Impaired escape performance and enhanced conditioned fear in rats following exposure to an uncontrollable stressor are mediated by glutamate and nitric oxide in the dorsal raphe nucleus.

Exposure to uncontrollable aversive events produces a variety of behavioral consequences that do not occur if the aversive event is controllable. Accumulating evidence suggests that exaggerated excitation of serotonin (5-HT) neurons in the dorsal raphe nucleus (DRN) is sufficient to cause these same behaviors, such as poor shuttlebox escape performance and enhanced conditioned fear that occur 24 h after exposure to inescapable tailshock (IS). The aim of the present studies was to explore the possibility that N-methyl-D-aspartate (NMDA) receptor activation and nitric oxide (NO) formation within the DRN might be involved in mediating the behavioral consequences of IS. To this end, either the NMDA receptor antagonist 2-amino-5-phosphonovaleric acid (APV) or the nitric oxide synthase inhibitor Nw-nitro-L-arginine methyl ester (L-NAME), was microinjected into the DRN before IS or before testing 24 h later. Blocking NMDA receptors with APV in the DRN during IS prevented the usual impact of IS on escape responding and conditioned fear. However, injection of APV at the time of testing only reduced these effects. The DRN was shown to be the critical site mediating blockade of these behavioral changes since injection of APV lateral to the DRN did not alter the behavioral consequences of IS. Conversely, L-NAME was most effective in reversing the effects of IS when administered at the time of testing. These results suggest that there is glutamatergic input to the DRN at the time of IS that produces long-lasting changes in DRN sensitivity. This plasticity in the DRN is discussed as a possible mechanism by which IS leads to changes in escape performance and conditioned fear responding.

Opioid-dependent effects of inescapable shock on escape behavior and conditioned fear responding are mediated by the dorsal raphe nucleus.

Manipulations of the dorsal raphe nucleus (DRN) modulate the behavioral effects of exposure to inescapable shock (IS). Opiate agonists and antagonists also influence the impact of IS, but the role of the DRN in mediating these effects is unknown. The opiate antagonist naltrexone micro-injected into the region of the DRN immediately prior to IS prevented both the escape deficit and the enhancement of fear conditioning that occur 24 h later. Intra-DRN naltrexone administered at the time of later behavioral testing reduced, but did not eliminate, these effects of prior IS. Conversely, the opiate agonist morphine, in combination with a subthreshold number of 20 IS trials, induced an escape deficit and enhanced conditioned fear 24 h later. Microinjections of naltrexone into the dorsolateral periaqueductal gray area did not alter the effects of IS and electrolytic lesions of the DRN prevented the effect of the morphine-20 IS trial combination. The role of opioids in mediating the behavioral effects of IS is discussed.

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.

Inescapable shock-induced potentiation of morphine analgesia in rats: involvement of opioid, GABAergic, and serotonergic mechanisms in the dorsal raphe nucleus.

Exposure of rats to inescapable shock (IS) potentiated the analgesic response to a low dose (1 mg/kg) of morphine 24 hr later. This effect was blocked by naltrexone (10 micrograms), diazepam (5 micrograms), or 8-hydroxy-2-(di-n-propylamine)-tetralin (8-OH-DPAT; 1 microgram) microinjected into the dorsal raphe nucleus (DRN) 15 min before IS. When microinjected into the DRN at the time of tail-flick testing, 8-OH-DPAT also effectively prevented this effect. Further, intra-DRN administration of a beta-carboline mimicked the effects of IS, because rats treated with methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (1 microgram) and simply restrained displayed potentiated morphine analgesia 24 hr later. These data suggest that this phenomenon shares mechanisms in common with other effects of IS at the level of the DRN.

Evidence that brief stress may induce the acute phase response in rats.

Exposing rats to a single session of inescapable tail shock (IS) reduces corticosteroid binding globulin (CBG) 24 h later (Fleshner et al., Endocrinology 136: 5336-5342, 1995). The present experiments examined whether reductions in CBG are differentially affected by controllable vs. identical uncontrollable tail shock, are mediated by IS-induced glucocorticoid elevation, or reflect IS-induced activation of the acute phase response and whether IS produces fever. The results demonstrate that 1) equivalent reductions in CBG are observed in response to escapable tail shock or yoked IS, 2) IS-induced CBG reduction is not blocked by adrenalectomy in rats that receive basal corticosteroid replacement or by pretreatment with RU-38486, and 3) IS appears to activate the acute phase response, since IS reduces serum levels of an acute-phase negative reactant (CBG), increases serum levels of acute-phase positive reactants (haptoglobin and alpha 1-acid glycoprotein), and increases core body temperature 20-24 h later.

