David A. Morilak

Effects of Chronic Antidepressant Treatments on Serotonin Transporter Function, Density, and mRNA Level

To investigate functional changes in the brain serotonin transporter (SERT) after chronic antidepressant treatment, several techniques were used to assess SERT activity, density, or its mRNA content. Rats were treated by osmotic minipump for 21 d with the selective serotonin reuptake inhibitors (SSRIs) paroxetine or sertraline, the selective norepinephrine reuptake inhibitor desipramine (DMI), or the monoamine oxidase inhibitor phenelzine. High-speedin vivo electrochemical recordings were used to assess the ability of the SSRI fluvoxamine to modulate the clearance of locally applied serotonin in the CA3 region of hippocampus in drug- or vehicle-treated rats. Fluvoxamine decreased the clearance of serotonin in rats treated with vehicle, DMI, or phenelzine but had no effect on the clearance of serotonin in SSRI-treated rats. SERT density in the CA3 region of the hippocampus of the same rats, assessed by quantitative autoradiography with tritiated cyanoimipramine ([3H]CN-IMI), was decreased by 80–90% in SSRI-treated rats but not in those treated with phenelzine or DMI. The serotonin content of the hippocampus was unaffected by paroxetine or sertraline treatment, ruling out neurotoxicity as a possible explanation for the SSRI-induced decrease in SERT binding and alteration in 5-HT clearance. Levels of mRNA for the SERT in the raphe nucleus were also unaltered by chronic paroxetine treatment. Based on these results, it appears that the SERT is downregulated by chronic administration of SSRIs but not other types of antidepressants; furthermore, the downregulation is not caused by decreases in SERT gene expression.

Chronic Unpredictable Stress Induces a Cognitive Deficit and Anxiety-Like Behavior in Rats that is Prevented by Chronic Antidepressant Drug Treatment

Chronic stress is a risk factor for the development of many psychopathological conditions in humans, including major depression and anxiety disorders. There is a high degree of comorbidity of depression and anxiety. Moreover, cognitive impairments associated with frontal lobe dysfunction, including deficits in cognitive set-shifting and behavioral flexibility, are increasingly recognized as major components of depression, anxiety disorders, and other stress-related psychiatric illnesses. To begin to understand the neurobiological mechanisms underlying the cognitive and emotional consequences of chronic stress, it is necessary to employ an animal model that exhibits similar effects. In the present study, a rat model of chronic unpredictable stress (CUS) consistently induced a cognitive impairment in extradimensional set shifting capability in an attentional set shifting test, suggesting an alteration in function of the medial prefrontal cortex. CUS also increased anxiety-like behavior on the elevated plus-maze. Further, chronic treatment both with the selective norepinephrine reuptake blocker, desipramine (7.5 mg/kg/day), and the selective serotonin reuptake blocker, escitalopram (10 mg/kg/day), beginning 1 week before CUS treatment and continuing through the behavioral testing period, prevented the CUS-induced deficit in extradimensional set-shifting. Chronic desipramine treatment also prevented the CUS-induced increase in anxiety-like behavioral reactivity on the plus-maze, but escitalopram was less effective on this measure. Thus, CUS induced both cognitive and emotional disturbances that are similar to components of major depression and anxiety disorders. These effects were prevented by chronic treatment with antidepressant drugs, consistent also with clinical evidence that relapse of depressive episodes can be prevented by antidepressant drug treatment.

Noradrenergic modulation of cognitive function in rat medial prefrontal cortex as measured by attentional set shifting capability.

