Stress duration modulates the spatiotemporal patterns of spine formation in the basolateral amygdala Rupshi Mitra*, Shantanu Jadhav*���, Bruce S. McEwen�����, Ajai Vyas*, and Sumantra Chattarji*�� *National Centre for Biological Sciences, Bangalore 560065, India and ���Laboratory of Neuroendocrinology, The Rockefeller University, 1230 York Avenue, New York, NY 10021 Contributed by Bruce S. McEwen, May 13, 2005 It has long been hypothesized that morphological and numerical alterations in dendritic spines underlie long-term structural encod- ing of experiences. Here we investigate the efficacy of aversive experience in the form of acute immobilization stress (AIS) and chronic immobilization stress (CIS) in modulating spine density in the basolateral amygdala (BLA) of male rats. We find that CIS elicits a robust increase in spine density across primary and secondary branches of BLA spiny neurons. We observed this CIS-induced spinogenesis in the BLA 1 d after the termination of CIS. In contrast, AIS fails to affect spine density or dendritic arborization when measured 1 d later. Strikingly, the same AIS causes a gradual increase in spine density 10 d later but without any effect on dendritic arbors. Thus, by modulating the duration of immobiliza- tion stress, it is possible to induce the formation of new spines without remodeling dendrites. However, unlike CIS-induced spine formation, the gradual increase in spine density 10 d after a single exposure to AIS is localized on primary dendrites. Finally, this delayed induction of BLA spinogenesis is paralleled by a gradual development of anxiety-like behavior on the elevated plus-maze 10 d after AIS. These findings demonstrate that stressful experi- ences can lead to the formation of new dendritic spines in the BLA, which is believed to be a locus of storage for fear memories. Our results also suggest that stress may facilitate symptoms of chronic anxiety disorders like post-traumatic stress disorder by enhancing synaptic connectivity in the BLA. anxiety dendritic remodeling immobilization synapse rats Tplasticity he search for cellular substrates underlying experience-related has focused on dendritic spines ever since Ramo ��n y Cajal (1, 2) proposed that the storage of long-term memory involves strengthening of synaptic connections (or even a building of new connections) among central neurons. More recently, the focus has shifted toward understanding the physiological and molecular basis of synaptic plasticity mechanisms, such as long-term potentiation (LTP), and their relationship to spine plasticity and ultimately behavioral memory (3, 4). A majority of these studies have exam- ined the hippocampus. Although the hippocampus is required for the acquisition and temporary storage of declarative memory, studies with human subjects and animal models suggest that more permanent morphological correlates of long-term memory storage are unlikely to reside in the hippocampus (5���7). In this context, the amygdala, for which the neural circuit underlying emotional mem- ory formation is well characterized (8), provides a significant advantage. The basolateral amygdala (BLA) is believed to be a site of storage for memories of fearful or stressful experiences (9���12). Furthermore, recent studies indicate that the synthesis of new proteins in the BLA is involved in the long-term consolidation of emotional memories (13). Thus, the BLA presents an attractive locus to investigate structural encoding of aversive experiences. Recent reports using rodent models of fear and stress are beginning to identify putative cellular and molecular determi- nants of structural plasticity in the amygdala (14). For example, there is evidence that the Rho GTPase pathway (15) and BDNF (brain-derived neurotrophic factor) signaling (16), both impor- tant regulators of neuronal structure, are involved in fear memory formation and consolidation in the lateral amygdala. Tissue plasminogen activator, which plays a key role in spine plasticity during visual cortical development (17), is also a critical component in the sequence of molecular events linking repeated restraint stress-induced neuronal remodeling in the amygdala with the development of anxiety-like behavior (18). These recent findings, taken together with earlier reports on the role of NMDA receptors in the BLA in fear (19, 20) and anxiety (21), all provide plausible mechanisms that can lead to spine forma- tion as a result of aversive experience. However, there is no direct morphological evidence for experience-induced spine formation in the BLA. In the present study we hypothesize that because animal models of chronic stress potentiate both fear (22, 23) and anxiety (24), they are likely to serve as a useful tool for amplifying molecular mechanisms underlying amygdalar spino- genesis, thereby eliciting robust and detectable changes in spines in the BLA. Hence, here we investigate whether aversive expe- rience, in the form of chronic immobilization stress, leads to numerical alterations in dendritic spines in the rat BLA. Materials and Methods Experimental Animals. Male Wistar rats were used for chronic immobilization stress (CIS) and acute immobilization stress (AIS) protocols. At the beginning of the experiments, CIS animals were between 3 and 3.5 mo of age, whereas the age of AIS and chronic unpredictable stress (CUS) animals at the onset of experiments was 2.5���3 mo. All animals (National Centre for Biological Sciences, Bangalore, India) were housed in groups of three with access to food and water ad libitum, unless specified otherwise in stress protocols. Control animals, which were littermates of the stress-treated animals, were housed in separate cages. Animals were maintained in a temperature-controlled room, with a light dark cycle of 12 h (lights on at 7:00 a.m.). All procedures related to animal maintenance and experimentation were approved by the Institutional Animal Ethics Committee (National Centre for Biological Sciences). Stress Protocols. Rats, randomly assigned to experimental groups, were subjected to CIS, AIS, or CUS. CIS consisted of complete immobilization (2 h d, 10 a.m.���noon) in rodent immobilization bags without access to either food or water, for 10 consecutive d (25, 26). AIS consisted of a single immobilization session of 2 h, after which either 1 d (AIS-1) or 10 d (AIS-10) were allowed to Abbreviations: AIS, acute immobilization stress BLA, basolateral amygdala CIS, chronic immobilization stress CUS, chronic unpredictable stress LTP, long-term potentiation CV, coefficient of variance. ���Present address: Computational Neurobiology Program, 9500 Gilman Drive, University of California at San Diego, La Jolla, CA 92093. ��To whom correspondence may be addressed. E-mail: mcewen@mail.rockefeller.edu or shona@ncbs.res.in. �� 2005 by The National Academy of Sciences of the USA www.pnas.org cgi doi 10.1073 pnas.0504011102 PNAS June 28, 2005 vol. 102 no. 26 9371���9376 NEUROSCIENCE

