternating patterns of limbic activity in different types of stress reaction. Mol. Psychiatr. 21 (5), 630–641. Sanacora, G., Mason, G.F., Krystal, J.H., 2000. Impairment of GABAergic transmission in depression: new insights from neuroimaging studies. Crit. Rev. Neurobiol. 14 (1), 23–45. Sanacora, G., Mason, G.F., Rothman, D.L., Behar, K.L., Hyder, F., Petroff, O.A., Berman, R.M., Charney, D.S., Krystal, J.H., 1999. Reduced cortical gamma-aminobutyric acid levels in depressed patients determined by proton magnetic resonance spectroscopy. Arch. Gen. Psychiatr. 56 (11), 1043–1047. Seidel, K., Helmeke, C., Poeggel, G., Braun, K., 2008. Repeated neonatal separation stress alters the composition of neurochemically characterized interneuron subpopulations in the rodent dentate gyrus and basolateral amygdala. Developmental Neurobiol. 68 (9), 1137–1152.
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Anhedonic behavior and γ-amino butyric acid during a sensitive period in female rats exposed to early adversity
T
Jodi L. Lukkesa,b,1, Shirisha Medaa, Kevin J. Normana, Susan L. Andersena,b,∗ a b
Laboratory for Developmental Neuropharmacology, McLean Hospital, MA, 02478, United States Harvard Medical School, United States
A R T I C L E I N F O
A B S T R A C T
Keywords: Adolescence Amygdala Depression Helplessness Parvalbumin Prefrontal cortex sensitive period
Early life adversity increases depressive behavior that emerges during adolescence. Sensitive periods have been associated with fewer GABAergic interneurons, especially parvalbumin (PV), brain derived growth factor, and its receptor, TrkB. Here, maternal separation (MS) and social isolation (ISO) were used to establish a sensitive period for anhedonic depression using the learned helplessness (LH) paradigm. Female Sprague-Dawley rat pups underwent MS for 4-h/day or received typical care (CON) between postnatal days 2–20; for the ISO condition, separate cohorts were individually housed between days 20–40 or served as controls (CON2). Anhedonia was defined by dichotomizing subjects into two groups based on one standard deviation of the mean number of escapes for the CON group (< 14). This approach categorized 22% of CON subjects and 44% of MS subjects as anhedonic (p < 0.05), similar to the prevalence in maltreated human populations. Only 12.5% of ISO rats met criterion versus 28.5% in CON2 rats. Levels of PV and TrkB were reduced in the amygdala and prelimbic prefrontal cortex (PFC) in MS rats with < 14 escapes, but elevated in behaviorally resilient MS rats (> 13 escapes). The number of escapes in MS subjects significantly correlated with PV and TrkB levels (PFC: r = 0.93 and 0.91 and amygdala: r = 0.63 and 0.81, respectively; n = 9), but not in CON/ISO/CON2 subjects. Calretinin, but not calbindin, was elevated in the amygdala of MS subjects. These data suggest that low levels of PV and TrkB double the risk for anhedonia in females with an MS history compared to normal adolescent females.
1. Introduction Exposure to early life adversity (ELA) in the form of physical/sexual abuse, neglect, loss of a parent or caregiver or a natural disaster affects approximately 33% of the population. The lifetime prevalence rate of depression in an ELA-exposed population is 67% (Andersen, 2015; Andersen and Teicher, 2008; Teicher et al., 2009; Widom et al., 2007) compared to 20% in the general population. Elevated emotionality has been associated with increased amygdala activity in children younger than 11 years old with an ELA history (Malter Cohen et al., 2013; Marusak et al., 2015; Tottenham et al., 2011). More mature connectivity (e.g., resting state) between the amygdala and frontal cortex following ELA has also been described (Gee et al., 2013). In humans, elevated depressive/anhedonic behavior in adults and adolescents has been associated with less GABA in the PFC (Gabbay et al., 2012; Sanacora et al., 1999). However, the relationship of ELA, GABA, and depression during adolescence in animals on an individual basis is not known. During the preteen years, abused and non-abused children begin to ∗
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diverge in developmental trajectories (Widom et al., 2007). Depression emerges earlier than the general population, and coincides with increased prevalence of depression during a sensitive period of adolescent development (Andersen, 2015; Andersen and Teicher, 2008). A sensitive period is defined as a maturational stage when experience can exert maximal effect on development, but is not as necessary as it is during a critical period (Andersen, 2003; Greenough et al., 1987). Critical periods have been defined by changes in growth factors, such as brainderived growth factor (BDNF) and its receptor, tropomyosin receptor kinase B (TrkB), and the GABAergic interneuron that expresses the calcium-binding protein of parvalbumin (PV) (Huang et al., 1999; Morishita et al., 2015). While critical and sensitive periods are defined differently, sensitive periods of affective development may utilize the same underlying mechanisms. The sensitive period for the amygdala has been characterized as starting during the second week of postnatal (P) development in the rodent and ending at postnatal day (P)25 (Gogolla et al., 2009). Indeed, rats exposed to the ethologically relevant rodent model of maternal separation (MS) (Andersen, 2015; Lehmann and Feldon, 2000) show an increase in PV in the basolateral amygdala
Corresponding author. 115 Mill Street, Mailstop 333, Belmont, MA, 02478, United States. E-mail address: [email protected] (S.L. Andersen). Department of Psychiatry, Indiana University School of Medicine, Indianapolis, IN, United States.
https://doi.org/10.1016/j.jpsychires.2018.02.005 Received 4 November 2017; Received in revised form 22 December 2017; Accepted 8 February 2018 0022-3956/ © 2018 Elsevier Ltd. All rights reserved.
