c-Fos Expression In Serotonergic Neurons Research Articles

Effects of repeated voluntary or forced exercise on brainstem serotonergic systems in rats

Voluntary exercise increases stress resistance by modulating stress-responsive neurocircuitry, including brainstem serotonergic systems. However, it remains unknown how exercise produces adaptations to serotonergic systems. Recruitment of serotonergic systems during repeated, daily exercise could contribute to the adaptations in serotonergic systems following exercise, but whether repeated voluntary exercise recruits serotonergic systems is unknown. In this study, we investigated the effects of six weeks of voluntary or forced exercise on rat brain serotonergic systems. Specifically, we analyzed c-Fos and FosB/ΔFosB as markers of acute and chronic cellular activation, respectively, in combination with tryptophan hydroxylase, a marker of serotonergic neurons, within subregions of the dorsal raphe nucleus using immunohistochemical staining. Compared to sedentary controls, rats exposed to repeated forced exercise, but not repeated voluntary exercise, displayed decreased c-Fos expression in serotonergic neurons in the rostral dorsal portion of the dorsal raphe nucleus (DRD) and increased c-Fos expression in serotonergic neurons in the caudal DR (DRC), and interfascicular part of the dorsal raphe nucleus (DRI) during the active phase of the diurnal activity rhythm. Similarly, increases in c-Fos expression in serotonergic neurons in the DRC, DRI, and ventral portion of the dorsal raphe nucleus (DRV) were observed in rats exposed to repeated forced exercise, compared to rats exposed to repeated voluntary exercise. Six weeks of forced exercise, relative to the sedentary control condition, also increased FosB/ΔFosB expression in DRD, DRI, and DRV serotonergic neurons. While both voluntary and forced exercise increase stress resistance, these results suggest that repeated forced exercise, but not repeated voluntary exercise, increases activation of DRI serotonergic neurons, an effect that may contribute to the stress resistance effects of forced exercise. These results also suggest that mechanisms of exercise-induced stress resistance may differ depending on the controllability of the exercise.

Prior cold water swim stress alters immobility in the forced swim test and associated activation of serotonergic neurons in the rat dorsal raphe nucleus

Prior adverse experience alters behavioral responses to subsequent stressors. For example, exposure to a brief swim increases immobility in a subsequent swim test 24h later. In order to determine if qualitative differences (e.g. 19°C versus 25°C) in an initial stressor (15-min swim) impact behavioral, physiological, and associated neural responses in a 5-min, 25°C swim test 24h later, rats were surgically implanted with biotelemetry devices 1week prior to experimentation then randomly assigned to one of six conditions (Day 1 (15min)/Day 2 (5min)): (1) home cage (HC)/HC, (2) HC/25°C swim, (3) 19°C swim/HC, (4) 19°C swim/25°C swim, (5) 25°C swim/HC, (6) 25°C swim/25°C swim. Core body temperature (Tb) was measured on Days 1 and 2 using biotelemetry; behavior was measured on Day 2. Rats were transcardially perfused with fixative 2h following the onset of the swim on Day 2 for analysis of c-Fos expression in midbrain serotonergic neurons. Cold water (19°C) swim on Day 1 reduced Tb, compared to both 25°C swim and HC groups on Day 1, and, relative to rats exposed to HC conditions on Day 1, reduced the hypothermic response to the 25°C swim on Day 2. The 19°C swim on Day 1, relative to HC exposure on Day 1, increased immobility during the 5-min swim on Day 2. Also, 19°C swim, relative to HC conditions, on Day 1 reduced swim (25°C)-induced increases in c-Fos expression in serotonergic neurons within the dorsal and interfascicular parts of the dorsal raphe nucleus. These results suggest that exposure to a 5-min 19°C cold water swim, but not exposure to a 5-min 25°C swim alters physiological, behavioral and serotonergic responses to a subsequent stressor.

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 24 h 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.

