Matthew W. Hale

Serotonergic Systems, Anxiety, and Affective Disorder

Depressed suicide patients have elevated expression of neuronal tryptophan hydroxylase 2 (TPH2) mRNA and protein in midbrain serotonergic neurons, as well as increases in brain serotonin turnover. The mechanisms underlying these changes are uncertain, but increased TPH2 expression and serotonin turnover could result from genetic influences, adverse early life experiences, or acute stressful life events, all of which can alter serotonergic neurotransmission and have been implicated in determining vulnerability to major depression. Emerging evidence suggests that there are several different stress‐related subsets of serotonergic neurons, each with a unique role in the integrated stress response. Here we review our current understanding of how genetic and environmental factors may influence TPH2 mRNA expression and serotonergic neurotransmission, focusing in particular on the dorsomedial part of the dorsal raphe nucleus. This subdivision of the dorsal raphe nucleus is selectively innervated by key forebrain structures implicated in regulation of anxiety states, it gives rise to projections to a distributed neural system mediating anxiety states, and serotonergic neurons within this subdivision are selectively activated by a number of stress‐ and anxiety‐related stimuli. A better understanding of the anatomical and functional properties of specific stress‐ or anxiety‐related serotonergic systems should aid our understanding of the neural mechanisms underlying the etiology of anxiety and affective disorders.

Functional topography of midbrain and pontine serotonergic systems: implications for synaptic regulation of serotonergic circuits

RationaleDysfunction of serotonergic systems is thought to play an important role in a number of neurological and psychiatric disorders. Recent studies suggest that there is anatomical and functional diversity among serotonergic systems innervating forebrain systems involved in the control of physiologic and behavioral responses, including the control of emotional states.ObjectiveHere, we highlight the methods that have been used to investigate the heterogeneity of serotonergic systems and review the evidence for the unique anatomical, hodological, and functional properties of topographically organized subpopulations of serotonergic neurons in the midbrain and pontine raphe complex.ConclusionThe emerging understanding of the topographically organized synaptic regulation of brainstem serotonergic systems, the topography of the efferent projections of these systems, and their functional properties, should enable identification of novel therapeutic approaches to treatment of neurological and psychiatric conditions that are associated with dysregulation of serotonergic systems.

Differential effects of exposure to low-light or high-light open-field on anxiety-related behaviors; relationship to c-Fos expression in serotonergic and non-serotonergic neurons in the dorsal raphe nucleus

Serotonergic systems arising from the mid-rostrocaudal and caudal dorsal raphe nucleus (DR) have been implicated in the facilitation of anxiety-related behavioral responses by anxiogenic drugs or aversive stimuli. In this study we attempted to determine a threshold to engage serotonergic neurons in the DR following exposure to aversive conditions in an anxiety-related behavioral test. We manipulated the intensity of anxiogenic stimuli in studies of male Wistar rats by leaving them undisturbed (CO), briefly handling them (HA), or exposing them to an open-field arena for 15-min under low-light (LL: 8-13 lux) or high-light (HL: 400-500 lux) conditions. Rats exposed to HL conditions responded with reduced locomotor activity, reduced time spent exploring the center of the arena, a lower frequency of rearing and grooming, and an increased frequency of facing the corner of the arena compared to LL rats. Rats exposed to HL conditions had small but significant increases in c-Fos expression within serotonergic neurons in subdivisions of the rostral DR. Exposure to HL conditions did not alter c-Fos responses in serotonergic neurons in any other DR subdivision. In contrast, rats exposed to the open-field arena had increased c-Fos expression in non-serotonergic cells throughout the DR compared to CO rats, and this effect was particularly apparent in the dorsolateral part of the DR. We conclude that exposure to HL conditions, compared to LL conditions, increased anxiety-related behavioral responses in an open-field arena but this stimulus was at or below the threshold required to increase c-Fos expression in serotonergic neurons.

