Prefrontal cortex is a target for glucocorticoids, shows neurochemical changes in response to stress and mediates many of the behaviors that are altered by chronic corticosterone administration. Three weeks of either daily corticosterone injections or 3 h daily restraint stress result in dendritic changes in pyramidal neurons in medial prefrontal cortex. Interestingly, vehicle injection results in similar but less pronounced changes. Thus, the mild stress of daily injections alone may alter morphology of medial prefrontal cortex, suggesting an exquisite sensitivity to chronic stress. To further examine this morphological sensitivity, we assessed the effect of 1 week of daily brief restraint stress on dendritic morphology in medial prefrontal cortex. Male rats were restrained 10 min per day for one week, handled daily or left unhandled. Rats were then overdosed and brains were stained using a Golgi–Cox procedure. Layer II–III pyramidal neurons in medial prefrontal cortex were drawn and dendritic morphology was quantified. One week of daily brief restraint resulted in selective remodeling of apical dendrites, with atrophy of up to 22–35% in distal branches and sparing of proximal branches. This pattern of reorganization is similar to that seen after either corticosterone injections or 3 weeks of daily 3 h restraint stress. Thus, the stress-induced dendritic changes in medial prefrontal cortex occur rapidly, and in response to a mild stressor.
Chronic stress adversely affects both physiology and behavior. A variety of studies have documented its association with increased risk for illness, the development of psychological disorders and changes in cognition. For instance, chronic exposure to a stressor results in the development of gastric ulcers (Henke, 1990). In addition, individuals experiencing increased numbers of stressful life events are more likely to develop respiratory infections (Stone et al., 1987). Likewise, depressed individuals are more likely than nondepressed individuals to have experienced at least one stressful life event prior to diagnosis (Brown and Harris, 1989), and stressful life events appear to increase the probability of a psychotic episode in schizophrenics (Ventura et al., 1989). Animal studies have also demonstrated detrimental effects of stress on many behaviors. For instance, several studies have demonstrated stress-induced deficits on a variety of cognitive tasks, including shuttle escape (Seligman and Maier, 1967), water maze (Altenor et al., 1977), appetitively motivated operant conditioning (Rosellini, 1978) and radial maze tasks (Luine et al., 1994).
Many of the effects of chronic stress are thought to be mediated by increases in glucocorticoids (e.g. Uno et al., 1989), and in fact, both chronic stress and chronic elevations of glucocorticoids have been shown to produce a variety of cognitive deficits. For instance, rats exposed to daily corticosterone injections for 8 weeks demonstrated decreased spontaneous alternation on a T maze (Bardgett et al., 1994). Likewise, 21 day corticosterone implants that produced a 2- to 4-fold increase in serum corticosterone levels in rats impaired acquisition of a passive-avoidance task (Bisagno et al., 2000). In addition, chronic corticosterone treatment has been shown to impair both acquisition of a radial arm maze task (Dachir et al., 1993) and accuracy of recall of spatial information in the Morris water maze (Sousa et al., 2000) in rats. Finally, chronic administration of stress levels of cortisol has been shown to impair response inhibition in squirrel monkeys (Lyons et al., 2000).
The behavioral deficits induced by chronic corticosterone administration have typically been attributed to corticosterone-induced changes in the hippocampus, which is a primary neural target of glucocorticoids (Gerlach and McEwen, 1972) and is involved in many of the behaviors altered by chronic corticosterone administration. Both chronic corticosterone administration and chronic stress result in extensive atrophy of apical dendrites of pyramidal neurons in hippocampal area CA3 (Woolley et al., 1990; Watanabe et al., 1992; Magariños et al., 1996), and administration of cyanoketone, which blocks stress-induced increases in corticosterone, prevents the stress-induced atrophy of CA3 apical dendrites (Magariños and McEwen, 1995).