The brain noradrenergic system is thought to facilitate neuronal processes that promote behavioral activation, alertness, and attention. One region in which norepinephrine may exert such effects is the medial prefrontal cortex, which has been implicated in many cognitive functions including arousal, attention, motivation, working memory, response inhibition, and behavioral flexibility. The present study addressed the modulatory influence of noradrenergic neurotransmission in medial prefrontal cortex on cognitive function in rats, as measured by performance in an attentional set shifting task. In experiment 1, we tested effects of increasing and decreasing brain noradrenergic neurotransmission by systemic administration of the alpha2-adrenergic autoreceptor antagonist and agonist drugs, atipamezole and clonidine, respectively. Atipamezole pretreatment significantly improved performance on the stages of the attentional task requiring an extradimensional shift in attention, and those involving stimulus reversals, whereas clonidine had no effect at any stage. In experiment 2, we then tested effects of microinjecting alpha1- or beta-adrenergic receptor antagonists into medial prefrontal cortex on the enhancement of performance on the extradimensional task produced by atipamezole. The atipamezole-induced enhancement of performance on the extradimensional set shifting task was blocked by alpha1-, but not beta-adrenergic receptor antagonists in medial prefrontal cortex. Neither antagonist alone had any effect on extradimensional set shift performance in the absence of atipamezole-induced enhancement. These results indicate that elevating noradrenergic activity at alpha1-receptors in medial prefrontal cortex facilitates cognitive performance of rats in an attentional set-shifting task, which may contribute to the role of norepinephrine in behavioral state changes such as arousal, or to the beneficial cognitive effects of psychotherapeutic drugs that target noradrenergic neurotransmission.

Modulatory effects of norepinephrine in the lateral bed nucleus of the stria terminalis on behavioral and neuroendocrine responses to acute stress.

The brain noradrenergic system is activated by stress, and modulates the activity of forebrain regions involved in behavioral and neuroendocrine responses to stress, such as the lateral bed nucleus of the stria terminalis (BSTL). This region of the limbic forebrain receives dense noradrenergic innervation, and has been implicated in both anxiety and regulation of the hypothalamic-pituitary-adrenal axis. We hypothesized that stress-induced release of norepinephrine in the BSTL modulates anxiety-like behavioral responses to stress and activation of the hypothalamic-pituitary-adrenal stress axis. Using microdialysis, we showed that release of norepinephrine was increased in the BSTL of male Sprague-Dawley rats during immobilization stress. In the next experiment, we then microinjected noradrenergic antagonists into the BSTL immediately prior to acute immobilization stress to examine noradrenergic modulation of behavioral stress reactivity. Either the alpha(1)-receptor antagonist benoxathian, or a cocktail of beta(1)- and beta(2)-receptor antagonists (betaxolol+ICI 118,551) blocked the anxiety-like reduction in open-arm exploration on the elevated plus-maze, but not the reduction in social behavior induced in the social interaction test. In a third experiment, benoxathian reduced plasma levels of adrenocorticotropic hormone following stress, but beta-receptor antagonists had no effect. From these results we suggest that stress-induced norepinephrine release acts on both alpha(1)- and beta-receptors in the BSTL to facilitate anxiety-like behavioral responses on the plus-maze but not the social interaction test, and modulates hypothalamic-pituitary-adrenal axis activation via alpha(1)-receptors only. Together with previous results in which adrenergic antagonists in central amygdala attenuated behavioral responses on the social interaction test but not the plus-maze, these observations suggest the two behavioral tests measure different dimensions of stress reactivity, and that norepinephrine facilitates different components of the stress response by region- and receptor-specific mechanisms.

Antidepressants and brain monoaminergic systems: A dimensional approach to understanding their behavioural effects in depression and anxiety disorders

There is extensive comorbidity between depression and anxiety disorders. Dimensional psychiatric and psychometric approaches have suggested that dysregulation of a limited number of behavioural dimensions that cut across diagnostic categories can account for both the shared and unique symptoms of depression and anxiety disorders. Such an approach recognizes that anxiety, the emotional response to stress, is a key element of depression as well as the defining feature of anxiety disorders, and many antidepressants appear to be effective in the treatment of anxiety disorders as well as depression. Therefore, the pharmacological actions of these drugs must account for their efficacy in both. Brain noradrenergic and serotonergic systems, and perhaps to a more limited extent the dopaminergic system, regulate or modulate many of the same behavioural dimensions (e.g. negative or positive affect) that are affected in depression and anxiety disorders, and that are ameliorated by drug treatment. Whereas much recent research has focused on the regulatory effects of antidepressants on synaptic function and cellular proteins, less emphasis has been placed on monoaminergic regulation at a more global systemic level, or how such systemic alterations in monoaminergic function might alleviate the behavioural, cognitive, emotional and physiological manifestations of depression and anxiety disorders. In this review, we discuss how chronic antidepressant treatment might regulate the tonic activity and/or phasic reactivity of brain monoaminergic systems to account for their ability to effectively modify the behavioural dimensions underlying improvement in both depression and anxiety disorders.