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day 16 of gestation. The day of birth was designated as P0. One day after birth, litters were culled to 10 pups (5 males and 5 females), and randomly assigned to either a MS or animal facility reared control group (CON). Only one pup per litter was assigned to a single condition. We acknowledge that the stress of shipping may have influenced our findings (Ogawa et al., 2007), but was minimized by distributing subjects both CON and MS rats equally across all conditions. Maternal separation: Pups in the MS group (n = 9) were isolated from their peers and mother for 4 h per day between P2 and P20, and kept at a thermo neutral temperature, following our methodology (Andersen, 2015; Brenhouse and Andersen, 2011; Leussis et al., 2012) and others (Plotsky and Meaney, 1993). Pups in the CON (n = 18) group were not disturbed after day two except for routine weekly cage changes, during which pups were weighed. A small, but significant, reduction in weight occurs in MS animals relative to CON during early development (Freund et al., 2013). Rats were housed with food and water available ad libitum in constant temperature and humidity conditions on a 12-h light/dark cycle (light period 07:00–19:00). Rats were weaned on P21, and group-housed in same-sex caging in male- or female-only vivarium until behavioral testing. Only females were used in this study. Social isolation: Rats were born and housed at McLean, as described above. Between P21-40, subjects were individually housed within the vivarium (ISO; n = 7); only females were used and were housed in a female-only room. No other manipulation occurred during this time. The controls (CON2; n = 7) were group housed (3/cage) under the same conditions. The experimental timeline is outlined in Fig. 1A. These experiments were conducted in accordance with the 2011 Guide for the Care and Use of Laboratory Animals (Committee for the Update of the Guide for the Care and Use of Laboratory Animals, 2011), and were approved by the Institutional Animal Care and Use Committee at McLean Hospital. Learned helplessness: In an initial cohort, CON (n = 9) and MS females (n = 9) were tested for depressive-like behavior on P40 and P41. Our previous studies have shown that females exposed to MS exhibited increased escape latency in the no shock (NS) condition (Leussis et al., 2012; Maier and Watkins, 2005; Pryce et al., 2011), which is associated with motivational deficits (Pryce et al., 2011). Females were gently restrained in the testing apparatus on Day 1 while a rat underwent the escapable shock (ES) condition of LH in their presence. This exposure affects escape behavior relative to rats that were not exposed to an ES rat on Day 1 (Lukkes et al., 2017). On Day 2, rats were individually tested in the shuttle box for 30 trials. Subjects could terminate a one mA foot-shock by shuttling to the other side for trials 1–5, or by shuttling to the other side and back again for trials 6–30. This response was cued by a tone that preceded the shock by 2 s. The shock remained on for 30 s, or until terminated by escape. The number of escapes was measured for trials 6–30. A second cohort of n = 9 females was added to the CON condition to increase our sample size for behavior only to increase statistical power to determine the prevalence of anhedonia in a typical population. These subjects did not differ from the first CON cohort on any measure. Western Immunoblots: Ninety minutes following the onset of behavioral testing on Day 2, animals were decapitated and tissue was regionally dissected in the amygdala and PFC and stored at −80 °C until processed. Tissue was homogenized in 1% sodium dodecyl sulfate (SDS) solution containing a protease inhibitor cocktail (Pierce, Rockford, IL). Protein concentration was determined by the Bradford method (BioRad Laboratories, Hercules, CA; Bradford, 1976). Proteins from the amygdala and PFC were analyzed for PV, CB, CR, and TrkB with each condition represented on a single blot for the CON/MS cohort of subjects. This approach minimized variability for the correlational analyses. The second cohort CON2/ISO was run on a single blot together for PV and TrkB. Eighty μg of protein was mixed in 6× SDS, centrifuged, and boiled for 3 min prior to separation by 15% SDS-PAGE. Proteins were then transferred to a nitrocellulose membrane (Bio-Rad
(BLA) by P35 (Giachino et al., 2007), consistent with an end to the sensitive period. Defining the sensitive period in the PFC has not received as much attention as the amygdala. Levels of PFC PV are low during juvenility and rise during adolescence into adulthood in normal rats (Brenhouse and Andersen, 2011; Caballero et al., 2014; Leussis et al., 2012). These PV data suggest a closing of the PFC sensitive period during adolescence. An early and protracted sensitive period in individuals with a stress history (Andersen and Teicher, 2008) implies that the brain remains highly sensitive to environmental stimuli, and thus, vulnerable during adolescence and even into adulthood. To better determine whether a sensitive period is present for anhedonia, the current study compared MS to the social isolation (ISO (Lukkes et al., 2009a);) as each of these stressor occur at different developmental stages. Comparison of these two 20-day manipulations may shed light on the role of sensitive periods in depression. Depressive and GABA changes are found in MS rats. MS increases learned helplessness (LH) and impaired working memory in adolescent rats (Brenhouse and Andersen, 2011; Leussis et al., 2012; Matthews and Robbins, 2003). GABA-A receptors are reduced in the amygdala of rats with a history of MS (Caldji et al., 2003), which is likely to lower GABAergic activity. Lower GABA levels or GABAergic activity in the amygdala is associated with increased anxiety and depressive-like behavior (Caldji et al., 1998; Raineki et al., 2012; Ritov et al., 2016). Finally, human post-mortem studies found low amygdala BDNF and GABA mRNA in depressed women (Guilloux et al., 2012). Reduced GABA activity in the amygdala during development can affect cortical development (Berretta and Benes, 2006). Together, these findings support a role for amygdala GABA in affective dysfunction, but has neither established the relationship between the loss of GABA in the amygdala as a direct consequence of MS nor its influence on behavior. Significant decreases in PFC PV-immunoreactive interneurons (counted by stereology) or PV expression (Western immunoblot) have been described during adolescence following MS between P2-20 days of age (Brenhouse and Andersen, 2011; Leussis et al., 2012). Sex differences in PV