However, prefrontal cortex is involved in many of the tasks that are influenced by chronic stress and elevations of glucocorticoids. For instance, lesions of prefrontal cortex impair spontaneous alternation, radial maze performance and passive avoidance in rats (see Kolb, 1984) and inhibition of the line-of-sight response in primates (e.g. Dias et al., 1996). Importantly, prefrontal cortex is also a target for glucocorticoids involved in the stress response: [3H]dexamethasone binds to receptors in frontal and prefrontal cortex at ∼75% of the concentration found in hippocampus. In addition, [3H]dexamethasone binding in frontal cortex is altered by both corticosterone treatment and adrenalectomy, indicating the presence of endogenously regulated corticosterone receptors (Meaney and Aitken, 1985). Accordingly, we examined dendritic morphology of layer II–III pyramidal neurons in medial prefrontal cortex after chronic restraint stress (Cook and Wellman, 2004). The results of this study demonstrated that 3 weeks of 3 h daily restraint stress resulted in marked dendritic changes in the apical dendrites of pyramidal neurons in medial prefrontal cortex. While restraint stress failed to significantly alter basilar dendritic arbor, it resulted in significant atrophy of the distal portion of the apical arbor. This effect has been replicated using a more severe but equally prolonged stressor (6 h of daily restraint for 3 weeks; Radley et al., 2004).
The effect of chronic stress on prefrontal dendritic morphology is probably mediated by corticosterone: chronic corticosterone administration dramatically reorganized apical arbors of layer II–III neurons in medial prefrontal cortex, with an increase in dendritic material proximal to the soma, along with a decrease in dendritic material distal to the soma (Wellman, 2001). Interestingly, vehicle-treated animals showed similar but less pronounced changes in apical arbors. The similarity of the vehicle- and corticosterone-treated animals suggests that the stress of daily subcutaneous injections alone may have altered apical dendritic arbors of layer II–III pyramidal neurons in medial prefrontal cortex, and that this effect is corticosterone-mediated.
These findings contrast with the morphological changes seen in hippocampal area CA3 after chronic corticosterone administration. Whereas others have found that chronic injection of vehicle has no effect on the morphology of hippocampal neurons (Woolley et al., 1990), we found that these same vehicle injections resulted in changes in layer II–III neurons in medial prefrontal cortex that were parallel to, but less pronounced than, the corticosterone-induced changes (Wellman, 2001). Thus, the relatively mild stress of daily injections alone appears to alter the morphology of medial prefrontal cortex, suggesting an exquisite sensitivity to chronic stress — perhaps even greater than that seen in the hippocampus.
Moreover, our previous studies assessed the effects of 3 weeks of daily injections or restraint stress (Cook and Wellman, 2004; Wellman, 2001), a duration chosen because it has been shown to produce alterations in hippocampal neuron morphology (Woolley et al., 1990). However, if prefrontal cortical morphology is in fact extremely sensitive to mild chronic stress, then dendritic changes may occur after a shorter duration of stress. Our previous data suggest that the largest stress effects may in fact occur within 1 week: stress-induced reductions in weight gain and elevations in corticosterone were most pronounced in the first 7 days of chronic stress (Cook and Wellman, 2004). Therefore, to further examine the morphological sensitivity of prefrontal cortex to chronic stress, we assessed the effect of just 1 week of daily brief (10 min) restraint stress on dendritic morphology in medial prefrontal cortex.
Materials and Methods
Adult male Sprague–Dawley rats (175–200 g, ∼50 days old at the start of the experiment; n = 15) either were exposed to chronic brief restraint stress or served as controls. Restraint-stressed rats (n = 5) were placed in a plastic rat restrainer in their home cages for 10 min daily for 7 days. Another group of rats (n = 5) were handled twice daily for 7 days. In addition, stressed and handled rats were weighed every other day. The remainder of the rats (n = 5) served as unhandled controls, and were neither handled nor weighed during this period. All rats were group housed in a vivarium with a 12:12 h light/dark cycle (lights on at 7 a.m.), ambient temperature of 23–25°C, and free access to food and water. All experimental procedures occurred between 10:00 a.m. and 4:00 p.m., were carried out in accordance with the guidelines published in the NIH Guide for the Care and Use of Laboratory Animals, and were approved by the Bloomington Institutional Animal Care and Use Committee.