Evidence for in vivo thermosensitivity of serotonergic neurons in the rat dorsal raphe nucleus and raphe pallidus nucleus implicated in thermoregulatory cooling

The ability to sense and respond appropriately to increases in ambient and body temperatures is critical for the survival of all animals. Although evidence suggests that brain serotonergic systems play a role in thermoregulation, including thermoregulatory cooling, evidence for activation of brainstem serotonergic neurons in vivo, in unanesthetized animals, during heat exposure is lacking. In this experiment we tested the hypothesis that populations of serotonergic neurons in the midbrain and medullary raphe complex are activated following exposure to warm ambient temperature. Rats were exposed to an incubation chamber at either warm ambient temperature (37 °C) or room temperature (RT; 23 °C) for 105 min. Brains then were removed and processed for immunohistochemical detection of the protein product of the immediate-early gene c-fos (as a marker of neuronal activation) and tryptophan hydroxylase (as a marker of serotonergic neurons). Exposure to warm ambient temperature increased body temperature and c-Fos expression in topographically organized populations of serotonergic neurons in the dorsal raphe nucleus. Activation of the dorsal raphe nucleus serotonergic system was positively correlated with body temperature following exposure to the incubation chamber. In the medulla, exposure to warm ambient temperature, compared with exposure to RT, decreased c-Fos expression in serotonergic neurons in the raphe pallidus nucleus and in non-serotonergic cells in the rostral ventrolateral medulla. Together, these results provide evidence for multiple but anatomically discrete thermosensitive serotonergic systems that may have implications for the regulation of body temperature, as well as, via projections to forebrain targets, cognitive and affective functions.

Urocortin 2 increases c-Fos expression in serotonergic neurons projecting to the ventricular/periventricular system

Serotonin plays an important role in the regulation of anxiety states and physiological responses to aversive stimuli. Intracerebroventricular (i.c.v.) injection of the stress- and anxiety-related neuropeptide urocortin 2 (Ucn 2) increases c-Fos expression in serotonergic neurons in the dorsal (DRD) and caudal (DRC) parts of the dorsal raphe nucleus. These regions contain a subset of serotonergic neurons that projects via the dorsal raphe periventricular tract to periventricular structures, including the subfornical organ and ependymal layer, and to the ventricular system. To determine if Ucn 2 activates ventricle/periventricular-projecting serotonergic neurons in the midbrain raphe complex, we made i.c.v. injections of the retrograde tracer Fluoro-Gold into the lateral ventricle, followed 7 days later by i.c.v. injection of Ucn 2. The DRD at − 8.18 mm and the DRC at −8.54 mm and −9.16 mm bregma were analyzed using a combined bright field and immunofluorescence technique. Approximately 40% of the ventricle/periventricular-projecting neurons in the subdivisions sampled were serotonergic. Urocortin 2 increased c-Fos expression in ventricle/periventricular-projecting serotonergic neurons in the DRC and in non-ventricle/periventricular-projecting serotonergic neurons in the DRD and DRC. Of the total population of ventricle/periventricular-projecting serotonergic neurons in the DRC at −8.54 and −9.16 mm bregma, 35% expressed c-Fos following Ucn 2 injections. These data are consistent with previous studies showing that i.c.v. injection of Ucn 2 activates subpopulations of serotonergic neurons restricted to the mid-rostrocaudal DRD and DRC and further demonstrate that these include both subsets of serotonergic neurons that do and do not project to the ventricle/periventricular system.

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.

Disruption of GABAergic tone in the dorsomedial hypothalamus attenuates responses in a subset of serotonergic neurons in the dorsal raphe nucleus following lactate-induced panic.

Panic patients are vulnerable to induction of panic attacks by sub-threshold interoceptive stimuli such as intravenous (i.v.) sodium lactate infusions. Facilitation of serotonergic signaling with selective serotonin reuptake inhibitors can suppress anxiety and panic-like responses, but the mechanisms involved are not clearly defined. We investigated the effects of i.v. 0.5 M sodium lactate or saline, in control and panic-prone rats on c-Fos expression in serotonergic neurons within subdivisions of the midbrain/pontine raphe nuclei. Rats were chronically infused with either the GABA synthesis inhibitor l-allylglycine into the dorsomedial hypo thalamus to make them panic-prone, or the enantiomer d-allylglycine (d-AG) in controls. Lactate increased c-Fos expression in serotonergic neurons located in the ventrolateral part of the dorsal raphe nucleus (DRVL) and ventrolateral periaqueductal gray (VLPAG) of control, but not panic-prone, rats. The distribution of lactate-sensitive serotonergic neurons in d-AG-treated rats is virtually identical to previously defined pre-sympathomotor serotonergic neurons with multisynaptic projections to peripheral organs mediating 'fight-or-flight'-related autonomic and motor responses. We hypothesized that serotonergic neurons within the DRVL/VLPAG region represent a 'sympathomotor control system' that normally limits autonomic/behavioral responses to innocuous interoceptive and exteroceptive stimuli, and that dysfunction of this serotonergic system contributes to an anxiety-like state and increases vulnerability to panic in animals and humans.