loss following MS occur earlier in females than males (Holland et al., 2014), but PV loss secondary to MS has been found in both sexes in adolescence (Leussis et al., 2012). Notably, one study of MS failed to find changes in PV in octus degus, a precocial rodent species (Seidel et al., 2008). Relatively little data is available on GABA in the cortex of ISO rats. Cortical chandelier cells (some expressing PV) decreased in males that underwent ISO (P28-84) (Bloomfield et al., 2008). However, magnetic resonance spectroscopy reported no differences in GABA levels between controls and ISO rats (Napolitano et al., 2014). The current study examined whether a sensitive period of ELA exists for anhedonic depression by comparing behavior, PV, and TrkB in MS and ISO rats during adolescence. As the prevalence of anhedonia was affected by MS, and by ISO, changes in other GABAergic interneurons that express calbindin (CB) and calretinin (CR) were only investigated in MS subjects. There is less evidence supporting their involvement in mood disorders (Sibille et al., 2011), even though the expression of CB changes during adolescence, but CR expression remains steady (Caballero et al., 2014). BDNF levels in cortical cell culture differentially modulated CB and CR (Fiumelli et al., 2000), raising the possibility that MS exposure will effect their expression. To better understand sensitive period-related changes, the inter-relationships between these GABAergic markers, TrkB, and depressive-like behavior were examined in the amygdala and the PFC across two different manipulations. 2. Methods and materials 2.1. Subjects Pregnant female multiparous Sprague-Dawley rats (250–275 g) were obtained from Charles River Laboratories (Wilmington, MA) on 9
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markers (PV, CR, CB, and TrkB) and the number of escapes in the CON/ MS group. All correlation data were subject to bootstrap analysis with 10,000 random resampling analyses; data were considered significant following this analysis if they met the adjusted P values (P < 0.05). The relationships between CON versus MS were then statistically compared with Fisher's z transformation followed by Bonferroni correction to determine if they were significantly different. Post-hoc analyses were adjusted with Bonferroni correction. Significance was set at P < 0.05. There were too few depressive subjects in the CON2 or ISO groups to perform correlational analyses on these data. 3. Results Dichotomized data analysis: Prior to dichotomization, rats in the MS group had an average number of 16 ± 2.9 escapes and CONs had 19.3 ± 1.9, escapes, which was not statistically different (P > 0.2). Isolation rats had an average of 19.6 ± 3.4 versus CON2 rats with 20.3 ± 3.6, and were not different (P > 0.5). Helplessness data were dichotomized based on one standard deviation from the mean (Ritov et al., 2016). As the average of the CON group was 20 ± 7 (1 SD), we used < 14 escapes as our cut-off to define anhedonia. Subjects with < 14 escapes were considered anhedonic and > 13 escapes considered non-anhedonic (Fig. 1B). Significant differences were found between the number of depressive versus non-depressive subjects overall (Mann-Whitney U, P < 0.001). The percentage of subjects who met criteria within each group are shown in Fig. 1C. Those meeting criterion were n = 4 of 18 and 4 of 9, for CON and MS respectively. The ISO group had a different profile. An n = 2 of 7 and 1 of 7 for CON2 and ISO, respectively, were anhedonic. Protein levels of PV and TrkB were dichotomized based on the number of escapes described above (Fig. 2A and B). In the amygdala, escape number significantly identified different levels of protein expression for both PV and TrkB in MS subjects (Kruskal-Wallis: P < 0.05 for both), but scores did not distinguish the data further between CON and MS within each dichotomized group (Fig. 2A). In the PFC, both PV and TrkB were significantly different in MS subjects between non-depressive and depressive groups; TrkB differed by escape number between CON and MS subjects in > 13 escapes (Kruskal-Wallis: P < 0.01). Individual comparisons were determined with a Dunn's test, which revealed that TrkB was significantly higher in MS subjects relative to the CON subjects in the non-depressive like group (P < 0.02). In CON2 and ISO animals, PV was significantly reduced in the < 14 depressive group in amygdala only. Correlational analysis: Since we found overall changes in PV expression levels in the amygdala, we investigated GABA's interactions with depressive behavior further by including other markers of GABA expression, such as CB and CR for the CON/MS groups. As the CON2 and ISO data are highly polarized between low and high escape values, this analysis is invalid. Dichotomizing the data minimizes individual differences that may offer additional insight into how GABA markers (PV, CB, and CR) and TrkB influence escape behavior. In Fig. 3A (top), Pearson's correlation with bootstrap adjustment indicated that MS subjects show a positive relationship with the number of escapes and PV expression in the amygdala (r = 0.63, P < 0.05), whereas the relationship was weaker in CON subjects, r = 0.36, P > 0.05. In the PFC, the correlation between escape number and PV was r = 0.14 (NS) in CON subjects. However, the strong relationship (r = 0.93, P < 0.001) in MS subjects indicates that fewer escapes were highly correlated with lower PV expression in this region. The correlations between the number of escapes and TrkB were similar to PV relationships. In the amygdala, higher levels of TrkB were associated with a less depressive phenotype (increased escapes); this relationship is significant in the MS group (r = 0.81; P < 0.01), but not the CON group (r = 0.41; P > 0.1). TrkB, however, was very distinctively associated with depressive scores in the MS group only (r = 0.91, P > 0.01) with no relationship in the CON group (r = −0.05; P > 0.6) in the PFC. Fig. 3
Fig. 1. Experimental design and prevalence of anhedonia. A) Timeline of treatments; LH: learned helplessness; P: postnatal day; maternal separation (MS), Control (CON) and social isolation (ISO) and CON2, the control group for ISO; B) Subjects were dichotomized based on one standard deviation from the number of escapes in the CON or CON2 group, for MS and ISO, respectively with < 14 suggesting anhedonia. Means ± SE presented, with the n for each group included; **P < 0.01. C) A pie chart represents the percentage of rats with a non-anhedonic phenotype and an anhedonic phenotype within the (left) CON group (n = 18) and the MS group (n = 9) and the (right) CON2 (n = 7) and ISO group (n = 7).