Stress reactivity of the brain noradrenergic system in three rat strains differing in their neuroendocrine and behavioral responses to stress: Implications for susceptibility to stress-related neuropsychiatric disorders

The brain noradrenergic system is activated by stress, modulating the activity of forebrain regions involved in behavioral and neuroendocrine responses to stress. In this study, we characterized brain noradrenergic reactivity to acute immobilization stress in three rat strains that differ in their neuroendocrine stress response: the inbred Lewis (Lew) and Wistar-Kyoto (WKY) rats, and outbred Sprague-Dawley (SD) rats. Noradrenergic reactivity was assessed by measuring tyrosine hydroxylase mRNA expression in locus coeruleus, and norepinephrine release in the lateral bed nucleus of the stria terminalis. Behavioral measures of arousal and acute stress responsivity included locomotion in a novel environment, fear-potentiated startle, and stress-induced reductions in social interaction and open-arm exploration on the elevated-plus maze. Neuroendocrine responses were assessed by plasma adrenocorticotropic hormone. Compared to SD, adrenocorticotropic hormone responses of Lew rats were blunted, whereas those of WKY were enhanced. The behavioral effects of stress were similar in Lew and SD rats, despite baseline differences. Lew had similar elevations of tyrosine hydroxylase mRNA, and initially greater norepinephrine release in the lateral bed nucleus of the stria terminalis during stress, although both noradrenergic responses returned toward baseline more rapidly than in SD rats. WKY rats showed depressed baseline startle and lower baseline exploratory and social behavior than SD. However, unlike the Lew or SD rats, WKY exhibited a lack both of fear potentiation of the startle response and of stress-induced reductions in exploratory and social behavior, indicating attenuated stress responsivity. Acute noradrenergic reactivity to stress, measured by either tyrosine hydroxylase mRNA levels or norepinephrine release, was also attenuated in WKY rats. Thus, reduced arousal and behavioral responsivity in WKY rats may be related to deficient brain noradrenergic reactivity. This deficit may alter their ability to cope with stress, resulting in the exaggerated neuroendocrine responses and increased susceptibility to stress-related pathology exhibited by this strain.

Modulatory effects of norepinephrine, acting on alpha1 receptors in the central nucleus of the amygdala, on behavioral and neuroendocrine responses to acute immobilization stress

The central nucleus of the amygdala (CeA) is a component of the limbic fear-anxiety circuit, and has also been implicated in regulation of the hypothalamic-pituitary-adrenal (HPA) stress axis. The CeA receives dense noradrenergic innervation, and is rich in expression of alpha(1)-adrenergic receptors. We hypothesized that norepinephrine (NE), acting on alpha(1) receptors in CeA, may modulate stress-induced anxiety-like behavioral responses and HPA activation. To investigate the role of alpha(1) adrenergic receptors in CeA on stress-induced behavioral reactivity, the alpha(1) antagonist benoxathian was microinjected bilaterally into CeA of male Sprague-Dawley rats, and anxiety-like behavioral responses to acute immobilization stress were measured on the Social Interaction (SI) test and on the Elevated Plus-maze (EPMZ). Benoxathian dose dependently blocked the reduction in SI time induced by immobilization stress, whereas beta-receptor antagonists had no effect, consistent with an absence of beta-receptors in CeA. By contrast, in separate experiments, benoxathian had no effect on stress-induced reduction in open-arm exploratory behavior on the EPMZ, nor on stress-induced plasma ACTH secretion. These results confirm that the SI test and EPMZ measure different aspects of behavioral stress reactivity that can be modulated independently, and likewise, that noradrenergic modulation of behavioral stress reactivity can occur independently of modulation of the HPA axis.