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 to 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: 8–13lx) or high-light (HL: 400–500lx) 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.

Serotonergic systems associated with arousal and vigilance behaviors following administration of anxiogenic drugs

Serotonergic systems play important roles in modulating behavioral arousal, including behavioral arousal and vigilance associated with anxiety states. To further our understanding of the neural systems associated with increases in anxiety states, we investigated the effects of multiple anxiogenic drugs on topographically organized subpopulations of serotonergic neurons using double immunohistochemical staining for c-Fos and tryptophan hydroxylase combined with topographical analysis of the rat dorsal raphe nucleus (DR). Anxiogenic drugs with diverse pharmacological properties including the adenosine receptor antagonist caffeine, the serotonin 5-HT 2A/2C receptor agonist m-chlorophenyl piperazine (mCPP), the α 2-adrenoreceptor antagonist yohimbine, and the benzodiazepine receptor partial inverse agonist N-methyl-beta-carboline-3-carboxamide (FG-7142) induced increases in behavioral arousal and vigilance behaviors consistent with an increase in anxiety state. In addition, these anxiogenic drugs, excluding yohimbine, had convergent actions on an anatomically-defined subset of serotonergic neurons within the middle and caudal, dorsal subdivision of the DR. High resolution topographical analysis revealed that at the mid-rostrocaudal level, caffeine and FG-7142 had convergent effects on c-Fos expression in serotonergic neurons that were restricted to a previously undefined region, which we have named the shell region of the dorsal part of the dorsal raphe nucleus (DRDSh), that overlaps the anatomical border between the dorsal part of the dorsal raphe nucleus, the ventral part of the dorsal raphe nucleus (DRV), and the ventrolateral part of the dorsal raphe nucleus (DRVL). Retrograde tracing methods revealed that DRDSh contains large numbers of neurons projecting to the basolateral amygdaloid nucleus, a forebrain structure important for emotional appraisal and modulation of anxiety-related physiological and behavioral responses. Together these findings support the hypothesis that there is a functional topographical organization in the DR and are consistent with the hypothesis that anxiogenic drugs have selective actions on a subpopulation of serotonergic neurons projecting to a distributed central autonomic and emotional motor control system regulating anxiety states and anxiety-related physiological and behavioral responses.

Perspective on NO in brain function

The ability to sense and respond appropriately to increases in ambient and body temperatures is critical for the survival of all animals. Although evidence suggests that brain serotonergic systems play a role in thermoregulation, including thermoregulatory cooling, evidence for activation of brainstem serotonergic neurons in vivo, in unanesthetized animals, during heat exposure is lacking. In this experiment we tested the hypothesis that populations of serotonergic neurons in the midbrain and medullary raphe complex are activated following exposure to warm ambient temperature. Rats were exposed to an incubation chamber at either warm ambient temperature (37 °C) or room temperature (RT; 23 °C) for 105 min. Brains then were removed and processed for immunohistochemical detection of the protein product of the immediate-early gene c-fos (as a marker of neuronal activation) and tryptophan hydroxylase (as a marker of serotonergic neurons). Exposure to warm ambient temperature increased body temperature and c-Fos expression in topographically organized populations of serotonergic neurons in the dorsal raphe nucleus. Activation of the dorsal raphe nucleus serotonergic system was positively correlated with body temperature following exposure to the incubation chamber. In the medulla, exposure to warm ambient temperature, compared with exposure to RT, decreased c-Fos expression in serotonergic neurons in the raphe pallidus nucleus and in non-serotonergic cells in the rostral ventrolateral medulla. Together, these results provide evidence for multiple but anatomically discrete thermosensitive serotonergic systems that may have implications for the regulation of body temperature, as well as, via projections to forebrain targets, cognitive and affective functions.