To assess whether the brief restraint was stressful, plasma corticosterone titers were measured in a separate set of restraint-stressed animals (n = 60). Elevations in corticosterone titers due to restraint were assessed by collecting blood from the tail vein either immediately, or 10, 30 or 60 min after being placed in the restrainer [a duration sufficient to obtain peak plasma corticosterone levels (De Souza and Van Loon, 1982)] on days 1, 3 and 7. To ensure that the effects of mild restraint stress were not confounded with the effects of the potentially stressful blood-drawing procedure, a cross-sectional design was employed, in which each rat only had blood drawn once. To establish baseline corticosterone levels, on days 1, 3 and 7, rats were placed in restrainers and had blood collected immediately (n = 15; 5 per group). Other groups of rats were restrained and had blood drawn after 10 min (the duration of the mild stressor) on day 1, 3 or 7 (n = 15; 5 per group). Rats from which samples were taken at either 30 or 60 min (n = 30; 5 per group) were restrained for 10 min, returned to their home cages, and then replaced in the restrainers for blood collection. These final groups were used to assay peak elevations in corticosterone resulting from the 10 min of restraint stress. Tail vein blood was collected in heparinized microcapillary tubes, centrifuged at 2000 g for 15 min to obtain plasma, and corticosterone titers were assessed using a competitive enzyme immunoassay kit (R&D Systems, Minneapolis, MN). This assay has low cross-reactivity with other major steroid hormones, sensitivity typically <27.0 pg/ml, and coefficients of variation within and across assays of ∼7.7% and ∼9.7%, respectively. Stress-induced changes in corticosterone concentrations were evaluated across time using a two-way ANOVA (time after initiation of restraint × day) followed by appropriate planned comparisons. Planned comparisons consisted of two-group F-tests done within the context of the overall ANOVA (Hays, 1994), comparing average corticosterone concentration at either 10, 30 or 60 min to baseline (0 min).
Histology and Dendritic Analysis
On the last day of restraint stress, animals were overdosed with urethane and then perfused with 0.9% saline. Brains were removed and processed using Glaser and Van der Loos' modified Golgi stain (Glaser and Van der Loos, 1981). The tissue was immersed in Golgi–Cox solution (a 1:1 solution of 5% potassium dichromate and 5% mercuric chloride diluted 4:10 with 5% potassium chromate) for 14 days. Brains were then dehydrated in 1:1 absolute ethanol:acetone (3 h), followed by absolute ethanol and then 1:1 ethanol:ether (30 min each). Brains were then infiltrated with a graded series of celloidins before being embedded in 8% celloidin (8% v/v parlodion in 1:1 absolute ethanol:ether). Coronal sections were cut at 150 μm on a sliding microtome (Leica Histoslide 2000). Free-floating sections were then alkalinized in 18.7% ammonia, developed in Dektol (Kodak), fixed in Kodak Rapid Fix (prepared as per package instructions with Solution B omitted), dehydrated through a graded series of ethanols, cleared in xylene, mounted and coverslipped (Glaser and Van der Loos, 1981).
Pyramidal neurons in layer II–III of Cg1–3 area of medial prefrontal cortex (Zilles and Wree, 1995) were drawn. The Cg1–3 area of medial prefrontal cortex is readily identified by its position on the medial wall of rostral cortex, and its location dorsal to infralimbic cortex, which is markedly thinner than the Cg1–3 area and has fewer, less well-defined layers (Zilles and Wree, 1995). Within Cg1–3, layer II–III is readily identifiable in Golgi-stained material based on its characteristic cytoarchitecture. Its position is immediately ventral to the relatively cell-poor layer I (which also contains the distal dendritic tufts of layer II–III pyramidal cells) and immediately dorsal to layer IV; in medial prefrontal cortex, this boundary is pronounced because of the greater cell-packing density and smaller somata of pyramidal cells in layer II–III relative to layer IV (Cajal, 1995; Zilles and Wree, 1995). Pyramidal neurons were defined by the presence of a basilar dendritic tree, a distinct, single apical dendrite, and dendritic spines. Neurons with somata in the middle third of sections were chosen to minimize the number of truncated branches. In addition, to minimize both variability of apical dendritic data and the probability of potential artifactual differences due to sampling neurons from varying cortical depths, only neurons whose somata were between 150 and 455 μm from the pial surface were chosen. Thus, the pyramidal neurons drawn were located in either deep layer II or superficial layer III; short-shaft pyramidal cells from superficial layer II were excluded. For each animal, 10 neurons were drawn; this number yields a within-animal error of <15% (mean within-animal SEM for total branch length = 12.99 ± 0.61%), and thus was considered to provide a representative sample of deeper layer II–III pyramidal neurons in medial prefrontal cortex. All neurons were drawn at 600× and morphology of apical and basilar arbors was quantified in three dimensions using a computer-based neuron tracing system (Neurolucida; MicroBrightField, Williston, VT) with the experimenter blind to condition.
