Heidi E.W. Day

Freewheel Running Prevents Learned Helplessness/Behavioral Depression: Role of Dorsal Raphe Serotonergic Neurons

Serotonin (5-HT) neurons in the dorsal raphe nucleus (DRN) are implicated in mediating learned helplessness (LH) behaviors, such as poor escape responding and expression of exaggerated conditioned fear, induced by acute exposure to uncontrollable stress. DRN 5-HT neurons are hyperactive during uncontrollable stress, resulting in desensitization of 5-HT type 1A (5-HT1A) inhibitory autoreceptors in the DRN. 5-HT1A autoreceptor downregulation is thought to induce transient sensitization of DRN 5-HT neurons, resulting in excessive 5-HT activity in brain areas that control the expression of learned helplessness behaviors. Habitual physical activity has antidepressant/anxiolytic properties and results in dramatic alterations in physiological stress responses, but the neurochemical mediators of these effects are unknown. The current study determined the effects of 6 weeks of voluntary freewheel running on LH behaviors, uncontrollable stress-induced activity of DRN 5-HT neurons, and basal expression of DRN 5-HT1A autoreceptor mRNA. Freewheel running prevented the shuttle box escape deficit and the exaggerated conditioned fear that is induced by uncontrollable tail shock in sedentary rats. Furthermore, double c-Fos/5-HT immunohistochemistry revealed that physical activity attenuated tail shock-induced activity of 5-HT neurons in the rostral–mid DRN. Six weeks of freewheel running also resulted in a basal increase in 5-HT1A inhibitory autoreceptor mRNA in the rostral–mid DRN. Results suggest that freewheel running prevents behavioral depression/LH and attenuates DRN 5-HT neural activity during uncontrollable stress. An increase in 5-HT1A inhibitory autoreceptor expression may contribute to the attenuation of DRN 5-HT activity and the prevention of LH in physically active rats.

Distribution of α1a-, α1b- and α1d-adrenergic receptor mRNA in the rat brain and spinal cord

The technique of in situ hybridization with specific ribonucleotide probes was used to determine the distribution patterns of mRNA encoding the alpha 1a-, alpha 1b- and alpha 1d-adrenoceptor (AR) subtypes in rat brain and spinal cord. The expression pattern of alpha 1a-AR mRNA has not been reported previously, and was found to be widespread throughout the rat central nervous system. High levels were found in regions of the olfactory system, several hypothalamic nuclei, and regions of the brainstem and spinal cord, particularly in areas related to motor function. Regions expressing moderate levels of mRNA for this receptor were the septum, bed nucleus of the stria terminalis, cerebral cortex, amygdala, cerebellum and pineal gland. Low expression levels were detected in the hippocampal formation. Most nuclei in the basal ganglia and thalamus expressed extremely low or undetectable levels of alpha 1a-AR mRNA. The expression patterns of the alpha 1b- and alpha 1d-AR mRNAs were similar to those described using oligonucleotide probes in earlier studies. High expression of alpha 1b-AR mRNA was noted in the pineal gland, most thalamic nuclei, lateral nucleus of the amygdala and dorsal and median raphe nuclei. Moderate expression levels were noted throughout the cerebral cortex, and in some olfactory, septal, and brainstem regions. The distribution of alpha 1d-AR mRNA was the most discrete of the three receptors examined. Expression was strong in the olfactory bulb, cerebral cortex, hippocampus, reticular thalamic nucleus, regions of the amygdala, motor nuclei of the brainstem, inferior olivary complex and spinal cord. Comparison of the distributions of the alpha 1a-, alpha 1b- and alpha 1d-AR mRNA suggests unique functional roles for each of these receptors.

