Paul J. Gasser

Radiation-induced assembly of Rad51 and Rad52 recombination complex requires ATM and c-Abl

Cells from individuals with the recessive cancer-prone disorder ataxia telangiectasia (A-T) are hypersensitive to ionizing radiation (I-R). ATM (mutated in A-T) is a protein kinase whose activity is stimulated by I-R. c-Abl, a nonreceptor tyrosine kinase, interacts with ATM and is activated by ATM following I-R. Rad51 is a homologue of bacterial RecA protein required for DNA recombination and repair. Here we demonstrate that there is an I-R-induced Rad51 tyrosine phosphorylation, and this induction is dependent on both ATM and c-Abl. ATM, c-Abl, and Rad51 can be co-immunoprecipitated from cell extracts. Consistent with the physical interaction, c-Abl phosphorylates Rad51 in vitro and in vivo. In assays using purified components, phosphorylation of Rad51 by c-Abl enhances complex formation between Rad51 and Rad52, which cooperates with Rad51 in recombination and repair. After I-R, an increase in association between Rad51 and Rad52 occurs in wild-type cells but not in cells with mutations that compromise ATM or c-Abl. Our data suggest signaling mediated through ATM, and c-Abl is required for the correct post-translational modification of Rad51, which is critical for the assembly of Rad51 repair protein complex following I-R.

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.

Corticosterone-Sensitive Monoamine Transport in the Rat Dorsomedial Hypothalamus: Potential Role for Organic Cation Transporter 3 in Stress-Induced Modulation of Monoaminergic Neurotransmission

Glucocorticoid hormones act within the brain to alter physiological and behavioral responses to stress-related stimuli. Previous studies indicated that acute stressors can increase serotonin [5-hydroxytryptamine (5-HT)] concentrations in the dorsomedial hypothalamus (DMH), a midline hypothalamic structure involved in the integration of physiological and behavioral responses to stress. The current study tests the hypothesis that rapid, stress-induced accumulation of 5-HT is attributable to the inhibition of 5-HT transport via organic cation transporters (OCTs). OCTs are a family of high-capacity, bidirectional, multispecific transporters of organic cations (including 5-HT, dopamine, and norepinephrine) only recently described in brain. In peripheral tissues, organic cation transport via some OCTs is inhibited by corticosterone. We examined the expression and function of OCTs in the periventricular medial hypothalamus of male Sprague Dawley rats using reverse-transcriptase (RT)-PCR, immunohistochemistry, and in vitro transport assays. RT-PCR revealed expression of OCT3 mRNA, but not OCT1 or OCT2 mRNA, in the medial hypothalamus. OCT3-like immunoreactivity was observed in ependymal and glial-like cells in the DMH. Acutely prepared minces of rat medial hypothalamic tissue accumulated the OCT substrates [3H]-histamine and [3H]-N-methyl-4-phenylpyridinium ([3H]-MPP+). Consistent with the pharmacological profile of OCT3, corticosterone, 5-HT, estradiol, and the OCT inhibitor decynium22 dose-dependently inhibited histamine accumulation. Corticosterone and decynium22 also inhibited efflux of [3H]-MPP+ from hypothalamic minces. These data support the hypothesis that corticosterone-induced inhibition of OCT3 mediates stress-induced accumulation of 5-HT in the DMH and suggest that corticosterone may acutely modulate physiological and behavioral responses to stressors by altering serotonergic neurotransmission in this brain region.

Distribution of organic cation transporter 3, a corticosterone-sensitive monoamine transporter, in the rat brain

Organic cation transporter 3 (OCT3) is a high‐capacity, low‐affinity transporter that mediates bidirectional, sodium‐independent transport of dopamine, norepinephrine, epinephrine, serotonin, and histamine. OCT3‐mediated transport is directly inhibited by corticosterone, suggesting a potential role for the transporter in mediating some of the effects of stress and glucocorticoids on monoaminergic neurotransmission. To elucidate the importance of OCT3 in clearance of extracellular monoamines in the brain, we used immunohistochemical techniques to describe the distribution of OCT3‐like‐immunoreactive (OCT3‐ir) cells throughout the rostrocaudal extent of adult male rat brains. OCT3‐ir cell bodies were widely distributed throughout the brain, with the highest densities observed in the superior and inferior colliculi, islands of Calleja, subiculum, lateral septum, lateral and dorsomedial hypothalamic nuclei, and granule cell layers of the main and accessory olfactory bulbs, the cerebellum, and the retrosplenial granular cortex. OCT3‐ir cells and/or fibers were also observed in circumventricular organs, and OCT3‐ir ependymal cells were observed in the linings of all cerebral ventricles. The widespread distribution of OCT3‐ir cell bodies, including regions receiving dense monoaminergic projections, suggests an important role for this transporter in regulating extracellular concentrations of monoamines in the rat brain and is consistent with the hypothesis that corticosterone‐induced inhibition of OCT3‐mediated transport may contribute to effects of acute stress or corticosterone on monoaminergic neurotransmission. J. Comp. Neurol. 512:529–555, 2009.

