Elsevier

Brain Research

Volume 826, Issue 1, 24 April 1999, Pages 35-43
Brain Research

Research report
Activation of serotonin-immunoreactive cells in the dorsal raphe nucleus in rats exposed to an uncontrollable stressor

https://doi.org/10.1016/S0006-8993(99)01208-1Get rights and content

Abstract

The dorsal raphe nucleus (DRN) and its serotonergic terminal regions have been suggested to be part of the neural substrate by which exposure to uncontrollable stressors produces poor escape responding and enhanced conditioned fear expression. Such stressor exposure is thought to selectively activate DRN serotonergic neurons in such a way as to render them transiently sensitized to further input. As a result of this sensitized state, behavioral testing procedures are thought to cause excess serotonergic activity in brain regions that control these behaviors. The present studies were conducted to investigate activity in the DRN following exposure to escapable and yoked, inescapable tailshock. Neural activity was characterized using immunohistochemistry to detect the immediate early gene product Fos in serotonin-immunoreactive cells in the DRN. Inescapable tailshock led to greater serotonergic neural activity than did escapable tailshock, supporting the hypothesis that uncontrollable stressors preferentially activate serotonergic neurons in the DRN.

Introduction

The nature of the physiological and behavioral reactions to an aversive event depends on whether or not the organism can control the event. Reactions that only occur subsequent to uncontrollable events but not to controllable events are referred to as learned helplessness effects. Learned helplessness, in the form of impaired escape performance, was initially demonstrated in dogs that were given a series of inescapable shocks 62, 71.

Extensive characterization of learned helplessness in rodents has revealed that the effects of uncontrollable stressors, relative to controllable stressors, extend beyond escape responding to affect a broad constellation of behaviors. Other defensive behaviors that are affected by uncontrollable stressors include expression of conditioned fear 48, 59, defensive burying [87], defeat posturing [88]and territorial defense [86]. Basic physiological processes, such as temperature regulation [40], eating [14], and sleeping [1]have also been shown to be sensitive to the effects of uncontrollable stressor exposure. Subjects exposed to uncontrollable stressors show exaggerated reactions to amphetamine [47]and morphine 27, 34. Altered pain sensitivity following exposure to uncontrollable stressors has been studied extensively 18, 19, 20, 38, 51, 52, 57. Finally, uncontrollable stressors produce increased anxiety, observed as decreased social interaction in rats [74].

The behavioral effects of a particular uncontrollable stressor, inescapable tailshock (IS), have been extensively characterized (see Maier for a review [49]). Recent efforts to understand the mechanisms which mediate these phenomena have focused on serotonergic (5-HT) neurons in the dorsal raphe nucleus (DRN). The DRN provides a large portion of the 5-HT projections ascending to cortex, hippocampus, hypothalamus, other forebrain and midbrain structures 79, 83. In addition, single neurons project to the cortex and spinal cord [41]. The ubiquitous nature of DRN projections provides a framework for 5-HT influence over the widespread effects of IS on behavior. 5-HT is known to modulate escape behavior [25], sleep [39], circadian rhythmicity [2], feeding [16], anxiety [37], and pain sensitivity [84]. The proximal brain sites that mediate these behaviors all receive projections from the DRN [83]. Activation of the DRN is sufficient to blunt pain sensitivity and release of 5-HT in DRN projection regions (i.e., nucleus raphe magnus) reduces pain sensitivity [36]. In addition, activation of the DRN reduces activity in the dorsolateral periaqueductal gray, an area known to mediate escape responding [44]. Indeed, 5-HT injected into this region impairs escape behavior [25]. In addition, the DRN projects to the amygdala 35, 45where 5-HT can increase neural activity [11]and produce anxious behavior [24]. Given that activity in the amygdala is necessary for fear conditioning [13], increased activity in this area would be expected to enhance fear conditioning. In addition, behaviors that are considered indices of anxiety can be directly modulated by DRN manipulations 29, 30, 31.

The foregoing suggests that activity in 5-HT neurons within the DRN could produce a constellation of outcomes similar to those produced by IS. Since behavioral testing typically occurs 24 h after exposure to IS, these behaviors would occur if IS sensitized DRN 5-HT neurons for a period of time so that they would respond in exaggerated fashion to input induced by the behavioral testing conditions (e.g., footshock during escape testing). Consistent with this possibility, lesion of the DRN and pharmacological inhibition of DRN 5-HT activity before behavioral testing reverses the effects of IS on behavior 50, 53, 56.

IS could sensitize DRN 5-HT neurons via a number of mechanisms but all assume that IS selectively activates DRN 5-HT neurons relative to ES [49]. Pharmacological data support this view. Pharmacological inhibition of DRN 5-HT activity at the time of IS prevents learned helplessness effects 55, 56, 81, and pharmacological activation of these neurons in the absence of IS produces behavioral sequelae similar to those of IS 50, 81. These studies suggest, but do not provide direct evidence, that IS selectively activates DRN 5-HT neurons.