Laboratories), blocked with Odyssey blocking buffer (LI-COR Biosciences, Lincoln, NE) in PBS for 60 min at room temperature, and incubated with primary polyclonal antibodies to PV (14–17 kDa; 1:10,000; Thermo Scientific, Rockford, IL), CB (28 kDa; 1:3000; Sigma), CR (31 kDa; 1:1000; Millipore), or TrkB (140 kDa; 1:1000; Cell Signaling Technology) in Odyssey blocking buffer (LI-COR Biosciences) in PBS containing 0.1–0.2% Tween (PBS-T) overnight at 4 °C. The membranes were rinsed in PBS-T and incubated for 1 h at room temperature in IRDye 800-conjugated anti-rabbit (LI-COR Biosciences) or anti-rat (LI-COR Biosciences) in 1:20,000 Odyssey blocking buffer in 0.1% PBST. Control for protein loading was achieved with β-tubulin (55 kDa; anti-mouse; 1:10,000, Covance Laboratories, Dedham, MA) visualized with a LI-COR secondary in PBS-T. Proteins were detected using the Odyssey infrared imaging system (excitation/emission filters at 780 nm/820 nm range). All protein values were normalized to β-tubulin expression. Statistical Analyses: Following dichotomization into depressive versus non-depressive groups based on one standard deviation from the CON mean, categorical data for helplessness were analyzed with the non-parametric analysis of Mann-Whitney U. Dichotomized Western data were analyzed with a Kruskal-Wallis for non-parametric data followed by a Dunn's test when significant overall differences were found (SPSS v. 22.0 Chicago, IL). Correlational analyses (Pearson's r) were also used to examine individual relationships between biochemical
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Fig. 3. A) Correlations between PV (top) and TrkB (middle) expression and depressivelike behavior in the (left) amygdala and the (right) prefrontal cortex (PFC) in the CON and MS groups. Too few depressive subjects (< 14) in the CON2/ISO group prevented a meaningful analysis. Pearson's r values are presented; with Fisher's Z transformation showing significance * P < 0.05, **P < 0.001. B) Representative Western immunoblots of tubulin (a loading control), PV, and TrkB in the amygdala (left) and the PFC (right).
between the regions. The correlation between the amygdala/PFC in CON subjects was not significant for PV or TrkB (r = −0.29 and 0.24, respectively, P > 0.1), but they were for MS subjects (PV and TrkB: r = 0.62 and 0.89, respectively, P > 0.05). The relationships between the number of escapes and CB and CR were also investigated. Both CB and CR levels in the amygdala demonstrated significant correlations with the number of escapes in the MS subjects (Fig. 4A; r = 0.64 and −0.64; P < 0.05), but not in CON subjects (P > 0.1 based on Fisher's z transformation). When the data were dichotomized, group differences between MS and CON females were observed for CR in the non-depressive condition for both regions (Fig. 4B), but not for CB (not shown). Significant differences were observed for CR in the amygdala (Kruskal-Wallis: P < 0.02), with Dunn's test determining that group differences were present in both non-depressive and depressive groups (P < 0.02). However, while significant differences were observed in the PFC between depressive groups (Kruskal-Wallis: P < 0.02), these differences in PFC CR were mainly driven by the non-depressive group (Dunn's: P < 0.01).
Fig. 2. Parvalbumin (PV) and TrkB protein levels for the A) amygdala and B) prefrontal cortex; (left) CON and MS subjects and (right) CON2 and ISO subjects dichotomized based on depression scores that were based on one standard deviation from CON and CON2 means. Western blot data were then normalized to the > 13 CON and CON2 values within each period. Means ± SE presented, with * P < 0.05; **P < 0.01.
B shows representative immunoblots of TrkB and PV. Fisher's z transformation was used to determine whether these correlations differed from each other by taking into account multiple comparisons. The MS and CON group correlations were significantly different for PV in the amygdala (z = −0.64, P < 0.05), but not for TrkB. In the PFC, the PV correlations between groups were different (z = −3.1, P < 0.001) as were the group comparisons for TrkB (z = −3.3, P < 0.001). Inter-correlations between the PFC and amygdala of individual subjects overall was investigated to determine any relationships
4. Discussion Individual changes in the GABA markers as they relate to helplessness in the amygdala and the PFC have not been examined previously in MS and ISO females. The strong correlation between escape number and PV following MS or in CON subjects within the PFC 11
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why ISO alone in weaned rats was insufficient to produce anhedonia. Exposure to ISO between P20-40 did not alter the prevalence of anhedonia, although we have recently observed an increase in helplessness in ISO females (Lukkes and Andersen, unpublished observation). It is also possible that the testing of subjects two days after isolation was insufficient to allow the stress effects to incubate. Whether the effects of MS and ISO produce similar levels of stress is unclear. When comparisons between MS and ISO are made directly within the same laboratory, the effects of MS versus ISO are either different (Biggio et al., 2014) or the same (Vargas et al., 2016). In a study by Biggio et al. (2014), ISO starting at P51 in male rats produced a significantly greater decrease in baseline levels of plasma corticosterone relative to MS subjects. In contrast, ISO that is initiated at P21, like our study, found plasma corticosterone levels were the same across these two conditions and depression in MS, but not ISO, animals (Vargas et al., 2016). These data suggest that MS has a greater effect on depressive effects than ISO when ISO is initiated at the time of weaning. While ELA is associated with a delay in depression, maltreatment during adolescence typically results in depression during adolescence (Teicher et al., 2009). A possible protective effect of ISO is suggested by a decrease in the prevalence of anhedonia from 44% in MS rats to 22–28% in controls to 12% in ISO rats. Two differences of significance exist between these groups. First, PV levels are elevated in the MS rats that exhibited > 13 escapes relative to those subjects that were depressive-like (< 14). The > 13 group could represent either rats that were resilient to MS, exhibited a different abnormal behavior associated with elevated PV levels, or both. Second, PV and TrkB levels were significantly lower in the PFC of MS, but not ISO, subjects who met the criterion of < 14 escapes for depressive behavior. Reduced PV and TrkB that are found in this < 14 group are consistent with the correlations in Fig. 3, where low PV and TrkB were strongly associated with depressive-like