Stress-related Serotonergic Systems: Implications for Symptomatology of Anxiety and Affective Disorders

Previous studies have suggested that serotonergic neurons in the midbrain raphe complex have a functional topographic organization. Recent studies suggest that stimulation of a bed nucleus of the stria terminalis-dorsal raphe nucleus pathway by stress- and anxiety-related stimuli modulates a subpopulation of serotonergic neurons in the dorsal part of the dorsal raphe nucleus (DRD) and caudal part of the dorsal raphe nucleus (DRC) that participates in facilitation of anxiety-like responses. In contrast, recent studies suggest that activation of a spinoparabrachial pathway by peripheral thermal or immune stimuli excites subpopulations of serotonergic neurons in the ventrolateral part of the dorsal raphe nucleus/ventrolateral periaqueducal gray (DRVL/VLPAG) region and interfascicular part of the dorsal raphe nucleus (DRI). Studies support a role for serotonergic neurons in the DRVL/VLPAG in inhibition of panic-like responses, and serotonergic neurons in the DRI in antidepressant-like effects. Thus, data suggest that while some subpopulations of serotonergic neurons in the dorsal raphe nucleus play a role in facilitation of anxiety-like responses, others play a role in inhibition of anxiety- or panic-like responses, while others play a role in antidepressant-like effects. Understanding the anatomical and functional properties of these distinct serotonergic systems may lead to novel therapeutic strategies for the prevention and/or treatment of affective and anxiety disorders. In this review, we describe the anatomical and functional properties of subpopulations of serotonergic neurons in the dorsal raphe nucleus, with a focus on those implicated in symptoms of anxiety and affective disorders, the DRD/DRC, DRVL/VLPAG, and DRI.

Exposure to high- and low-light conditions in an open-field test of anxiety increases c-Fos expression in specific subdivisions of the rat basolateral amygdaloid complex.

Anxiety states and anxiety-related behaviors appear to be regulated by a distributed and highly interconnected system of forebrain structures including the basolateral amygdaloid complex (basolateral amygdala). Despite a wealth of research examining the role of the basolateral amygdala in anxiety-related behaviors and anxiety states, the specific subdivisions of the basolateral amygdala that are involved in responses to anxiogenic stimuli have not been examined. In this study, we investigated the effects of exposure to a novel open-field environment, with either low- or high-levels of illumination, on expression of the protein product of the immediate-early gene c-Fos in subdivisions of the rat basolateral amygdala. The subdivisions studied included the lateral, ventrolateral and ventromedial parts of the lateral amygdaloid nucleus, the anterior, posterior and ventral parts of the basolateral amygdaloid nucleus and the anterior and posterior part of the basomedial amygdaloid nucleus. Small increases in the number of c-Fos-immunoreactive cells were observed in several, but not all, of the subdivisions of the basolateral amygdala studied following exposure of rats to either the high- or low-light conditions, compared to home cage or handled control groups. Open-field exposure in both the high- and low-light conditions resulted in a marked increase in c-Fos expression in the anterior part of the basolateral amygdaloid nucleus compared to either home cage or handled control groups. These findings point toward anatomical and functional heterogeneity within the basolateral amygdaloid complex and an important role of the anterior part of the basolateral amygdaloid nucleus in the neural mechanisms underlying physiological or behavioral responses to this anxiety-related stimulus.

Immunization with a heat-killed preparation of the environmental bacterium Mycobacterium vaccae promotes stress resilience in mice

