Dendritic morphology of amygdala and hippocampal neurons in more and less predator stress responsive rats and more and less spontaneously anxious handled controls

Abstract

We investigated the neurobiological bases of variation in response to predator stress (PS). Sixteen days after treatment (PS or handling), rats were grouped according to anxiety in the elevated plus maze (EPM). Acoustic startle was also measured. We examined the structure of dendritic trees of basolateral amygdala (BLA) output neurons (stellate and pyramidal cells) and of dorsal hippocampal (DHC) dentate granule cells of less anxious (LA) and more (extremely) anxious (MA) stressed animals (PSLA and PSMA). Handled controls (HC) which were less anxious (HCLA) and spontaneously more anxious (HCMA) equivalently to predator stressed subgroups were also studied. Golgi analysis revealed BLA output neurons of HCMA rats exhibited longer, more branched dendrites with higher spine density than the other groups of rats, which did not differ. Finally, spine density of DHC granule cells was equally depressed in HCMA and PSMA rats relative to HCLA and PSLA rats. Total dendritic length of BLA pyramidal and stellate cells (positive predictor) and DHC spine density (negative predictor) together accounted for 96% of the variance of anxiety of handled rats. DHC spine density was a negative predictor of PSMA and PSLA anxiety, accounting for 70% of the variance. Data are discussed in the context of morphological differences as phenotypic markers of a genetic predisposition to anxiety in handled controls, and a possible genetic vulnerability to predator stress expressed as reduced spine density in the DHC. Significance of findings for animal models of anxiety and hyperarousal comorbidities of PTSD are discussed.

Introduction

Individuals respond to stress and trauma differently. In some, traumatic experience leads to post traumatic stress disorder (PTSD) while others are less affected [1], [2], [3]. Relatively little is known about the molecular and neural substrates of individual differences in response to trauma [1]. However, correlational behavioral research implicates a variety of factors, including personality traits [4], [5] as well as interaction of genetic factors and experiential factors, such as reduced functioning polymorphisms in the serotonin transporter (5-HTTLPR), and life stress or social support at the time of stress [6], [7], [8]. Moreover, the 5-HTTLPR exerts a potent modulatory effect on amygdala reactivity to environmental threat [6]. Since this genetically driven effect exists in healthy subjects, Hariri and colleagues suggest that the 5-HTTLPR may represent a susceptibility factor for affective disorders by biasing the functional reactivity of the human amygdala in the context of stressful life experiences and/or deficient cortical regulatory input [6]. Support for this idea comes from studies associating PTSD with 5-HTTLPR [8], [9], [10], [11], [12], [13]. So, factors affecting functional amygdala reactivity may be important contributors to vulnerability to stress. In this context, it is important to note that right amygdala reactivity to both trauma reminders and general negative stimuli is enhanced in humans diagnosed with PTSD [14], [15].

One way to identify putative causal substrates is to study the effects of stress on brain and behavior of more and less stress vulnerable animals. A useful paradigm in this regard is exposure of rodents to brief predator stress, a putative model of hyperarousal and generalized anxiety characteristics of PTSD [16], [17], [18]. Domesticated strains of laboratory rats retain the fear of predators like a cat, even if they have never been exposed to predators [19], [20]. On exposure to a cat (predator stress – PS) or cat stimuli (predator scent stress – PSS), laboratory rats and mice develop long-lasting (3 weeks or longer) hyperarousal (enhanced acoustic startle response) and anxiety [18], [21], [22], [23], [24], [25]. Like humans following a traumatic event, not all stressed animals respond similarly. Some remain unaffected, showing little fear sensitization [18], [26], [27].

Consistent with human data, serotonin transporter gene knockout mice are more vulnerable to predator stress [7], [28]. This finding provides an interesting parallel to the human clinical literature involving the 5-HTTPLR, and in that context, implicates modulation of amygdala function in vulnerability to predator stress. It is of interest in this regard that predator stress induces a lasting enhancement of excitability of right rodent amygdala, detected as a potentiation of afferent and efferent transmission in basolateral (BLA) and central amygdala [29], [30]. Furthermore, degree of anxiogenic effect of predator stress is tightly predicted by degree of potentiation in amygdala circuitry [31]. Moreover, it has been inferred from electrophysiological evidence that one mechanism mediating predator stress potentiation of amygdala circuitry could be changes in dendritic morphology [29]. In these studies, potentiation of afferent input to BLA from the ventral hippocampus was accompanied by an increase in electrophysiological estimates of maximal receptor binding (Bmax). Among the possible explanations of this finding discussed was an alteration in dendritic morphology resulting in increased receptor binding due to synaptic restructuring or synaptic proliferation. Structural variation which alters neural transmission in BLA could then alter fearful response which highly correlates with BLA transmission.