Corticosterone Acts in the Nucleus Accumbens to Enhance Dopamine Signaling and Potentiate Reinstatement of Cocaine Seeking

Stressful life events are important contributors to relapse in recovering cocaine addicts, but the mechanisms by which they influence motivational systems are poorly understood. Studies suggest that stress may “set the stage” for relapse by increasing the sensitivity of brain reward circuits to drug-associated stimuli. We examined the effects of stress and corticosterone on behavioral and neurochemical responses of rats to a cocaine prime after cocaine self-administration and extinction. Exposure of rats to acute electric footshock stress did not by itself reinstate drug-seeking behavior but potentiated reinstatement in response to a subthreshold dose of cocaine. This effect of stress was not observed in adrenalectomized animals, and was reproduced in nonstressed animals by administration of corticosterone at a dose that reproduced stress-induced plasma levels. Pretreatment with the glucocorticoid receptor antagonist RU38486 did not block the corticosterone effect. Corticosterone potentiated cocaine-induced increases in extracellular dopamine in the nucleus accumbens (NAc), and pharmacological blockade of NAc dopamine receptors blocked corticosterone-induced potentiation of reinstatement. Intra-accumbens administration of corticosterone reproduced the behavioral effects of stress and systemic corticosterone. Corticosterone treatment acutely decreased NAc dopamine clearance measured by fast-scan cyclic voltammetry, suggesting that inhibition of uptake2-mediated dopamine clearance may underlie corticosterone effects. Consistent with this hypothesis, intra-accumbens administration of the uptake2 inhibitor normetanephrine potentiated cocaine-induced reinstatement. Expression of organic cation transporter 3, a corticosterone-sensitive uptake2 transporter, was detected on NAc neurons. These findings reveal a novel mechanism by which stress hormones can rapidly regulate dopamine signaling and contribute to the impact of stress on drug intake.

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.

The Rainbow Trout Pineal Organ: An Endocrine Photometer

Like the pineal organs of other vertebrates, those of teleost fish synthesize and secrete melatonin (N-acetyl-5-methoxytryptamine) (Fenwick, 1970; Gern et al., l978a,b; Falcon et al., 1989a; Kezuka et al., 1989; Zachmann et al., 1991). Unlike mammals, pineal organs in non-mammalian vertebrates are directly photoreceptive and these processes are involved in regulating melatonin synthesis. Pineal melatonin synthesis in chickens (Binkely et al., 1978; Kasal et al., 1979; Takahashi et al., 1980; Takahashi et al., 1989), anoline lizards (Menaker & Wisner, 1983) and probably many other nonmammalian vertebrates (Falcon et al., 1989b; Kezuka et al., 1989) is associated with an endogenous circadian clock. In mammals, experimental evidence does not indicate the presence of a clock within the pineal. Rather in mammals a clock, present in the suprachiasmatic nucleus, regulates pineal melatonin synthesis via neuronal connections from the SCN (Moore & Klein, 1974).

Stress-Induced Cocaine Seeking Requires a Beta-2 Adrenergic Receptor-Regulated Pathway from the Ventral Bed Nucleus of the Stria Terminalis That Regulates CRF Actions in the Ventral Tegmental Area

The ventral bed nucleus of the stria terminalis (vBNST) has been implicated in stress-induced cocaine use. Here we demonstrate that, in the vBNST, corticotropin releasing factor (CRF) is expressed in neurons that innervate the ventral tegmental area (VTA), a site where the CRF receptor antagonist antalarmin prevents the reinstatement of cocaine seeking by a stressor, intermittent footshock, following intravenous self-administration in rats. The vBNST receives dense noradrenergic innervation and expresses β adrenergic receptors (ARs). Footshock-induced reinstatement was prevented by bilateral intra-vBNST injection of the β-2 AR antagonist, ICI-118,551, but not the β-1 AR antagonist, betaxolol. Moreover, bilateral intra-vBNST injection of the β-2 AR agonist, clenbuterol, but not the β-1 agonist, dobutamine, reinstated cocaine seeking, suggesting that activation of vBNST β-2 AR is both necessary for stress-induced reinstatement and sufficient to induce cocaine seeking. The contribution of a β-2 AR-regulated vBNST-to-VTA pathway that releases CRF was investigated using a disconnection approach. Injection of ICI-118,551 into the vBNST in one hemisphere and antalarmin into the VTA of the contralateral hemisphere prevented footshock-induced reinstatement, whereas ipsilateral manipulations failed to attenuate stress-induced cocaine seeking, suggesting that β-2 AR regulate vBNST efferents that release CRF into the VTA, activating CRF receptors, and promoting cocaine use. Last, reinstatement by clenbuterol delivered bilaterally into the vBNST was prevented by bilateral vBNST pretreatment with antalarmin, indicating that β-2 AR-mediated actions in the vBNST also require local CRF receptor activation. Understanding the processes through which stress induces cocaine seeking should guide the development of new treatments for addiction.