behavior. The preclinical data of low PV in the amygdala of MS subjects are consistent with observations of increased emotionality and blood flow responses to emotional stimuli in children with an ELA history. Independent of condition, rats with fewer escapes had low levels of PV in the amygdala. These results are similar to reports of reduced forced swim immobility following muscimol in the amygdala (Raineki et al., 2012), but forced swim changes in ISO animals have not been found (Hall et al., 1998), (Raz, 2013), but see (Gilles and Polston, 2017). A decrease in amygdala PV was observed in a subset of CON2 and ISO subjects, consistent with reports of less c-fos activity in amygdalar GABAergic interneurons following ISO (Lukkes et al., 2012) and greater anxiety (Lukkes et al., 2009b). Finally, low BDNF in the amygdala has been found in rats that receive less maternal care (Macri et al., 2010). Low PV levels in the PFC of CON or MS females with few escapes are common to reports of low GABA in humans with depressive symptoms (Gabbay et al., 2012; Klempan et al., 2009; Sanacora et al., 2000). Surprisingly, the results of this study show that low PV in the PFC can occur independently of an ELA history. ELA may further decrease PFC PV, doubling the prevalence of anhedonia. The strong amygdala/PFC correlation in MS, but not in CON, CON2 or ISO subjects, suggests that PV loss in both regions simultaneously is needed to increase depression risk (Fig. 5). Alternatively, the shock in the LH paradigm could produce the observed changes in PV and TrkB. Our previous observations of PV loss in MS animals that were not exposed to the LH paradigm argue against this possibility (Brenhouse and Andersen, 2011; Leussis et al., 2012). Inflammation may also enhance PV vulnerability in MS animals (Brenhouse and Andersen, 2011; Canetta et al., 2016; Dugan et al., 2009; Hennessy et al., 2011; Wieck et al., 2013). The dichotomization of behavioral data into anhedonia and nonanhedonia resulted in a distribution of 22–28% of the subset of CON/ CON2 rats versus 44% of the MS rats. The lifetime prevalence rate of depression is 20% in the general population (Kessler et al., 1994) and 67% in an ELA-exposed population (Andersen, 2015; Teicher et al., 2009). While we categorized groups by one standard deviation of the
Fig. 4. A) Correlations between calbindin (top) and calretinin (middle) expression and depressive-like behavior in the amygdala and the prefrontal cortex (PFC). Pearson's r values are presented; *P < 0.05. B) Calretinin data from the amygdala (left) and PFC (right) from CON/MS subjects dichotomized for anhedonia scores. Means ± SE presented, with * P < 0.05; **P < 0.01; #P < 0.06.
supports the importance of GABAergic expression in depressive/anhedonic behavior, similar to reports in humans (Gabbay et al., 2012; Klempan et al., 2009; Sanacora et al., 1999). Changes in amygdala PV also correlated with escape behavior in MS subjects, although not as strongly as in the PFC. Significant correlations between the number of escapes and PV and TrkB in the PFC suggest that depressive-like behaviors following MS exposure are associated with a sensitive period. Exposure to the ISO stressor during a later developmental period (Lukkes et al., 2009b) failed to change anhedonia with this paradigm. Clinically, early life maltreatment accelerates the onset of depression (Teicher et al., 2009; Widom et al., 2007). We hypothesized that exposure to MS would facilitate depressive behavior by keeping the sensitive period open. Low PV/TrkB levels in both amygdala and PFC could be interpreted as an open sensitive period for emotionality and its cognitive control, where the environment would have greater influences on mood. This hypothesis requires additional testing, but a proposed model of how ELA alters sensitive periods is presented in Fig. 5. We include inflammation as an additional factor whereby exposure to MS during a sensitive period of development may render subjects more vulnerable to later insult. Inflammation is elevated in some animals with a MS (Brenhouse and Andersen, 2011; Lukkes et al., 2017; Wieck et al., 2013) or ISO history (Ko and Liu, 2016), although we did not observe changed in interleukin-6 (unpublished). As the dam may mediate the inflammation (Hennessy et al., 2013), this may explain 12
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Fig. 5. Proposed model of sensitive period changes following early life adversity that leads to an anhedonic depression. Evidence suggests that the sensitive period for anxiety as mediated by the amygdala opens during the juvenile period. As PV and TrkB levels naturally rise and close the amygdala sensitive period, the sensitive period in the prefrontal cortex (PFC) opens to environmental stimuli during adolescence. If allowed to develop normally, PV and TrkB levels rise and close the window at each stage. However, if inflammation or other mediating factors are present and PV and TrkB levels remain low, the sensitive period is extended and vulnerability to outside events remains. The result is anxiety and depression, two common observations following exposure to early life adversity. In contrast, animals exposed to social isolation demonstrate only decreased PV levels in the amygdala, with no changes in the PFC, and relatively little anhedonic depression.
from the number of immunoreactive cells (Caballero et al., 2014), although our studies in MS males demonstrated a 38% decrease in PVimmunoreactive neurons (Brenhouse and Andersen, 2011) and a 42 ± 8% decrease with Westerns (Leussis et al., 2012). Second, the design of our blots minimized variability by running groups of subjects on a single gel with CON/MS and CON2/ISO run simultaneously. Protein values were normalized to tubulin levels to further minimize variance. Third, we included different cohorts of animals as a replication sample. These additional subjects demonstrated reliability in the findings. For example, we observed a correlation of r = 0.97 in the first MS group (n = 6) and a correlation of r = 0.93 in the second cohort (n = 6 + 3 additional). The results of this study demonstrate the importance of individual differences in PV and TrkB levels in the expression of anhedonia. By comparing the results from MS subjects to ISO subjects, we revealed a sensitive period for anhedonia as indicated by a 1) doubled prevalence following MS, but not ISO; and 2) reductions in PV in both the amygdala and the PFC that was absent in ISO animals. Notably, CON and CON2 and ISO subjects with atypical escapes had lower PV levels in the amygdala. These data further suggest that ELA exposure may maintain heightened environmental sensitivity and advocate for earlier interventions in this vulnerable population.