Distinct neurochemical populations in the rat central nucleus of the amygdala and bed nucleus of the stria terminalis: Evidence for their selective activation by interleukin-1β

The lateral division of the central nucleus of the amygdala (CEAl) and the oval nucleus of the bed nucleus of the stria terminalis (BSTov) have been linked closely anatomically and functionally. To determine whether these regions may be subdivided further on a neurochemical basis, dual in situ hybridization was used to determine the colocalization of corticotropin‐releasing hormone (CRH), enkephalin (ENK), or neurotensin (NT) with glutamic acid decarboxylase isoforms 65 and 67 [used concurrently as a marker for γ‐aminobutyric acid GABA] in these nuclei. It was found that, for both regions, each peptide invariably was localized in a GABAergic cell. Although there was a similar overlap in the distribution of NT with ENK in the BSTov and CEAl, it was observed that CRH and ENK rarely were colocalized in either nucleus. To determine whether these distinct neuronal populations could be activated differentially, male rats were given a systemic injection of interleukin‐1β (IL‐1β; 5 μg/kg, i.p.), a stimulus that results in a robust increase in c‐fos mRNA expression in the BSTov and CEAl. The neurochemical identity of these activated neurons showed striking similarities between the BSTov and the CEAl; All IL‐1β‐responsive cells were GABAergic, the majority of c‐fos‐ positive cells expressed ENK mRNA (BSTov, 81%; CEAl, 94%), and some expressed NT mRNA (BSTov, 23%; CEAl, 22%), whereas very few expressed CRH mRNA (BSTov, 4%; CEAl, 1%). These data provide evidence for the existence of discrete neural circuits within the BSTov and CEAl, and the similarities in the patterns of neurochemical colocalization in these nuclei are consistent with the concept of an extended amygdala. Furthermore, these data indicate that intraperitoneal IL‐1β recruits neurochemically distinct pathways within the BSTov and CEAl, and it is suggested that this differential activation may mediate specific aspects of immune, limbic, and/or autonomic processes. J. Comp. Neurol. 413:113–128, 1999.

Long-term voluntary wheel running is rewarding and produces plasticity in the mesolimbic reward pathway.

The mesolimbic reward pathway is implicated in stress-related psychiatric disorders and is a potential target of plasticity underlying the stress resistance produced by repeated voluntary exercise. It is unknown, however, whether rats find long-term access to running wheels rewarding, or if repeated voluntary exercise reward produces plastic changes in mesolimbic reward neurocircuitry. In the current studies, young adult, male Fischer 344 rats allowed voluntary access to running wheels for 6 weeks, but not 2 weeks, found wheel running rewarding, as measured by conditioned place preference (CPP). Consistent with prior reports and the behavioral data, 6 weeks of wheel running increased ΔFosB/FosB immunoreactivity in the nucleus accumbens (Acb). In addition, semi quantitative in situ hybridization revealed that 6 weeks of wheel running, compared to sedentary housing, increased tyrosine hydroxylase (TH) mRNA levels in the ventral tegmental area (VTA), increased delta opioid receptor (DOR) mRNA levels in the Acb shell, and reduced levels of dopamine receptor (DR)-D2 mRNA in the Acb core. Results indicate that repeated voluntary exercise is rewarding and alters gene transcription in mesolimbic reward neurocircuitry. The duration-dependent effects of wheel running on CPP suggest that as the weeks of wheel running progress, the rewarding effects of a night of voluntary wheel running might linger longer into the inactive cycle thus providing stronger support for CPP. The observed plasticity could contribute to the mechanisms by which exercise reduces the incidence and severity of substance abuse disorders, changes the rewarding properties of drugs of abuse, and facilitates successful coping with stress.

Differential expression of 5HT-1A, α1b adrenergic, CRF-R1, and CRF-R2 receptor mRNA in serotonergic, γ-aminobutyric acidergic, and catecholaminergic cells of the rat dorsal raphe nucleus

The dorsal raphe nucleus (DR) has a topographic neuroanatomy consistent with the idea that different parts of this nucleus subserve different functions. Here we use dual in situ hybridization to describe the rostral‐caudal neurochemical distribution of three major cell groups, serotonin (5‐hydroxytryptamine; 5‐HT), γ‐aminobutyric acid (GABA), and catecholamine, and their relative colocalization with each other and mRNA encoding four different receptor subtypes that have been described to influence DR responses, namely, 5HT‐1A, α1b adrenergic (α1b ADR), and corticotropin‐releasing factor type 1 (CRF‐R1) and 2 (CRF‐R2) receptors. Serotonergic and GABAergic neurons were distributed throughout the rostral‐caudal extent of the DR, whereas catecholaminergic neurons were generally restricted to the rostral half of the nucleus. These phenotypes essentially represent distinct cell populations, because the neurochemical markers were rarely colocalized. Both 5HT‐1A and α1b ADR mRNA were highly expressed throughout the DR, and the vast majority of serotonergic neurons expressed both receptors. A smaller percentage of GABAergic neurons also expressed 5HT‐1A or α1b ADR mRNA. Very few catecholaminergic cells expressed either 5HT‐1A or α1b ADR mRNA. CRF‐R1 mRNA was detected only at very low levels within the DR, and quantitative colocalization studies were not technically feasible. CRF‐R2 mRNA was mainly expressed at the middle and caudal levels of the DR. At midlevels, CRF‐R2 mRNA was expressed exclusively in serotonin neurons, whereas, at caudal levels, approximately half the CRF‐R2 mRNA was expressed in GABAergic neurons. The differential distribution of distinct neurochemical phenotypes lends support to the idea of functional differentiation of the DR. J. Comp. Neurol. 474:364–378, 2004.