Fos immunohistochemistry has become a popular technique to assay neural activity [17]. The protein Fos is the product of the immediate early gene c-fos and is one of many transcription factors found in most cells [61]. Transcription factors bind to DNA sequences to modulate transcription of genes. The same sequence can occur on many genes, allowing the induction of a transcription factor to orchestrate the transcription of a number of genes in response to demands made on the cell. Since the immediate early gene products are thought to mediate the metabolic challenge imposed on the cell by strong activation [61], Fos and other products of these genes are considered `third-messenger' cellular signals [60].

The use of Fos as a marker for DRN neural activation following IS is warranted for several reasons. Serotonin cells can be identified and characterized as being activated using double-labeling in which nuclear Fos and cytoplasmic serotonin can be visualized. The cellular signals known to induce Fos, such as the second messenger cAMP [32], are known to occur in serotonin neurons in the DRN. The signals proposed to activate the DRN as a result of IS have all been shown to induce Fos in other brain regions. For example, NMDA receptor activation is well characterized as an inducer of Fos [76]and is also involved in the DRN-dependent effects of IS (Grahn et al., unpublished data). Other signals known to induce Fos expression, including opioid-receptor activation 9, 23, interference with GABA inhibition [69], and activation of noradrenergic receptors 5, 80are thought to be processed by the DRN during IS 26, 50, 54, 56. All of these signals have been shown to change levels of Fos in various brain regions, although the DRN has not been the focus of such investigations.

Surprisingly, given the extent to which Fos has been employed as an activation marker, only a handful of reports have demonstrated double-labeling of serotonin and Fos in the DRN 15, 43, 46, 90. The neural circuitry activated during various stressors has been studied extensively using Fos. These studies have focused on brain sites that are traditionally considered to be involved in the effects of stressors, such as the components which activate the HPA axis (hippocampus, cortex, hypothalamus) 12, 65, 70, 89or sites included in `fear' circuitry (amygdala, cortex, septum, periaqueductal gray) 7, 10, 21, 75, and lower brainstem regions (locus coeruleus, nucleus tractus solitarius, ventrolateral medulla) 8, 65, 67, 75. When the DRN has been examined, it has been reported to have very little basal Fos expression. Some, but not all stressors, increase Fos expression in the DRN 3, 4, 42, 72.

The aim of the present studies was to investigate a neural consequence of exposure to stressor conditions that are identical to those that are known to cause learned helplessness behavior. Recently, IS was shown to produce a large accumulation of extracellular 5-HT within the DRN relative to ES [58]. However, the release of serotonin within the DRN has been shown to occur from dendrites and soma and can be independent of depolarization [82]. Release of serotonin is also modulated by activation of somatodendritic and terminal autoreceptors in such a way that neural activation and release do not always correlate [66]. Further, it is possible that serotonin released in the DRN at the time of IS originates in other raphe nuclei which project to the DRN [78]. The present studies therefore employed the activation marker Fos to characterize DRN activity following IS or ES. Double-labeling of neurons for serotonin and Fos was conducted to assess activation specifically in serotonin neurons.

Section snippets

Materials and methods

Male Sprague–Dawley rats, 80–100 days old, served as subjects in all experiments. They were housed individually with free access to standard lab chow and water. All procedures were in accordance with NIH animal care guidelines and were conducted with approval of the University of Colorado Institutional Animal Care and Use Committee.

Results

Photomicrographs of the DRN examined from an IS-treated rat 2 h after stressor exposure are shown in Fig. 1. Panels A–C represent the rostral, middle and caudal sections examined for each rat. Panels D–G are higher magnifications showing each type of particle analyzed. As shown in Table 1, the mean number of 5-HT-stained nuclei was not different between IS and ES conditions for any timepoint. Table 1 also shows the number of Fos-stained particles observed in three regions of the DRN for each

Discussion

The present results indicate that Fos expression in DRN 5-HT neurons is preferentially induced by exposure to IS, relative to ES. Fos expression in these experiments exhibited the expected time-course with maximal expression 2–3 h after IS or ES and decreased expression 4 h after stressor exposure. It is notable that there would be no controllability effect if expression of Fos alone were examined. As shown in Table 1, Fos expression 2 h after stressor exposure was similar in both IS- and

Acknowledgements

Support was provided to SFM by NIMH MH50479 and RSA MH00314. Additional support was provided to MBM by the University of Colorado Undergraduate Research Opportunity Program. We would like to thank Jack Yamuy and Ron Gaykema for helpful advice regarding serotonin immunohistochemistry.

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