Several aspects of dendritic morphology were examined. To assess overall changes in dendritic morphology, total length and number of basilar and apical dendrites were compared across groups using one-way ANOVAs. To assess differences in the amount and location of dendritic material, a three-dimensional version of a Sholl analysis (Sholl, 1956) was performed. A Sholl analysis estimates the amount and distribution of dendritic material by counting the number of intersections of dendrites with an overlay of concentric rings centered on the soma. In the present study, the number of intersections of dendrites with concentric spheres at 10 μm radii (10 μm from soma radius, 20 μm from soma radius, etc., to encompass the most distal dendrites) was counted; for statistical and graphical purposes, the counts of intersections were summed over pairs of radii (i.e. 10 and 20 μm radii, 30 and 40 μm radii, etc.). These data were compared using two-way repeated-measures ANOVAs (group × distance from soma) followed by appropriate planned comparisons. In addition, the number and length of terminal branches were compared across groups using one-way ANOVAs followed by appropriate planned comparisons. For all analyses, planned comparisons consisted of two-group F-tests done within the context of the overall ANOVA (Hays, 1994), comparing unstressed versus either handled or stressed groups at each interval.
Chronic brief restraint stress resulted in significant increases in corticosterone titers (Fig. 1). Two-way ANOVA (time after initiation of restrain × day) revealed a significant effect on plasma corticosterone titers of time after initiation of restraint [F(3,48) = 24.14, P < 0.01] but not day of restraint [F(2,48) = 1.43, NS], as well as a significant interaction of time post-restraint and day [F(6,48) = 2.33, P < 0.05]. Planned comparisons revealed that, averaged across days, corticosterone titers were significantly increased relative to baseline at 10 and 30 min after being placed in the restrainer [F(1,28) = 51.47 and 25.36, respectively, P < 0.01], but had returned to baseline by 60 min [F(1,28) = 0.38, NS].
In all treatment groups, complete impregnation (defined as the absence of dendrites trailing off into a series of dots: see Buell, 1982; Coleman and Flood, 1987; Williams et al., 1978) of numerous cortical pyramidal neurons was apparent (Fig. 2), and both Cg1–3 and layer II–III were readily identifiable. Because relatively thick sections were taken through prefrontal cortex, the apical and basilar arbors of essentially all neurons selected were completely contained within a single section.
To rule out potential artifactual differences in dendritic morphology due to differential sampling in layer II and III, the distance from the soma to the pial surface of cortex was measured in each neuron. Average distance to the cortical surface was then compared across groups using one-way ANOVA. Average distance to the cortical surface did not vary across groups [for unhandled rats, mean = 283.58 ± 6.16 μm; for handled rats, mean = 257.75 ± 8.48 μm; for restraint-stressed rats, mean = 253.72 ± 13.42 μm; F(2,12) = 2.66, NS]. Thus, neurons were sampled from equivalent laminar depths across groups.
To assess overall changes in dendritic morphology, total length and number of apical dendrites were compared across groups. One-way ANOVAs revealed that chronic brief restraint stress significantly altered both branch number and length [F(2,12) = 4.81 and 3.75, respectively, P ≤ 0.05]. Planned comparisons indicated that whereas handling did not significantly alter apical branch number or length compared to unstressed controls [F(1,8) = 0.65 and.64, respectively, NS], 10 min restraint stress significantly decreased apical branch number and length by ∼20–28% relative to both handled and unhandled rats [all Fs(1,8) > 6.10, P < 0.05; Figs 3 and 4).
