Sayamwong E. Hammack

Activation of serotonin-immunoreactive cells in the dorsal raphe nucleus in rats exposed to an uncontrollable stressor.

The dorsal raphe nucleus (DRN) and its serotonergic terminal regions have been suggested to be part of the neural substrate by which exposure to uncontrollable stressors produces poor escape responding and enhanced conditioned fear expression. Such stressor exposure is thought to selectively activate DRN serotonergic neurons in such a way as to render them transiently sensitized to further input. As a result of this sensitized state, behavioral testing procedures are thought to cause excess serotonergic activity in brain regions that control these behaviors. The present studies were conducted to investigate activity in the DRN following exposure to escapable and yoked, inescapable tailshock. Neural activity was characterized using immunohistochemistry to detect the immediate early gene product Fos in serotonin-immunoreactive cells in the DRN. Inescapable tailshock led to greater serotonergic neural activity than did escapable tailshock, supporting the hypothesis that uncontrollable stressors preferentially activate serotonergic neurons in the DRN.

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.

Controlling neuropathic pain by adeno-associated virus driven production of the anti-inflammatory cytokine, interleukin-10

Despite many decades of drug development, effective therapies for neuropathic pain remain elusive. The recent recognition of spinal cord glia and glial pro-inflammatory cytokines as important contributors to neuropathic pain suggests an alternative therapeutic strategy; that is, targeting glial activation or its downstream consequences. While several glial-selective drugs have been successful in controlling neuropathic pain in animal models, none are optimal for human use. Thus the aim of the present studies was to explore a novel approach for controlling neuropathic pain. Here, an adeno-associated viral (serotype II; AAV2) vector was created that encodes the anti-inflammatory cytokine, interleukin-10 (IL-10). This anti-inflammatory cytokine is known to suppress the production of pro-inflammatory cytokines. Upon intrathecal administration, this novel AAV2-IL-10 vector was successful in transiently preventing and reversing neuropathic pain. Intrathecal administration of an AAV2 vector encoding beta-galactosidase revealed that AAV2 preferentially infects meningeal cells surrounding the CSF space. Taken together, these data provide initial support that intrathecal gene therapy to drive the production of IL-10 may prove to be an efficacious treatment for neuropathic pain.

Chemical lesion of the bed nucleus of the stria terminalis blocks the behavioral consequences of uncontrollable stress

Uncontrollable or inescapable shock (IS) produces behavioral changes that are characterized by a sensitized fear system and a deficit in fight-flight responding. These behavioral changes have been argued to represent an anxiety-like state produced by the uncontrollability of the stressor. The bed nucleus of the stria terminalis (BNST) has been implicated in the mediation of long-duration responses to unpredictable stressors, which have also been argued to represent anxiety. In the present study, the effects of BNST chemical lesion on the IS-induced sensitization of freezing to an environment previously paired with shock and the IS-induced impairment of escape responding were investigated. BNST chemical lesion blocked the potentiation of freezing and the increases in escape latency that normally follow IS.

Low doses of corticotropin-releasing hormone injected into the dorsal raphe nucleus block the behavioral consequences of uncontrollable stress

The behavioral consequences of uncontrollable stress that are collectively called learned helplessness (LH) are mediated in part by increased levels of serotonin (5-HT) activity in the dorsal raphe nucleus (DRN) and its projection regions. Recently, corticotropin-releasing hormone (CRH) within the DRN has been implicated in the development of LH because intra-DRN CRH produces LH at very high doses, and because intra-DRN antagonists for the CRH 2 receptor (CRHR2) block LH. Since these behavioral effects are mediated by both 5-HT excitation and CRHR2 activation, we have suggested that CRHR2 mediates excitation of DRN 5-HT neurons. However, CRH has been shown to inhibit DRN 5-HT neurons at low doses that are expected to bind to CRHR1. Since CRHR1 antagonists were ineffective in blocking LH, we have further suggested that CRHR1 might mediate the inhibition of DRN 5-HT neurons. In support of this hypothesis, although low doses of CRH that preferentially bind CRHR1 inhibit DRN 5-HT activity, higher doses at which CRH would be expected to bind both receptor subtypes no longer inhibit DRN 5-HT. In addition, high doses of CRH are required to produce LH, which is known to be mediated by 5-HT excitation, and the CRHR2 agonist urocortin II (UCN II) produces LH at much lower doses than does CRH. The present studies show that intra-DRN CRH microinjection blocks the behavioral effects produced by DRN UCN II, but only at doses that have been shown to inhibit DRN 5-HT activity. Indeed, a higher dose of CRH that has been shown to no longer inhibit DRN 5-HT activity did not affect the behavioral consequences of DRN UCN II. In a separate experiment, the effective dose of CRH blocked the usual behavioral consequences of uncontrollable stress.