Behavioral reactivity to stress: Amplification of stress-induced noradrenergic activation elicits a galanin-mediated anxiolytic effect in central amygdala

Brain norepinephrine (NE) modulates many aspects of the stress response. The interaction between NE and neuropeptides such as galanin, with which it is closely associated and which may be released from noradrenergic terminals under conditions of high activity, has not been well studied. We therefore investigated the modulatory effects of galanin in the central nucleus of the amygdala (CeA) on behavioral responsivity to stress when activation of the noradrenergic system was amplified using the adrenergic autoreceptor antagonist yohimbine (2.5 mg/kg ip). Either immobilization stress or yohimbine alone had anxiogenic effects on rat behavior in the elevated plus maze. However, yohimbine pretreatment before stress produced a paradoxical anxiolytic response, which we hypothesized was attributable to galanin release in CeA. Microdialysis verified that yohimbine amplified NE release in CeA during immobilization stress, and also showed that whereas there was no detectable change in galanin release in CeA during stress alone, there was an increase during immobilization stress after yohimbine pretreatment. Bilateral administration of the galanin antagonist M40 into CeA before stress blocked the anxiolytic influence of yohimbine pretreatment. Exogenous galanin mimicked the anxiolytic effect of yohimbine pretreatment, and this too was blocked by M40. These results suggest that amplifying the noradrenergic response to stress can recruit galanin release in CeA, which buffers the anxiety-like behavioral response to acute stress. The balance between noradrenergic and peptidergic neurotransmission may be modified by prior stress, drug treatment or genetic variability, and may represent a novel target for treatment of stress-related neuropsychiatric disorders.

Behavioural assays to model cognitive and affective dimensions of depression and anxiety in rats.

Animal models have been used extensively to investigate neuropsychiatric disorders, such as depression, and their treatment. However, the aetiology and pathophysiology of many such disorders are largely unknown, which makes validation of animal models particularly challenging. Furthermore, many diagnostic symptoms are difficult to define, operationalise and quantify, especially in experimental animals such as rats. Thus, rather than attempting to model complex human syndromes such as depression in their entirety, it can be more productive to define and model components of the illness that may account for clusters of co‐varying symptoms, and that may share common underlying neurobiological mechanisms. In preclinical investigations of the neural regulatory mechanisms linking stress to depression and anxiety disorders, as well as the mechanisms by which chronic treatment with antidepressant drugs may exert their beneficial effects in these conditions, we have employed a number of behavioural tests in rats to model specific cognitive and anxiety‐like components of depression and anxiety disorders. In the present study, we review the procedures for conducting four such behavioural assays: the attentional set‐shifting test, the elevated‐plus maze, the social interaction test and the shock‐probe defensive burying test. The purpose is to serve as a guide to the utility and limitations of these tools, and as an aid in optimising their use and productivity.

Overexpression of the α 1B -adrenergic receptor causes apoptotic neurodegeneration: Multiple system atrophy

Progress toward elucidating the function of α1B-adrenergic receptors (α1BARs) in the central nervous system has been constrained by a lack of agonists and antagonists with adequate α1B-specificity. We have obviated this constraint by generating transgenic mice engineered to overexpress either wild-type or constitutively active α1BARs in tissues that normally express the receptor, including the brain. All transgenic lines showed granulovacular neurodegeneration, beginning in α1B-expressing domains of the brain and progressing with age to encompass all areas. The degeneration was apoptotic and did not occur in non-transgenic mice. Correspondingly, transgenic mice showed an age-progressive hindlimb disorder that was parkinsonian-like, as demonstrated by rescue of the dysfunction by 3, 4-dihydroxyphenylalanine and considerable dopaminergic-neuronal degeneration in the substantia nigra. Transgenic mice also had a grand mal seizure disorder accompanied by a corresponding dysplasia and neurodegeneration of the cerebral cortex. Both behavioral phenotypes (locomotor impairment and seizure) could be partially rescued with the α1AR antagonist terazosin, indicating that α1AR signaling participated directly in the pathology. Our results indicate that overstimulation of α1BAR leads to apoptotic neurodegeneration with a corresponding multiple system atrophy indicative of Shy-Drager syndrome, a disease whose etiology is unknown.