To more closely examine changes in the distribution of dendritic material, Sholl analyses were performed. Chronic brief stress significantly altered the distribution of apical dendritic material [for main effect of group, F(2,12) = 3.66, P ≤ 0.06; for interaction of group and distance from soma, F(2,22) = 2.61, P ≤ 0.01]. Planned comparisons indicated that handling alone failed to significantly alter apical dendritic material at any distance from the soma [all Fs(1,8) ≤ 3.37, NS]. On the other hand, relative to unhandled controls, chronic brief restraint stress significantly decreased apical dendritic material in the middle portion of the apical arbor by 22–31% [for 90–100, 110–120, and 130–140 μm from the soma, all Fs(1,8) ≥ 6.47, P ≤ 0.04]; decreases in dendritic material more distally approached significance [for 150–160 μm from the soma, F(1,8) = 3.57, P ≤ 0.09; for 210–220 and >220 μm from the soma, Fs(1,8) ≥ 4.44, P ≤ 0.07; all other Fs(1,13) ≤ 2.75, NS; Fig. 5]. Similarly, dendritic material in the middle portion of the apical arbor in restraint-stressed rats was decreased relative to handled rats [for 70–80, 90–100, 110–120, and 130–140 μm from the soma, all Fs(1,8) ≥ 7.71, P ≤ 0.02], but was not significantly different either closer to or further away from the soma [all Fs(1,8) ≤ 3.16, NS; Fig. 5].
Finally, because terminal branches may be more plastic than other parts of the arbor (Coleman and Flood, 1987; Rosenzweig and Bennett, 1996), and thus may be more sensitive to the effects of stress, the length and number of terminal dendritic branches were also compared across groups. One-way ANOVA revealed a significant effect of group on apical branch number [F(2,12) = 4.71, P ≤ 0.03]. Planned comparisons demonstrated that while handling failed to significantly alter apical terminal branch number [F(1,8) = 0.61, NS], the number of apical terminal branches was significantly reduced in restraint-stressed animals relative to both unhandled and handled controls, by 26 and 19%, respectively [Fs(1,8) ≥ 6.94, P ≤ 0.03; Fig. 6]. Similarly, one-way ANOVA demonstrated a significant effect of group on apical terminal branch length [F(2,12) = 4.04, P ≤ 0.05], with subsequent planned comparisons demonstrating no difference between handled and unhandled rats [F(1,8) = 0.45, NS], but a significant reduction in the length of apical terminal branches in restraint-stressed animals relative to both unhandled and handled controls [29 and 23%, respectively; Fs(1,8) ≥ 6.94, P ≤ 0.03; Fig. 6].
In contrast, handling and brief restraint stress failed to significantly alter any measure of basilar dendritic arbor. Mean basilar branch number and length were not significantly altered by either handling or restraint stress [for branch number, F(2,12) = 2.35, NS; for branch length, F(2,12) = 1.72, NS; Figs 3 and 4]. Furthermore, the distribution of basilar dendritic material did not vary across groups [main effect of group, F(2,12) = 1.92, NS; interaction of group by distance from soma, F(2,10) = 1.52, NS; Fig. 5]. Likewise, basilar terminal branches were not significantly altered by stress [for basilar terminal number, F(2,12) = 2.29, NS; for basilar terminal length, F(2,12) = 0.98, NS; Fig. 6].
The present study demonstrates pronounced changes in the dendritic morphology of layer II–III pyramidal neurons in medial prefrontal cortex as a result of a short-duration, mild stressor: 1 week of daily brief restraint resulted in a selective remodeling of apical dendrites, with atrophy of up to 31% occurring in more distal branches and relative sparing of proximal branches. These effects replicate our previous work using a longer-term, more severe stressor (3 weeks of daily 3 h restraint). In our previous study, the longer-term, more severe stress produced a pronounced decrease in both branch number and branch length in the apical dendrites of layer II–III pyramidal neurons in medial prefrontal cortex (Cook and Wellman, 2004). Sholl analyses demonstrated that this decrease was restricted to the more distal portion of the apical arbor, where the amount of dendritic material was reduced by up to 83%. This restriction may reflect the differential atrophy of terminal branches: terminal branch number and length throughout the arbor were decreased by 19 and 35%, respectively (Cook and Wellman, 2004). In the present study, a short-term mild stressor produced a similar pattern of dendritic changes. As with the effects of the longer-term, more severe stressor used in the previous study, the dendritic atrophy resulting from the short-term, mild stressor used in the current study was restricted to relatively distal portions of the apical arbor and appeared to differentially affect terminal branches: apical terminal branch number and length were decreased by 26 and 29%, respectively, relative to unhandled rats. Thus, our results suggest that the morphology of medial prefrontal cortex is exquisitely sensitive to stress, with changes occurring in as little as 1 week in response to even a mild stressor.