Natural and synthetic corticosteroids inhibit uptake2-mediated transport in CNS neurons

In addition to exerting actions via mineralocorticoid and glucocorticoid receptors, corticosteroids also act by inhibiting uptake(2), a high-capacity monoamine transport system originally described in peripheral tissues. Recent studies have demonstrated that uptake(2) transporters are expressed in the brain and play roles in monoamine clearance, suggesting that they mediate some corticosteroid effects on physiological and behavioral processes. However, the sensitivity of brain uptake(2) to many natural and synthetic corticosteroids has not been characterized. Cultured rat cerebellar granule neurons (CGNs) were previously shown to exhibit corticosterone-sensitive accumulation of the uptake(2) substrate 1-methyl-4-phenylpyridinium (MPP(+)). We examined the expression of uptake(1) and uptake(2) transporters in CGNs, and tested the effects of a variety of natural and synthetic corticosteroids on accumulation of [(3)H]-MPP(+) by these cells. Cultured rat CGNs expressed mRNA for three uptake(2)-like transporters: organic cation transporters 1 and 3, and the plasma membrane monoamine transporter. They did not express mRNA for the dopamine or norepinephrine transporters, and expressed very little mRNA for the serotonin reuptake transporter. Accumulation of [(3)H]-MPP(+) by CGNs was dose-dependently inhibited by corticosterone and decynium-22, known inhibitors of uptake(2). Accumulation of MPP(+) was also dose-dependently inhibited, with varying efficacies, by aldosterone, 11-deoxycorticosterone, cortisol, and cortisone, and by the synthetic glucocorticoids betamethasone, dexamethasone and prednisolone, and the glucocorticoid receptor antagonist RU38486. These studies demonstrate that uptake(2) in the CNS is inhibited by a variety of natural and synthetic corticosteroids, and suggest that inhibition of uptake(2)-mediated monoamine clearance may underlie some behavioral and physiological effects of these hormones.

Organic cation transporter 3 is densely expressed in the intercalated cell groups of the amygdala: Anatomical evidence for a stress hormone-sensitive dopamine clearance system

The intercalated cell groups of the amygdala (ITCs) are clusters of GABAergic neurons which exert powerful modulatory control of amygdala output, and are thought to play key roles in the extinction of conditioned fear responses. Dopamine, acting through D1 receptors, inhibits ITC neuronal activity, an action that has the potential to disinhibit amygdala activity, leading to changes in behavioral responses. Dopaminergic neurotransmission in the ITC occurs through a combination of synaptic and volume transmission. Thus, mechanisms, including transport mechanisms, that regulate extracellular dopamine concentrations in the ITC, are likely to be important determinants of amygdala function. We have recently demonstrated the expression of organic cation transporter 3 (OCT3), a high-capacity transporter for dopamine and other monoamines, throughout the rat brain. In this study, we used immunohistochemical and immunofluorescence techniques to examine the distribution of OCT3 in the ITC, to identify the phenotype of OCT3-expressing cells, and to describe the spatial relationships of OCT3 to dopaminergic terminals and dopamine D1 receptors in these areas. We observed high densities of OCT3-immunoreactive perikarya and punctae throughout the D1 receptor-rich main, anterior and paracapsular ITCs, in contrast with the basolateral amygdala, where OCT3 immunoreactive perikarya and puncta were observed at much lower density. OCT3-immunoreactive perikarya in the ITC were identified as neurons. Tyrosine hydroxylase-immunoreactive fibers in the ITC were immunonegative for OCT3, though OCT3-immunoreactive punctae were observed in close proximity to TH+ terminals. Punctate OCT3-immunoreactivity in the ITCs was observed in very close proximity (<1 μm) to D1 receptor immunoreactivity. These anatomical data are consistent with the hypothesis that OCT3 plays a central role in regulating dopaminergic neurotransmission in the ITC, and that it represents a post- or peri-synaptic dopamine clearance mechanism. Inhibition of OCT3-mediated transport by corticosterone may represent a mechanism by which acute stress alters dopaminergic neurotransmission in the amygdala, leading to alterations in fear and anxiety-like behavior.