CON mean (Ritov et al., 2016), others have a priori defined a few missed escapes as ‘depressive’ (Shirayama et al., 2002) or did not dichotomize (Leussis et al., 2012). Regardless, the doubling of anhedonia prevalence is similar to clinical prevalence levels, and thus may further aid in determining common causes of depression where ELA plays a permissive role. Increased levels of PV and TrkB in MS relative to CON females may signal resilience, or possibly, another outcome of ELA such as an externalizing disorder (D'Andrea et al., 2012). In depression-resilient humans, greater PFC activity and less amygdala activity facilitate adapting to stress quickly (Gee et al., 2013). As changes in GABA inversely correlate with blood oxygenated levels in the brain (Northoff et al., 2007), the observation that elevated PFC PV levels were associated with more escapes is consistent with its role in adapting to stress. Calretinin levels were increased in the amygdala, but not PFC, of MS subjects with greatest increases in the anhedonia phenotype. A positive relationship was observed between TrkB levels and CB expression (not shown), consistent with earlier findings in cell culture (Fiumelli et al., 2000). However, non-significant relationships were observed for CR neurons and TrkB in both regions, where BDNF is reported to decrease CR expression (Fiumelli et al., 2000). We readily acknowledge caveats to our methodological/analytical approaches. Proteins were quantified with Westerns, without quantifying the number of neurons. Functional protein expression may differ 13
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Conflicts of interest
Gilles, Y.D., Polston, E.K., 2017. Effects of social deprivation on social and depressive-like behaviors and the numbers of oxytocin expressing neurons in rats. Behav. Brain Res. 328, 28–38. Gogolla, N., Caroni, P., Luthi, A., Herry, C., 2009. Perineuronal nets protect fear memories from erasure. Science 325 (5945), 1258–1261. Greenough, W.T., Black, J.E., Wallace, C.S., 1987. Experience and brain development. Child Dev. 58 (3), 539–559. Guilloux, J.P., Douillard-Guilloux, G., Kota, R., Wang, X., Gardier, A.M., Martinowich, K., Tseng, G.C., Lewis, D.A., Sibille, E., 2012. Molecular evidence for BDNF- and GABArelated dysfunctions in the amygdala of female subjects with major depression. Mol. Psychiatr. 17 (11), 1130–1142. Hall, F.S., Huang, S., Fong, G.F., Pert, A., 1998. The effects of social isolation on the forced swim test in Fawn hooded and Wistar rats. J. Neurosci. Meth. 79 (1), 47–51. Hennessy, M.B., Jacobs, S., Schiml, P.A., Hawk, K., Stafford, N., Deak, T., 2013. Maternal inhibition of infant behavioral response following isolation in novel surroundings and inflammatory challenge. Dev. Psychobiol. 55 (4), 395–403. Hennessy, M.B., Paik, K.D., Caraway, J.D., Schiml, P.A., Deak, T., 2011. Proinflammatory activity and the sensitization of depressive-like behavior during maternal separation. Behav. Neurosci. 125 (3), 426–433. Holland, F.H., Ganguly, P., Potter, D.N., Chartoff, E.H., Brenhouse, H.C., 2014. Early life stress disrupts social behavior and prefrontal cortex parvalbumin interneurons at an earlier time-point in females than in males. Neurosci. Lett. 566, 131–136. Huang, Z.J., Kirkwood, A., Pizzorusso, T., Porciatti, V., Morales, B., Bear, M.F., Maffei, L., Tonegawa, S., 1999. BDNF regulates the maturation of inhibition and the critical period of plasticity in mouse visual cortex. Cell 98 (6), 739–755. Kessler, R.C., McGonagle, K.A., Zhao, S., Nelson, C.B., Hughes, M., Eshleman, S., Wittchen, H.U., Kendler, K.S., 1994. Lifetime and 12-month prevalence of DSM-III-r psychiatric disorders in the United States. Results from the National comorbidity survey. Arch. Gen. Psychiatr. 51 (1), 8–19. Klempan, T.A., Sequeira, A., Canetti, L., Lalovic, A., Ernst, C., ffrench-Mullen, J., Turecki, G., 2009. Altered expression of genes involved in ATP biosynthesis and GABAergic neurotransmission in the ventral prefrontal cortex of suicides with and without major depression. Mol. Psychiatr. 14 (2), 175–189. Ko, C.Y., Liu, Y.P., 2016. Disruptions of sensorimotor gating, cytokines, glycemia, monoamines, and genes in both sexes of rats reared in social isolation can be ameliorated by oral chronic quetiapine administration. Brain Behav. Immun. 51, 119–130. Lehmann, J., Feldon, J., 2000. Long-term biobehavioral effects of maternal separation in the rat: consistent or confusing? Rev. Neurosci. 11 (4), 383–408. Leussis, M.P., Freund, N., Brenhouse, H.C., Thompson, B.S., Andersen, S.L., 2012. Depressive-like behavior in adolescents after maternal separation: sex differences, controllability, and GABA. Dev. Neurosci. 34 (2–3), 210–217. Lukkes, J.L., Burke, A.R., Zelin, N.S., Hale, M.W., Lowry, C.A., 2012. Post-weaning social isolation attenuates c-Fos expression in GABAergic interneurons in the basolateral amygdala of adult female rats. Physiol. Behav. 107 (5), 719–725. Lukkes, J.L., Meda, S., Thompson, B.S., Freund, N., Andersen, S.L., 2017. Early life stress and later peer distress on depressive behavior in adolescent female rats: effects of a novel intervention on GABA and D2 receptors. Behav. Brain Res. 330, 37–45. Lukkes, J.L., Mokin, M.V., Scholl, J.L., Forster, G.L., 2009a. Adult rats exposed to earlylife social isolation exhibit increased anxiety and conditioned fear behavior, and altered hormonal stress responses. Horm. Behav. 