Significance The hygiene, or “old friends,” hypothesis proposes that lack of exposure to immunoregulatory microorganisms in modern urban societies is resulting in an epidemic of inflammatory disease, as well as psychiatric disorders in which chronic, low-level inflammation is a risk factor. An important determinant of immunoregulation is the microbial community occupying the host organism, collectively referred to as the microbiota. Here we show that stress disrupts the homeostatic relationship between the microbiota and the host, resulting in exaggerated inflammation. Treatment of mice with a heat-killed preparation of an immunoregulatory environmental microorganism, Mycobacterium vaccae, prevents stress-induced pathology. These data support a strategy of “reintroducing” humans to their old friends to promote optimal health and wellness. The prevalence of inflammatory diseases is increasing in modern urban societies. Inflammation increases risk of stress-related pathology; consequently, immunoregulatory or antiinflammatory approaches may protect against negative stress-related outcomes. We show that stress disrupts the homeostatic relationship between the microbiota and the host, resulting in exaggerated inflammation. Repeated immunization with a heat-killed preparation of Mycobacterium vaccae, an immunoregulatory environmental microorganism, reduced subordinate, flight, and avoiding behavioral responses to a dominant aggressor in a murine model of chronic psychosocial stress when tested 1–2 wk following the final immunization. Furthermore, immunization with M. vaccae prevented stress-induced spontaneous colitis and, in stressed mice, induced anxiolytic or fear-reducing effects as measured on the elevated plus-maze, despite stress-induced gut microbiota changes characteristic of gut infection and colitis. Immunization with M. vaccae also prevented stress-induced aggravation of colitis in a model of inflammatory bowel disease. Depletion of regulatory T cells negated protective effects of immunization with M. vaccae on stress-induced colitis and anxiety-like or fear behaviors. These data provide a framework for developing microbiome- and immunoregulation-based strategies for prevention of stress-related pathologies.

Repeated social defeat increases reactive emotional coping behavior and alters functional responses in serotonergic neurons in the rat dorsal raphe nucleus

Chronic stress is a vulnerability factor for a number of psychiatric disorders, including anxiety and affective disorders. Social defeat in rats has proven to be a useful paradigm to investigate the neural mechanisms underlying physiologic and behavioral adaptation to acute and chronic stress. Previous studies suggest that serotonergic systems may contribute to the physiologic and behavioral adaptation to chronic stress, including social defeat in rodent models. In order to test the hypothesis that repeated social defeat alters the emotional behavior and the excitability of brainstem serotonergic systems implicated in control of emotional behavior, we exposed adult male rats either to home cage control conditions, acute social defeat, or social defeat followed 24h later by a second social defeat encounter. We then assessed behavioral responses during social defeat as well as the excitability of serotonergic neurons within the dorsal raphe nucleus using immunohistochemical staining of tryptophan hydroxylase, a marker of serotonergic neurons, and the protein product of the immediate-early gene, c-fos. Repeated social defeat resulted in a shift away from proactive emotional coping behaviors, such as rearing (explorative escape behavior), and toward reactive emotional coping behaviors such as freezing. Both acute and repeated defeat led to widespread increases in c-Fos expression in serotonergic neurons in the dorsal raphe nucleus. Changes in behavior following a second exposure to social defeat, relative to acute defeat, were associated with decreased c-Fos expression in serotonergic neurons within the dorsal and ventral parts of the mid-rostrocaudal dorsal raphe nucleus, regions that have been implicated in 1) serotonergic modulation of fear- and anxiety-related behavior and 2) defensive behavior in conspecific aggressive encounters, respectively. These data support the hypothesis that serotonergic systems play a role in physiologic and behavioral responses to both acute and repeated social defeat.

Exposure to an open-field arena increases c-Fos expression in a subpopulation of neurons in the dorsal raphe nucleus, including neurons projecting to the basolateral amygdaloid complex.