Blockade of alpha1 adrenoreceptors in the dorsal raphe nucleus prevents enhanced conditioned fear and impaired escape performance following uncontrollable stressor exposure in rats

Previous research has shown that the effect of exposure to uncontrollable stressors on conditioned fear responding and escape behavior in rats is dependent on serotonergic neural activity in the dorsal raphe nucleus (DRN). The role that norepinephrine released in the DRN plays in producing the behavioral consequences of exposure to inescapable tail shock in rats was investigated in the present study. The selective alpha1 adrenoreceptor antagonist benoxathian was injected into the DRN before exposure to inescapable tail shock or before behavioral testing conducted 24 h later. Benoxathian prevented the impairment of escape responding produced by inescapable shock, but did not reverse this effect when given before testing. The enhancement of conditioned fear produced by prior inescapable shock was attenuated by benoxathian administered before inescapable shock or before behavioral testing. These results support the view that noradrenergic input to the DRN is necessary to produce the behavioral effects of inescapable tail shock.

Identification of cell-type-specific promoters within the brain using lentiviral vectors

The development of cell-type-specific mini-promoters for genetic studies is complicated by a number of issues. Here, we describe a general method for the relatively rapid screening of specific promoter activity in cell culture, in acute brain slice preparations and in vivo. Specifically, we examine the activity of an ∼3 kb promoter region from the neuroactive peptide cholecystokinin (CCK) compared to the commonly used cytomegalovirus promoter. We find a high degree of cell-type selectivity in vivo using lentiviral approaches in rats and traditional transgenic approaches in mice. Appropriate colocalization of Cre-recombinase and CCK gene expression is found within the hippocampus, when the CCK promoter is driving either the expression of Cre-recombinase or green fluorescent protein. We also demonstrate fluorescent identification of CCK-positive interneurons that allows for cell-type-specific electrophysiologic studies in rats and mice. In conclusion, these studies identify a functional mini-promoter for the CCK gene and outline a novel and sensitive general method to test activity of selective promoters in vitro and in vivo. This approach may allow for the more rapid identification of specific promoters for use with transgenic animals, in genetically modified viruses, and in the design of targeted, therapeutic gene-delivery systems.

Inescapable shock-induced potentiation of morphine analgesia in rats: sites of action.

Inescapable shock (IS) enhances analgesia to systemic morphine (MOR) 24 hr later. IS activates serotonin neurons in the dorsal raphe nucleus (DRN), rendering them hyperexcitable. These studies tested whether IS potentiates the analgesic effect of MOR microinjected in the DRN, as predicted by this hypothesis. To test site specificity, the effect of previous IS was examined on MOR microinjected lateral to the DRN and into 2 other sites that support MOR analgesia, the nucleus raphe magnus (NRM) and spinal cord. Twenty-four hours after IS, potentiated analgesia was observed after 0.5 microg MOR microinjected into, but not lateral to, the DRN. Potentiated analgesia was also observed after NRM (1.0 microg) and spinal cord (3.0 microg) MOR microinjections. These data suggest that IS-induced excitability changes within the DRN synergize with opiates microinjected in other analgesia areas and that this potentiates the responses to opiates 24 hr after IS.