lapse before behavioral or morphological analysis was con- ducted. CUS, as described earlier, involved exposing rats to several types of stressors, which varied from day to day, for a period of 10 d (26, 27). Control animals were not subjected to any type of stress. Elevated Plus-Maze. The elevated plus-maze, consisting of two opposite open arms (60 15 cm) and two enclosed arms (60 15 cm, surrounded by a 15-cm-high opaque wall), was elevated 75 cm from the ground. The animals were tested on the maze 24 h after the termination of the stress paradigm. Individual trials lasted for 5 min each and were videotaped for subsequent off-line analysis. At the beginning of each trial, animals were placed at the center of the maze, facing an enclosed arm. All trials were conducted between 10 a.m. and 2 p.m., and the maze was cleaned with 5% (vol vol) ethanol solution after each trial. Tissue Preparation. After completion of stress protocols, animals were killed under deep anesthesia. The brain was removed quickly, and blocks of tissue containing the amygdala were dissected and processed for rapid Golgi staining as described (26, 28). Coronal sections (120 m thick) were prepared as described (26). Slides were coded before quantitative analysis, and the code was broken only after the analysis was completed. Analysis of Dendritic Arborization. To be selected for analysis, Golgi-impregnated neurons (Fig. 1A) had to satisfy the following criteria that have been applied in similar morphometric studies (26, 29���31): (i) presence of untruncated dendrites, (ii) consistent and dark impregnation along the entire extent of all dendrites, and (iii) relative isolation from neighboring impregnated neu- rons to avoid interfering with analysis. Both spiny pyramidal-like and stellate neurons from the BLA were selected for analysis on the basis of morphological criteria described in the literature (26, 32, 33). As described (26), our analysis of BLA neurons was restricted to those located between bregma 2.0 mm and 3.2 mm. To analyze effects of AIS on dendritic length and the number of branch points, we carried out morphometry in six neurons per animal in each experimental group (control, AIS-1, and AIS-10). To this end, 3D reconstructions of the selected neurons were accomplished by using the NeuroLucida image analysis system (MicroBrightField, Wiliston, VT) attached to an Olympus BX61 microscope (40 , 0.75 numerical aperture). Analysis of Dendritic Spine Density. By using the same NeuroLucida system (100 , 1.3 numerical aperture, Olympus BX61), all protrusions, irrespective of their morphological characteristics, were counted as spines if they were in direct continuity with the dendritic shaft (Fig. 1A Inset and C). For the purpose of this study, dendrites directly originating from cell soma were classi- fied as primary dendrites, and those originating from primary dendrites were classified as secondary dendrites (Fig. 1B). Moreover, we always selected the first branch that emerged from the primary branch (Fig. 1B) and designated it as the secondary branch to be analyzed. Starting from the origin of the branch, and continuing away from the cell soma, spines were counted along an 80- m stretch of the dendrite. This total length of 80 m was further subjected to a detailed segmental analysis, which consisted of counting the number of spines in successive steps of 8 m each, for a total of 10 steps. The values for number of spines from each 8- m segment, at a given distance from the origin of the branch, were then averaged across all neurons in a particular experimental group. For spine density analysis in chronic stress experiments, five neurons per animal were used in each exper- imental group (control, CIS, and CUS). Finally, it may be noted that our analysis, like all those involving Golgi staining, is likely to lead to a systematic underestimation of spine density because it is not possible to visualize spines pointing directly toward the surface or extending beneath the dendrite (34���36). In the present study, however, no attempt was made to correct for these hidden spines, because of previously reported validation (37) of the use of visible spine counts for comparison between different experimental conditions. Statistical Analysis. Values are reported as mean SEM [along with coefficient of variance (CV)], and percentage changes are calculated with respect to corresponding control values. In all cases, n refers to the number of neurons used for morphometry, Fig. 1. Effects of CIS on spiny BLA neurons. (A) Low- power photomicrograph of a Golgi stain-impregnated pyramidal neuron in the BLA. (Scale bar, 20 m.) (Inset) High-power image of spines on a secondary dendrite from the same neuron. (Scale bar, 20 m.) (B) Sche- matic drawing classifying types of apical dendrites se- lected for spine density analysis. In our analysis, a den- dritic branch emanating directly from the cell soma (��) was defined as a primary branch, whereas a dendrite originating from a primary branch was defined as a secondary branch. Spines were counted, starting from the origin of a branch, in 10 consecutive segments of 8 m each (small tick marks). (C) Photomicrographs of representative segments of primary (Left) and second- ary (Right) branches from control and CIS neurons, demonstrating an increase in the number of spines. (Scale bar, 5 m.) (D) Mean ( SEM) values for spine- density (calculated as the average number of spines per 10 m) of primary (Left) and secondary (Right) branches of spiny BLA neurons from CIS and control groups. *, P 0.05, compared with control, Student���s t test control, n 20 neurons CIS, n 20 neurons. (E) Segmental analysis of the mean ( SEM) number of spines in each successive 8- m segment along primary (Left) and secondary (Right) branches as a function of the distance of that segment from the origin of the branch. *, P 0.05 **, P 0.01, compared with control, Student���s t test. 9372 www.pnas.org cgi doi 10.1073 pnas.0504011102 Mitra et al.