Importantly, this remodeling of the apical arbors of prefrontal pyramidal neurons is likely due to the stressful nature of the restraint: 1 week of brief restraint produced robust increases in corticosterone levels, which did not habituate over the course of the study. Interestingly, in a previous study (Wellman, 2001), we found that daily vehicle injections also resulted in atrophy of the distal portion of the apical arbor of layer II–III neurons in medial prefrontal cortex (Wellman, 2001). This atrophy was less pronounced than that seen in the present study. Thus, the relatively mild stress of daily injections alone appears to alter the morphology of medial prefrontal cortex, and this morphological alteration is more pronounced with the more severe stressor used in the present study. Indeed, our present data, combined with our previous studies, suggest a consistent dose–effect relationship between stress, stress-induced corticosterone levels and dendritic changes in medial prefrontal cortex. Daily vehicle injections, which presumably would produce relatively small increases in corticosterone titers, produced moderate decreases in apical arbor (Wellman, 2001); 10 min of daily restraint, resulting in average peak corticosterone titers of ∼28 μg/dl, produced similarly moderate but robust alterations in apical arbor; 3 h of daily restraint, resulting in average peak corticosterone titers of ∼74 μg/dl, produced more pronounced decreases in distal apical arbor (Cook and Wellman, 2004); and daily corticosterone administration, which typically produces average peak corticosterone titers of ∼95 μg/dl (Hauger et al., 1987), produced similarly pronounced decreases in distal dendritic material accompanied by increases in dendritic material proximal to the soma (Wellman, 2001).
However, it is important to note that, while our results suggest that the chronic stress effects we have documented in medial prefrontal cortex are corticosterone-mediated, we have not yet directly tested this hypothesis. In fact, stress produces a variety of neurochemical and physiological changes in prefrontal cortex (e.g. Moghaddam, 1993; Gresch et al., 1994; Mark et al., 1996; Mizoguchi et al., 2000; Rocher et al., 2004; Tan et al., 2004), which are not necessarily mediated by corticosterone. Indeed, stress-induced changes in dendritic morphology of hippocampal area CA3 neurons have been shown to be mediated at least in part by glutamatergic NMDA receptors (Magariños and McEwen, 1995). Thus, the dendritic reorganization in medial prefrontal cortex resulting from chronic stress could be due to stress-induced processes not mediated by corticosterone.
Finally, dendrites are a major site of synaptic connectivity, with adult cortical neurons receiving ∼15 000 synaptic inputs (Huttenlocher, 1994). Given that the geometry of the dendritic arbor (e.g. dendritic branching patterns, distribution and overall shape) determines many functional properties of neurons (e.g. Rall et al., 1992; Mainen and Sejnowksi, 1996; Koch and Segev, 2000; Lu et al., 2001; Grudt and Perl, 2002), the dendritic changes resulting from even the short-term, mild stressor documented here probably result in important functional changes and may have consequences for the behaviors mediated by medial prefrontal cortex. Given that both stress (e.g. Brown and Birley, 1968; Brown and Harris, 1989; Ventura et al., 1989) and dysfunction of prefrontal cortex (e.g. Baxter et al., 1989; Mayberg, 1997; Berman and Weinberger, 1999) are hypothesized to play important roles in disorders such as depression and schizophrenia, the morphological sensitivity of prefrontal cortex to chronic stress has important implications for the etiology of these disorders.
We thank Ms Anthea C. Diamondis for her technical assistance and Dr Dale R. Sengelaub for his helpful comments on the manuscript. This research was supported by the HHMI Biological Sciences Education Program (S.M.B.) and MH067607 (C.L.W.).
1Department of Psychology, Indiana University, Bloomington, IN, USA and 2Program in Neural Science, Indiana University, Bloomington, IN, USA