Lynn G. Kirby

Serotonin1A receptor acts during development to establish normal anxiety-like behaviour in the adult.

Serotonin is implicated in mood regulation, and drugs acting via the serotonergic system are effective in treating anxiety and depression. Specifically, agonists of the serotonin1A receptor have anxiolytic properties, and knockout mice lacking this receptor show increased anxiety-like behaviour. Here we use a tissue-specific, conditional rescue strategy to show that expression of the serotonin1A receptor primarily in the hippocampus and cortex, but not in the raphe nuclei, is sufficient to rescue the behavioural phenotype of the knockout mice. Furthermore, using the conditional nature of these transgenic mice, we suggest that receptor expression during the early postnatal period, but not in the adult, is necessary for this behavioural rescue. These findings show that postnatal developmental processes help to establish adult anxiety-like behaviour. In addition, the normal role of the serotonin1A receptor during development may be different from its function when this receptor is activated by therapeutic intervention in adulthood.

Serotonergic mediation of the effects of fluoxetine, but not desipramine, in the rat forced swimming test

Abstract Rationale: The forced swimming test (FST) is a behavioral test in rodents that predicts the clinical efficacy of many types of antidepressant treatments. Recently, a behavior sampling technique was developed that scores individual response categories, including swimming, climbing and immobility. Although all antidepressant drugs reduce immobility in the FST, at least two distinct active behavioral patterns are produced by pharmacologically selective antidepressant drugs. Serotonin-selective reuptake inhibitors increase swimming behavior, while drugs acting primarily to increase extracellular levels of norepinephrine or dopamine increase climbing behavior. Distinct patterns of active behaviors in the FST may be mediated by distinct neurotransmitters, but this has not been shown directly. Objectives: The present study examined the role of serotonin in mediating active behaviors in the forced swimming test after treatment with two antidepressant drugs, the selective serotonin reuptake inhibitor, fluoxetine and the selective norepinephrine reuptake inhibitor, desipramine. Methods: Endogenous serotonin was depleted by administering para-cholorophenylalanine (PCPA, 150 mg/kg, IP.) to rats 72 h and 48 h prior to the swim test. Fluoxetine (10 mg/kg, SC) or desipramine (10 mg/kg, SC) was given three times over a 24-h period prior to the FST. Behavioral responses, including immobility, swimming and climbing, were counted during the 5-min test. Results: Pretreatment with PCPA blocked fluoxetine-induced reduction in immobility and increase in swimming behavior during the FST. In contrast, PCPA pretreatment did not interfere with the ability of desipramine to reduce immobility and increase climbing behavior. Conclusions: Depletion of serotonin prevented the behavioral effects of the selective serotonin reuptake inhibitor fluoxetine in the rat FST. Furthermore, depletion of serotonin had no impact on the behavioral effects induced by the selective norepinephrine reuptake inhibitor, desipramine. The effects of antidepressant drugs on FST-induced immobility may be exerted by distinguishable contributions from different neurotransmitter systems.

Effects of corticotropin-releasing factor on neuronal activity in the serotonergic dorsal raphe nucleus.

The present study examined the regional localization of corticotropin-releasing factor (CRF)- and 5-hydroxytryptamine (5-HT)-immunoreactive (IR) fibers within the rat dorsal raphe nucleus (DRN) using immunohistochemistry. Additionally, the effects of CRF, administered intracerebroventricularly (0.1–3.0 μg) or intraraphe (0.3–30 ng), on discharge rates of putative 5-HT DRN neurons were quantified using in vivo single unit recording in halothane-anesthetized rats. CRF-IR fibers were present at all rostrocaudal levels of the DRN and exhibited a topographical distribution. CRF produced predominantly inhibitory effects on DRN discharge at lower doses and these effects diminished or became excitatory at higher doses. Inhibition of DRN discharge by CRF was attenuated by the nonselective CRF antagonist, DPheCRF12–41 and the CRF-R1-selective antagonist, antalarmin, implicating the CRF-R1 receptor subtype in these electrophysiological effects. The present findings provide anatomical and physiological evidence for an impact of CRF on the DRN-5HT system.

Effects of Corticotropin-Releasing Factor on Brain Serotonergic Activity

The serotonergic dorsal raphe nucleus is innervated by corticotropin-releasing factor (CRF) and expresses CRF receptors, suggesting that endogenous CRF impacts on this system. The present study characterized interactions between CRF and the dorsal raphe serotonin (5-HT) system. The effects of intracerebroventricularly (i.c.v.) administered CRF on microdialysate concentrations of 5-HT in the lateral striatum of freely moving rats were determined. CRF had biphasic effects, with 0.1 and 0.3 μg decreasing, and 3.0 μg increasing 5-HT dialysate concentrations. I.C.V. administration of CRF inhibited neuronal activity of the majority of dorsal raphe neurons at both low (0.3 μg) and high (3 μg) doses. Likewise, intraraphe administration of CRF (0.3 and 1.0 ng) had predominantly inhibitory effects on discharge rate. Together, these results suggest that CRF is positioned to regulate the function of the dorsal raphe serotonergic system via actions within the cell body region. This regulation may play a role in stress-related psychiatric disorders in which 5-HT has been implicated.