Serotonergic systems in the dorsal raphe nucleus are thought to play an important role in the regulation of anxiety states. To investigate responses of neurons in the dorsal raphe nucleus to a mild anxiety-related stimulus, we exposed rats to an open-field, under low-light or high-light conditions. Treatment effects on c-Fos expression in serotonergic and non-serotonergic cells in the midbrain raphe nuclei were determined 2 h following open-field exposure or home cage control (CO) conditions. Rats tested under both light conditions responded with increases in c-Fos expression in serotonergic neurons within subdivisions of the midbrain raphe nuclei compared with CO rats. However, the total numbers of serotonergic neurons involved were small suggesting that exposure to the open-field may affect a subpopulation of serotonergic neurons. To determine if exposure to the open-field activates a subset of neurons in the midbrain raphe complex that projects to forebrain circuits regulating anxiety states, we used cholera toxin B subunit (CTb) as a retrograde tracer to identify neurons projecting to the basolateral amygdaloid complex (BL) in combination with c-Fos immunostaining to identify cells that responded to open-field exposure. Rats received a unilateral injection of CTb into the BL. Seven to 11 days following CTb injection rats were either, 1) exposed to an open-field in low-light conditions, 2) briefly handled or 3) left undisturbed in home cages. Dual immunostaining for c-Fos and CTb revealed an increase in the percentage of c-Fos-immunoreactive BL-projecting neurons in open-field-exposed rats compared with handled and control rats. Dual immunostaining for tryptophan hydroxylase and CTb revealed that a majority (65%) of BL-projecting neurons were serotonergic, leaving open the possibility that activated neurons were serotonergic, non-serotonergic, or both. These data are consistent with the hypothesis that exposure to anxiogenic stimuli activates a subset of neurons in the midbrain raphe complex projecting to amygdala anxiety circuits.

Topographic organization and chemoarchitecture of the dorsal raphe nucleus and the median raphe nucleus

The role of serotonergic systems in regulation of behavioral arousal and sleep-wake cycles is complex and may depend on both the receptor subtype and brain region involved. Increasing evidence points toward the existence of multiple topographically organized subpopulations of serotonergic neurons that receive unique afferent connections, give rise to unique patterns of projections to forebrain systems, and have unique functional properties. A better understanding of the properties of these subpopulations of serotonergic neurons may aid in the understanding of the role of serotonergic systems in regulation of behavioral arousal, sleep-wake cycles and other physiological and behavioral responses attributed to serotonin. In this chapter, we outline evidence for multiple serotonergic systems within the midbrain and pontine raphe complex that can be defined based on cytoarchitectonic and hodological properties. In addition, we describe how these topographically organized groups of serotonergic neurons correspond to the six major ascending serotonergic tracts innervating the forebrain.

Exposure to an open-field arena increases c-Fos expression in a distributed anxiety-related system projecting to the basolateral amygdaloid complex

Anxiety states and anxiety-related behaviors appear to be regulated by a distributed and highly interconnected system of brain structures including the basolateral amygdala. Our previous studies demonstrate that exposure of rats to an open-field in high- and low-light conditions results in a marked increase in c-Fos expression in the anterior part of the basolateral amygdaloid nucleus (BLA) compared with controls. The neural mechanisms underlying the anatomically specific effects of open-field exposure on c-Fos expression in the BLA are not clear, however, it is likely that this reflects activation of specific afferent input to this region of the amygdala. In order to identify candidate brain regions mediating anxiety-induced activation of the basolateral amygdaloid complex in rats, we used cholera toxin B subunit (CTb) as a retrograde tracer to identify neurons with direct afferent projections to this region in combination with c-Fos immunostaining to identify cells responding to exposure to an open-field arena in low-light (8-13 lux) conditions (an anxiogenic stimulus in rats). Adult male Wistar rats received a unilateral microinjection of 4% CTb in phosphate-buffered saline into the basolateral amygdaloid complex. Rats were housed individually for 11 days after CTb injections and handled (HA) for 2 min each day. On the test day rats were either, 1) exposed to an open-field in low-light conditions (8-13 lux) for 15 min (OF); 2) briefly HA or 3) left undisturbed (control). We report that dual immunohistochemical staining for c-Fos and CTb revealed an increase in the percentage of c-Fos-immunopositive basolateral amygdaloid complex-projecting neurons in open-field-exposed rats compared with HA and control rats in the ipsilateral CA1 region of the ventral hippocampus, subiculum and lateral entorhinal cortex. These data are consistent with the hypothesis that exposure to the open-field arena activates an anxiety-related neuronal system with convergent input to the basolateral amygdaloid complex.