55 (1), 248–256. Lukkes, J.L., Watt, M.J., Lowry, C.A., Forster, G.L., 2009b. Consequences of post-weaning social isolation on anxiety behavior and related neural circuits in rodents. Front. Behav. Neurosci. 3, 18. Macri, S., Laviola, G., Leussis, M.P., Andersen, S.L., 2010. Abnormal behavioral and neurotrophic development in the younger sibling receiving less maternal care in a communal nursing paradigm in rats. Psychoneuroendocrinology 35, 392–402. Maier, S.F., Watkins, L.R., 2005. Stressor controllability and learned helplessness: the roles of the dorsal raphe nucleus, serotonin, and corticotropin-releasing factor. Neurosci. Biobehav. Rev. 29 (4–5), 829–841. Malter Cohen, M., Jing, D., Yang, R.R., Tottenham, N., Lee, F.S., Casey, B.J., 2013. Earlylife stress has persistent effects on amygdala function and development in mice and humans. Proc. Natl. Acad. Sci. U.S.A. 110 (45), 18274–18278. Marusak, H.A., Martin, K.R., Etkin, A., Thomason, M.E., 2015. Childhood trauma exposure disrupts the automatic regulation of emotional processing. Neuropsychopharmacology 40 (5), 1250–1258. Matthews, K., Robbins, T.W., 2003. Early experience as a determinant of adult behavioural responses to reward: the effects of repeated maternal separation in the rat. Neurosci. Biobehav. Rev. 27 (1–2), 45–55. Morishita, H., Cabungcal, J.H., Chen, Y., Do, K.Q., Hensch, T.K., 2015. Prolonged period of cortical plasticity upon redox dysregulation in fast-spiking interneurons. Biol. Psychiatr. 78 (6), 396–402. Napolitano, A., Shah, K., Schubert, M.I., Porkess, V., Fone, K.C., Auer, D.P., 2014. In vivo neurometabolic profiling to characterize the effects of social isolation and ketamineinduced NMDA antagonism: a rodent study at 7.0 T. Schizophr. Bull. 40 (3), 566–574. Northoff, G., Walter, M., Schulte, R.F., Beck, J., Dydak, U., Henning, A., Boeker, H., Grimm, S., Boesiger, P., 2007. GABA concentrations in the human anterior cingulate cortex predict negative BOLD responses in fMRI. Nat. Neurosci. 10 (12), 1515–1517. Ogawa, T., Kuwagata, M., Hori, Y., Shioda, S., 2007. Valproate-induced developmental neurotoxicity is affected by maternal conditions including shipping stress and environmental change during early pregnancy. Toxicol. Lett. 174 (1–3), 18–24. Plotsky, P.M., Meaney, M.J., 1993. Early, postnatal experience alters hypothalamic corticotropin-releasing factor (CRF) mRNA, median eminence CRF content and stressinduced release in adult rats. Brain research. Mol. Brain Res. 18 (3), 195–200. Pryce, C.R., Azzinnari, D., Spinelli, S., Seifritz, E., Tegethoff, M., Meinlschmidt, G., 2011. Helplessness: a systematic translational review of theory and evidence for its
None. Funding The National Institute on Drug Abuse (DA-015403 and DA-026485 [SLA]), the Simches Family (SLA), and a 2013 NARSAD Young Investigator Award from the Brain and Behavior Research Foundation (JLL) supported the project. Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx. doi.org/10.1016/j.jpsychires.2018.02.005. References Andersen, S.L., 2003. Trajectories of brain development: point of vulnerability or window of opportunity? Neurosci. Biobehav. Rev. 27 (1–2), 3–18. Andersen, S.L., 2015. Exposure to early adversity: points of cross-species translation that can lead to improved understanding of depression. Dev. Psychopathol. 27 (2), 477–491. Andersen, S.L., Teicher, M.H., 2008. Stress, sensitive periods and maturational events in adolescent depression. Trends Neurosci. 31 (4), 183–191. Berretta, S., Benes, F.M., 2006. A rat model for neural circuitry abnormalities in schizophrenia. Nat. Protoc. 1 (2), 833–839. Biggio, F., Pisu, M.G., Garau, A., Boero, G., Locci, V., Mostallino, M.C., Olla, P., Utzeri, C., Serra, M., 2014. Maternal separation attenuates the effect of adolescent social isolation on HPA axis responsiveness in adult rats. European Neuropsychopharmacology. J. Eur. Coll. Neuropsychopharmacol. 24 (7), 1152–1161. Bloomfield, C., French, S.J., Jones, D.N., Reavill, C., Southam, E., Cilia, J., Totterdell, S., 2008. Chandelier cartridges in the prefrontal cortex are reduced in isolation reared rats. Synapse 62 (8), 628–631. Bradford, M., 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 7, 248–254. Brenhouse, H.C., Andersen, S.L., 2011. Nonsteroidal anti-inflammatory treatment prevents delayed effects of early life stress in rats. Biol. Psychiatr. 70 (5), 434–440. Caballero, A., Flores-Barrera, E., Cass, D.K., Tseng, K.Y., 2014. Differential regulation of parvalbumin and calretinin interneurons in the prefrontal cortex during adolescence. Brain Struct. Funct. 219 (1), 395–406. Caldji, C., Diorio, J., Meaney, M.J., 2003. Variations in maternal care alter GABA(A) receptor subunit expression in brain regions associated with fear. Neuropsychopharmacology 28 (11), 1950–1959. Caldji, C., Tannenbaum, B., Sharma, S., Francis, D., Plotsky, P.M., Meaney, M.J., 1998. Maternal care during infancy regulates the development of neural systems mediating the expression of fearfulness in the rat. Proc. Natl. Acad. Sci. U.S.A. 95 (9), 5335–5340. Canetta, S., Bolkan, S., Padilla-Coreano, N., Song, L.J., Sahn, R., Harrison, N.L., Gordon, J.A., Brown, A., Kellendonk, C., 2016. Maternal immune activation leads to selective functional deficits in offspring parvalbumin interneurons. Mol. Psychiatr. 