Regional differences in the effects of forced swimming on extracellular levels of 5-hydroxytryptamine and 5-hydroxyindoleacetic acid

The effects of forced swimming for 30 min on extracellular 5-hydroxytryptamine (5-HT) levels were examined in five brain regions in rats using in vivo microdialysis. A single dialysis probe was implanted under surgical anesthesia into either the striatum, ventral hippocampus, frontal cortex, amygdala, or lateral septum on the day before the study. Dialysate content of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA) was measured by HPLC. Forced swimming elevated extracellular levels of 5-HT in the striatum to a maximum of 90% above baseline. In contrast, forced swimming reduced 5-HT levels in the amygdala and lateral septum to 50 and 40% of baseline, respectively. In the hippocampus and frontal cortex, 5-HT levels were not altered significantly by forced swimming. In all five brain regions, forced swimming reduced 5-HIAA levels to 45-60% of baseline. These results suggest that forced swimming modulates 5-HT neurotransmission in a regionally specific manner. Aside from being a significant biological stressor, the forced swimming test is used as an animal behavioral model to detect antidepressant drugs, including drugs that alter 5-HT neurotransmission. It is possible that the alterations of extracellular levels of 5-HT produced by forced swimming in certain brain regions may be associated with the ability of antidepressant drugs to selectively alter behavioral performance during the forced swimming test.

The effects of different stressors on extracellular 5-hydroxytryptamine and 5-hydroxyindoleacetic acid.

The effects of application of five different stressors on extracellular 5-hydroxytryptamine and 5-hydroxyindoleacetic acid in the striatum and hippocampus were compared using in vivo microdialysis. Forced swimming for 30 min elevated extracellular 5-hydroxytryptamine to 90% above basal levels and reduced 5-hydroxyindoleacetic acid to 45% of basal levels in the striatum during the swim session. In contrast, hippocampal 5-hydroxytryptamine was not altered significantly by forced swimming but 5-hydroxyindoleacetic acid levels were reduced to 60% of basal levels. Tail pinch for 5 min elevated 5-hydroxytryptamine to 55% above basal levels in striatum and to 35% above basal levels in hippocampus. Tail pinch had no effect on 5-hydroxyindoleacetic acid in either brain region. In contrast to forced swimming and the tail pinch, the other three stressors, immobilization stress for 100 min, exposure to a cold environment (4 degrees C) for 2 h, and forced motor activity on a rotarod for 30 min, failed to alter extracellular 5-hydroxytryptamine in either the striatum or the hippocampus. All five stressors increased plasma corticosterone levels: tail pinch, 246%; cold stress, 432%; immobilization, 870%; forced motor activity, 1030%; and forced swimming, 1530%. These results suggest that individual stressors produce different effects on extracellular 5-hydroxytryptamine in different brain regions. In addition, there does not appear to be a relationship between the effects of stressors on the 5-hydroxytryptamine system and the magnitude of their ability to activate the hypothalamic-pituitary-adrenal axis.

Distinguishing characteristics of serotonin and non-serotonin-containing cells in the dorsal raphe nucleus: Electrophysiological and immunohistochemical studies

The membrane properties and receptor-mediated responses of rat dorsal raphe nucleus neurons were measured using intracellular recording techniques in a slice preparation. After each experiment, the recorded neuron was filled with neurobiotin and immunohistochemically identified as 5-hydroxytryptamine (5-HT)-immunopositive or 5-HT-immunonegative. The cellular characteristics of all recorded neurons conformed to previously determined classic properties of serotonergic dorsal raphe nucleus neurons: slow, rhythmic activity in spontaneously active cells, broad action potential and large afterhyperpolarization potential. Two electrophysiological characteristics were identified that distinguished 5-HT from non-5-HT-containing cells in this study. In 5-HT-immunopositive cells, the initial phase of the afterhyperpolarization potential was gradual (tau=7.3+/-1.9) and in 5-HT-immunonegative cells it was abrupt (tau=1.8+/-0.6). In addition, 5-HT-immunopositive cells had a shorter membrane time constant (tau=21.4+/-4.4) than 5-HT-immunonegative cells (tau=33.5+/-4.2). Interestingly, almost all recorded neurons were hyperpolarized in response to stimulation of the inhibitory 5-HT(1A) receptor. These results suggested that 5-HT(1A) receptors are present on non-5-HT as well as 5-HT neurons. This was confirmed by immunohistochemistry showing that although the majority of 5-HT-immunopositive cells in the dorsal raphe nucleus were double-labeled for 5-HT(1A) receptor-IR, a small but significant population of 5-HT-immunonegative cells expressed the 5-HT(1A) receptor. These results underscore the heterogeneous nature of the dorsal raphe nucleus and highlight two membrane properties that may better distinguish 5-HT from non-5-HT cells than those typically reported in the literature. In addition, these results present electrophysiological and anatomical evidence for the presence of 5-HT(1A) receptors on non-5-HT neurons in the dorsal raphe nucleus.