Indeed, variation in dendritic arbors of BLA neurons are related to the ability of restraint stress to generate anxiety [32], [33]. Anxiety generated by stress and stress hormone is accompanied by BLA dendritic hypertrophy [32], [33], [34], and experimental reduction of dendritic length results in reduction of anxiety [35]. Moreover, once generated, BLA dendritic hypertrophy is as long lasting as stress induced anxiety [33]. Finally recent findings suggest that dendritic hypotrophy in principle output neurons (stellate and pyramidal cells) of the right BLA may be a resilience marker against lasting anxiogenic effects of predator stress [27].

Studies in the dorsal hippocampus (DHC) add to our understanding of individual differences in response to severe stress. In DHC (area CA1), up regulation of ARC gene expression (mRNA) was found in rats unaffected by PSS [36], whereas down regulation of BDNF and up regulation of TrkB receptors was observed in rats made extremely fearful by PSS [37]. These data implicate the dorsal hippocampus in vulnerability to predator stress. Moreover, reduced hippocampal volumes in humans have been implicated as a predisposing factor to PTSD in twin studies [38]. In animal models of stress effects on hippocampal morphology, the loss of hippocampal volume arises from losses in the neuropil involving losses of dendrites and synapses [39] suggesting a substrate for loss of hippocampal volume in humans. In addition, psychosocial and restraint stress induce dendritic atrophy in DHC CA3 neurons of animals [40]. Finally, chronic immobilization stress in rats induces dendritic atrophy and debranching in dorsal hippocampal area CA3 neurons, while inducing hypertrophy of dendrites of BLA stellate and pyramidal neurons. Both effects are associated with increased anxiety in the elevated plus maze. In contrast chronic unpredictable stress had no effects on plus maze anxiety and no effects on hippocampal cells, while producing dendritic atrophy in amygdala bipolar cells [32]. These results indicate that types of stress inducing anxiety can cause contrasting patterns of dendritic remodeling in neurons of the amygdala and hippocampus. Moreover, not all stressors induce anxiety or remodeling of output neurons in the amygdala or DHC.

Given the above considerations, it is timely to ask in more detail if neurons of the BLA and their plasticity are involved in individual differences in lasting response to predator stress. In previous work only the right amygdala was studied and stellate and pyramidal cells were combined in the data analysis. In the present study dendritic morphology of stellate and pyramidal neurons were studied separately. In addition neurons were examined in both right and left amygdalas. Finally spine density was measured, which was not done previously [27]. A similar analysis was done in DHC dentate granule cells. If behavioral changes are dependent on right amygdala excitability, then dendritic morphology differences between predator stress responsive and non responsive rats should be localized to the right amygdala. Finally, unlike previous work, two controls were used. One was a handled control that showed average anxiety-like behavior in the elevated plus maze (EPM) characteristic of a large sample of handled controls used in previous studies (less anxious – LA). A second handled control was spontaneously more (extremely) anxious (MA). Attempts were successful in establishing groups of comparable LA and MA predator stressed rats and handled controls. These four groups were established to tease out dendritic morphology characteristics that reflect anxiety level per se or stress effects on morphology. Two hypotheses can be proposed to explain individual differences. First, in MA animals, stress causes neural expansion in BLA dendrites related to the enhanced anxiety they experience and/or increased spine density, while stress causes dendritic hypotrophy and/or reduction of dendritic spine density in DHC dentate cells. Second, stress causes amygdala dendritic retraction in LA animals and/or reduced spine density, while stress causes dendritic expansion and/or increased dendritic spine density in DHC dentate cells, and together these plastic changes prevent MA effects of trauma. The handled MA and LA groups of comparable anxiety levels provide a control that tested the predator stress specificity of the EPM anxiety related morphological differences. Given that predator stress has been proposed as an animal model for hyperarousal and anxiety aspects of PTSD [16], findings in this study should shed light on mechanisms of vulnerability to these aspects of PTSD.