21 (7), 956–968. Committee for the Update of the Guide for the Care and Use of Laboratory Animals, 2011. N.R.C. Guide for the Care and Use of Laboratory Animals Eighth Ed. The National Academies. (Washington, D.C). D'Andrea, W., Ford, J., Stolbach, B., Spinazzola, J., van der Kolk, B.A., 2012. Understanding interpersonal trauma in children: why we need a developmentally appropriate trauma diagnosis. Am. J. Orthopsychiatry 82 (2), 187–200. Dugan, L.L., Ali, S.S., Shekhtman, G., Roberts, A.J., Lucero, J., Quick, K.L., Behrens, M.M., 2009. IL-6 mediated degeneration of forebrain GABAergic interneurons and cognitive impairment in aged mice through activation of neuronal NADPH oxidase. PLoS One 4 (5), e5518. Fiumelli, H., Kiraly, M., Ambrus, A., Magistretti, P.J., Martin, J.L., 2000. Opposite regulation of calbindin and calretinin expression by brain-derived neurotrophic factor in cortical neurons. J. Neurochem. 74 (5), 1870–1877. Freund, N., Thompson, B.S., Denormandie, J., Vaccarro, K., Andersen, S.L., 2013. Windows of vulnerability: maternal separation, age, and fluoxetine on adolescent depressive-like behavior in rats. Neuroscience 249, 88–97. Gabbay, V., Mao, X., Klein, R.G., Ely, B.A., Babb, J.S., Panzer, A.M., Alonso, C.M., Shungu, D.C., 2012. Anterior cingulate cortex gamma-aminobutyric acid in depressed adolescents: relationship to anhedonia. Arch. Gen. Psychiatr. 69 (2), 139–149. Gee, D.G., Gabard-Durnam, L.J., Flannery, J., Goff, B., Humphreys, K.L., Telzer, E.H., Hare, T.A., Bookheimer, S.Y., Tottenham, N., 2013. Early developmental emergence of human amygdala-prefrontal connectivity after maternal deprivation. Proc. Natl. Acad. Sci. U.S.A. 110 (39), 15638–15643. Giachino, C., Canalia, N., Capone, F., Fasolo, A., Alleva, E., Riva, M.A., Cirulli, F., Peretto, P., 2007. Maternal deprivation and early handling affect density of calcium binding protein-containing neurons in selected brain regions and emotional behavior in periadolescent rats. Neuroscience 145 (2), 568–578.
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Journal of Psychiatric Research 100 (2018) 8–15
J.L. Lukkes et al.
Shirayama, Y., Chen, A.C., Nakagawa, S., Russell, D.S., Duman, R.S., 2002. Brain-derived neurotrophic factor produces antidepressant effects in behavioral models of depression. J. Neurosci. 22 (8), 3251–3261. Sibille, E., Morris, H.M., Kota, R.S., Lewis, D.A., 2011. GABA-related transcripts in the dorsolateral prefrontal cortex in mood disorders. Int. J. Neuropsychopharmacol. 14 (6), 721–734. Teicher, M.H., Samson, J.A., Polcari, A., Andersen, S.L., 2009. Length of time between onset of childhood sexual abuse and emergence of depression in a young adult sample: a retrospective clinical report. J. Clin. Psychiatr. 70 (5), 684–691. Tottenham, N., Hare, T.A., Millner, A., Gilhooly, T., Zevin, J.D., Casey, B.J., 2011. Elevated amygdala response to faces following early deprivation. Dev. Sci. 14 (2), 190–204. Vargas, J., Junco, M., Gomez, C., Lajud, N., 2016. Early life stress increases metabolic risk, HPA Axis Reactivity, and depressive-like behavior when combined with postweaning social isolation in rats. PLoS One 11 (9) e0162665. Widom, C.S., DuMont, K., Czaja, S.J., 2007. A prospective investigation of major depressive disorder and comorbidity in abused and neglected children grown up. Arch. Gen. Psychiatr. 64 (1), 49–56. Wieck, A., Andersen, S.L., Brenhouse, H.C., 2013. Evidence for a neuroinflammatory mechanism in delayed effects of early life adversity in rats: relationship to cortical NMDA receptor expression. Brain Behav. Immun. 28, 218–226.
relevance to understanding and treating depression. Pharmacol. Therapeut. 132 (3), 242–267. Raineki, C., Cortes, M.R., Belnoue, L., Sullivan, R.M., 2012. Effects of early-life abuse differ across development: infant social behavior deficits are followed by adolescent depressive-like behaviors mediated by the amygdala. J. Neurosci. 32 (22), 7758–7765. Raz, S., 2013. Ameliorative effects of brief daily periods of social interaction on isolationinduced behavioral and hormonal alterations. Physiol. Behav. 116–117, 13–22. Ritov, G., Boltyansky, B., Richter-Levin, G., 2016. A novel approach to PTSD modeling in rats reveals alternating patterns of limbic activity in different types of stress reaction. Mol. Psychiatr. 21 (5), 630–641. Sanacora, G., Mason, G.F., Krystal, J.H., 2000. Impairment of GABAergic transmission in depression: new insights from neuroimaging studies. Crit. Rev. Neurobiol. 14 (1), 23–45. Sanacora, G., Mason, G.F., Rothman, D.L., Behar, K.L., Hyder, F., Petroff, O.A., Berman, R.M., Charney, D.S., Krystal, J.H., 1999. Reduced cortical gamma-aminobutyric acid levels in depressed patients determined by proton magnetic resonance spectroscopy. Arch. Gen. Psychiatr. 56 (11), 1043–1047. Seidel, K., Helmeke, C., Poeggel, G., Braun, K., 2008. Repeated neonatal separation stress alters the composition of neurochemically characterized interneuron subpopulations in the rodent dentate gyrus and basolateral amygdala. Developmental Neurobiol. 68 (9), 1137–1152.
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