Evidence for corticotropin-releasing factor regulation of serotonin in the lateral septum during acute swim stress: adaptation produced by repeated swimming

AbstractRationale. Swim stress decreases extracellular serotonin (5-HT) levels in the rat lateral septum, and adaptation to this effect occurs with repeated swimming. Corticotropin-releasing factor (CRF) administered into the dorsal raphe nucleus (DRN) also decreases 5-HT release in the lateral septum, suggesting that CRF may mediate the effects of swim stress. Objectives. The hypothesis that endogenous CRF mediates the reduction of 5-HT levels in the lateral septum evoked by swim stress and is involved in the adaptation that occurs with repeated swim stress was tested. Methods. Extracellular 5-HT levels in rat lateral septum were quantified by means of in vivo microdialysis. Extracellular single unit activity was recorded from the DRN. Results. Intracerebroventricular (i.c.v.) administration of a CRF receptor antagonist prevented the ability of swim stress to decrease 5-HT release in the lateral septum. Prior exposure to swim stress reduced the ability of both CRF (i.c.v.) and a subsequent swim stress to decrease lateral septum 5-HT release (cross adaptation). Additionally, the effects of CRF, administered into the DRN, on DR neuronal discharge were attenuated in rats with a history of swim stress. Finally, administration of a CRF receptor antagonist (i.c.v.) between two swim stress sessions restored the neurochemical response to swim stress (i.e., 5-HT levels were reduced during the second exposure to swim). Conclusions. Endogenous CRF modulates 5-HT transmission during acute environmental stress and is also integral to adaptation of the 5-HT response produced by repeated stress. Modulation of the 5-HT system by CRF during acute stress may underlie certain coping behaviors, while stress-induced adaptation of this effect may be involved in psychiatric manifestations of repeated stress.

Corticotropin-Releasing Factor Increases GABA Synaptic Activity and Induces Inward Current in 5-Hydroxytryptamine Dorsal Raphe Neurons

Stress-related psychiatric disorders such as anxiety and depression involve dysfunction of the serotonin [5-hydroxytryptamine (5-HT)] system. Previous studies have found that the stress neurohormone corticotropin-releasing factor (CRF) inhibits 5-HT neurons in the dorsal raphe nucleus (DRN) in vivo. The goals of the present study were to characterize the CRF receptor subtypes (CRF-R1 and -R2) and cellular mechanisms underlying CRF–5-HT interactions. Visualized whole-cell patch-clamp recording techniques in brain slices were used to measure spontaneous or evoked GABA synaptic activity in DRN neurons of rats and CRF effects on these measures. CRF-R1 and -R2-selective agonists were bath applied alone or in combination with receptor-selective antagonists. CRF increased presynaptic GABA release selectively onto 5-HT neurons, an effect mediated by the CRF-R1 receptor. CRF increased postsynaptic GABA receptor sensitivity selectively in 5-HT neurons, an effect to which both receptor subtypes contributed. CRF also had direct effects on DRN neurons, eliciting an inward current in 5-HT neurons mediated by the CRF-R2 receptor and in non-5-HT neurons mediated by the CRF-R1 receptor. These results indicate that CRF has direct membrane effects on 5-HT DRN neurons as well as indirect effects on GABAergic synaptic transmission that are mediated by distinct receptor subtypes. The inhibition of 5-HT DRN neurons by CRF in vivo may therefore be primarily an indirect effect via stimulation of inhibitory GABA synaptic transmission. These results regarding the cellular mechanisms underlying the complex interaction between CRF, 5-HT, and GABA systems could contribute to the development of novel treatments for stress-related psychiatric disorders.

The Effect of Repeated Exposure to Forced Swimming on Extracellular Levels of 5-Hydroxytryptamine in the Rat

The effects of repeated exposure to forced swimming was examined on extracellular concentrations of 5-hydroxytryptamine (5-HT), behavioral, and endocrine responses in rats. Animals were exposed to a 15-min swimming session on two consecutive days. On the first day, the swimming session increased extracellular 5-HT by 80 % over baseline in the striatum and reduced 5-HT to 40 % below baseline in the lateral septum. On the second day, however, the swimming session produced no effect on 5-HT in either brain region. Ratings of behavior showed that rats demonstrated climbing and swimming behaviors and developed immobility during the initial swimming session and that ratings of immobility increased and swimming decreased during the second swimming session. Immobility was positively correlated and swimming was negatively correlated with changes in extracellular 5-HT in the lateral septum but not in the striatum. Plasma corticosterone was equally elevated by 950 % after either 1 or 2 days of swimming exposure. These results show that there is rapid adaptation to the effects of repeated forced swimming on the regionally-specific, bi-directional response of extracellular 5-HT. In addition, changes in extracellular 5-HT in the lateral septum may be related to the behaviors produced during the forced swimming test that underlie its utility as an animal model of depression.