The pattern of brain c-fos mRNA induced by a component of fox odor, 2,5-dihydro-2,4,5-trimethylthiazoline (TMT), in rats, suggests both systemic and processive stress characteristics.

Predators to rodents and their associated odors are increasingly chosen to study the neural mechanisms of stress and anxiety. Specifically, predatory odors are believed to elicit responses based on the perceived threat (psychological or processive), rather than to any direct systemic effects (pain, blood loss, infection, etc.) of the stimulus, which are mediated by distinct neural pathways. The hypothesis that a chemical component from fox feces, 2,5-dihydro-2,4,5-trimethylthiazoline (TMT), elicits stress responses by specific activation of processive neural pathways was tested. Different amounts of TMT (range: 0-600 micromol) or the control odor butyric acid (0-1200 micromol) were presented to male Sprague-Dawley rats for 30 min. Immediately after odor presentation, rats were sacrificed, blood levels of adrenocorticotropic hormone (ACTH) and corticosterone were measured, and brains were rapidly harvested to measure regional brain c-fos mRNA induction by in situ hybridization. Presentation of TMT (> or =75 micromol), but not butyric acid (up to 1200 micromol), significantly increased ACTH and corticosterone release. TMT presentation, especially with amounts (> or =75 micromol) producing endocrine activation, induced c-fos mRNA in several brain areas, including the olfactory bulb, lateral septal nucleus, septohypothalamic nucleus, anteromedial and oval nuclei of the bed nucleus of the stria terminalis, the central nucleus of the amygdala, the anteroventral, anterodorsal, and medial preoptic nuclei, the anterior, dorsomedial, lateral, supramammillary, dorsal premammillary and paraventricular hypothalamic nuclei, the external lateral parabrachial nucleus, the locus coeruleus, and the nucleus of the solitary tract. Interestingly, these brain regions represent a mix of regional c-fos mRNA induction pattern not reported previously with any other single stressor. These results suggest that TMT elicits stress responses through a relatively unique and complex mix of brain regions associated with both processive and systemic neural pathways, unlike those seen in response to cat odors.

Environmental modulation of amphetamine-induced c-fos expression in D1 versus D2 striatal neurons

We have reported previously that exposure to environmental novelty enhances the behavioral activating effects of amphetamine and its ability to induce the immediate early gene c-fos in the striatum and in other brain regions. In the present study, we used double in situ hybridization histochemistry to study the effect of amphetamine and/or novelty on c-fos expression in two populations of striatal neurons that preferentially express either D1 or D2 dopamine receptor mRNA. When given intraperitoneally to rats in their home cage, amphetamine (2.0 mg/kg) increased c-fos expression only in D1 neurons. In contrast, when the same dose of amphetamine was administered to rats in a novel environment, c-fos was increased in both D1 and D2 neurons. We conclude that the neural populations engaged by amphetamine vary as a function of the circumstances surrounding its administration.

Differential Pattern of c-fos mRNA in Rat Brain following Central and Systemic Administration of lnterleukin-1-Beta: Implications for Mechanism of Action

Interleukin-1-beta (IL-1 beta) is a potent activator of the hypothalamic-pituitary-adrenal (HPA) axis, resulting in the release of corticosteroids from the adrenal glands. This effect is evident after both central and peripheral administration, and controversy surrounds the mechanism(s) by which systemic administration of this peptide, which should not cross the blood-brain barrier, may activate the HPA axis. In the present study, IL-1 beta was administered systemically (5 micrograms/kg i.p.) or centrally (100 ng i.c.v.) to male rats. Both routes of administration of IL-1 beta resulted in significant and comparable activation of the HPA axis, as assessed by analysis of plasma conrticosterone. In addition, both routes of administration of IL-1 beta resulted in c-fos mRNA induction in specific regions, as determined by in situ hybridization. These included the meninges, cerebral vasculature, choroid plexus and circumventricular organs. Semiquantitative analysis revealed that both routes of administration resulted in significant and comparable induction of c-fos mRNA in the paraventricular nucleus of the hypothalamus, as compared with control animals. In contrast, in the nucleus tractus solitarius (NTS) and central nucleus of the amygdala (CeA), levels of c-fos mRNA were 3-4 times higher in animals treated intraperitoneally compared with intracerebroventricularly. A similar differential activation of c-fos mRNA was observed in the lateral divisions of the parabrachial nucleus (PBN) and bed nucleus of the stria terminalis (BNST). These data indicate that following systemic administration, IL-1 beta may activate specific brain areas through mechanisms distinct from those involved following central administration. The differential magnitude of the c-fos mRNA response in the NTS, PBN, CeA and BNST is consistent with vagal activation. Physiologically, these results suggest that IL-1 beta may have differential central effects depending on its source or point of entry to the brain.

Amphetamine and cocaine induce different patterns of c-fos mRNA expression in the striatum and subthalamic nucleus depending on environmental context

In the dorsal striatum, there are two major populations of medium spiny projection neurons. One population is positive for dynorphin mRNA (DYN+), and these cells project preferentially to the substantia nigra, forming the so‐called ‘direct pathway’. A second population is positive for enkephalin mRNA (ENK+), and these cells influence the substantia nigra indirectly, via the globus pallidus and subthalamic nucleus. Psychostimulant drugs, such as amphetamine and cocaine, are reported to induce immediate early genes (IEGs) in only one subpopulation of dorsal striatal projection neurons, DYN+ cells. However, this apparent selectivity appears to be a function of environmental context. We found that when given in the animals home cage, amphetamine and cocaine increased expression of the IEG, c‐fos, almost exclusively in DYN+ cells. However, when given in a novel environment, amphetamine and cocaine increased c‐fos mRNA in both DYN+ and ENK+ cells. Furthermore, amphetamine and cocaine increased c‐fos mRNA expression in the subthalamic nucleus when administered in the novel environment, but not when given at home. We conclude that the neural circuitry engaged by psychostimulant drugs, and their ability to induce specific patterns of gene expression, are determined by the environmental context in which they are experienced. This may be related to the ability of environmental novelty to facilitate psychostimulant drug‐induced neuroplasticity.

Wheel running alters serotonin (5-HT) transporter, 5-HT1A, 5-HT1B, and alpha1b-adrenergic receptor mRNA in the rat raphe nuclei

BACKGROUND Altered serotonergic (5-HT) neurotransmission is implicated in the antidepressant and anxiolytic properties of physical activity. In the current study, we investigated whether physical activity alters factors involved in the regulation of central 5-HT neural activity. METHODS In situ hybridization was used to quantify levels of 5-HT transporter (5-HTT), 5-HT(1A), 5-HT(1B), and alpha(1b)-adrenergic receptor (alpha(1b) ADR) messenger ribonucleic acids (mRNAs) in the dorsal (DRN) and median raphe (MR) nuclei of male Fischer rats after either sedentary housing or 3 days, 3 weeks, or 6 weeks of wheel running. RESULTS Wheel running produced a rapid and lasting reduction of 5-HT(1B) mRNA in the ventral DRN. Three weeks of wheel running decreased 5-HTT mRNA in the DRN and MR and increased alpha(1b) ADR mRNA in the DRN. After 6 weeks of wheel running, 5-HTT mRNA remained reduced, but alpha(1b) ADR mRNA returned to sedentary levels. Serotonin(1A) mRNA was increased in the MR and certain DRN subregions after 6 weeks only. CONCLUSIONS Data suggest that the central 5-HT system is sensitive to wheel running in a time-dependent manner. The observed changes in mRNA regulation in a subset of raphe nuclei might contribute to the stress resistance produced by wheel running and the antidepressant and anxiolytic effects of physical activity.