The elevated plus-maze is not sensitive to the effect of stressor controllability in rats.

The present experiments examined the sensitivity of the elevated plus-maze to the effects of stressor controllability. Previous work had established that inescapable but not an equal amount of escapable electric tail shock reduced social interaction. The present experiments demonstrate that prior exposure to shock alters elevated plus-maze behavior, but that this effect is not sensitive to the escapability of the shock. These experiments include a replication of the usual pharmacologic effects of benzodiazepine ligands (2 mg/kg diazepam; 0.4 mg/kg methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate) to demonstrate the sensitivity of the elevated plus-maze procedures used. The results provide additional support for the idea that the social interaction and elevated plus-maze measures of "anxiety" are sensitive to different processes.

The benzodiazepine receptor antagonists flumazenil and CGS8216 block the enhancement of fear conditioning and interference with escape behavior produced by inescapable shock.

Prior work has suggested that the mediation of the behavioral effects of inescapable shock (IS) might involve release of an endogenous beta-carboline-like ligand at the dorsal raphe nucleus (DRN) that binds to the benzodiazepine (BZ) recognition site on the GABAA complex, thereby disinhibiting the DRN. This was tested by microinjection of the BZ receptor antagonists flumazenil and CGS8216 in the region of the DRN, either before IS or before later behavioral testing. Both compounds blocked subsequent enhancement of fear conditioning and interference with shuttlebox escape when administered before IS, but had no effect when given before testing. In addition, flumazenil did not alter the behavior of escapably shocked subjects.

The dorsal raphe nucleus is a site of action mediating the behavioral effects of the benzodiazepine receptor inverse agonist DMCM.

Systemic administration of benzodiazepine receptor inverse agonists leads to behavioral changes similar to those produced by inescapable shock (IS). The dorsal raphe nucleus (DRN) is a critical structure mediating IS effects. The present experiments determined whether the DRN is a site mediating the behavioral changes produced by benzodiazepine receptor inverse agonists. Microinjection of the inverse agonist Methyl 6,7-Dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM) in the region of the DRN produced enhancement of fear conditioning as assessed by the amount of freezing in the presence of shock cues as well as interference with shuttlebox escape learning assessed 24 hr later. Furthermore, lesion of the DRN blocked the effects of systemic DMCM on fear conditioning and escape learning. These data suggest that the DRN is indeed critical in mediating these behavioral consequences of DMCM and further support a role for the DRN in producing the behavioral changes induced by IS.

8-OH-DPAT microinjected in the region of the dorsal raphe nucleus blocks and reverses the enhancement of fear conditioning and interference with escape produced by exposure to inescapable shock.

Prior work suggests that inhibition of the dorsal raphe nucleus (DRN) either during exposure to inescapable electric shock (IS) or during later behavioral testing might block the usual behavioral consequences of IS. The 5-HT1A agonist 8-OH-DPAT was microinjected into the region of the DRN either before exposure to IS or before testing for fear conditioning and escape learning conducted 24 hr later. IS potentiated fear conditioning and interfered with escape performance. These effects were completely prevented by intra-DRN administration of 8-OH-DPAT at either point. Low but not high systemic doses of 8-OH-DPAT had a similar effect, supporting the idea that the effective site of action is presynaptic. The relation between these data and other effects of 8-OH-DPAT is discussed.

Chlordiazepoxide microinjected into the region of the dorsal raphe nucleus eliminates the interference with escape responding produced by inescapable shock whether administered before inescapable shock or escape testing.

Systemic administration of benzodiazepines before exposure to inescapable shock (IS) blocks the enhanced fear conditioning and escape learning deficits that follow exposure to IS, whereas administration before the subsequent behavioral testing eliminates the enhanced fear but not the interference with escape. The failure of benzodiazepines to reduce the IS-produced escape learning deficit when given before testing is inconsistent with a recent proposal that interference with escape is mediated by an IS-induced sensitization of dorsal raphe nucleus (DRN) activity. The present experiments demonstrate that chlordiazepoxide will block both the enhancement of fear and interference with escape responding when given before either IS or testing if microinjected in the region of the DRN. This suggests that systemic benzodiazepines fail to block escape deficits when given before testing because action at a site distant from the DRN counters the effect of the drug at the DRN.

The role of the amygdala and dorsal raphe nucleus in mediating the behavioral consequences of inescapable shock.

It has been argued that exposure to inescapable shock produces later behavioral changes such as poor shuttle box escape learning because it leads to the conditioning of intense fear, which later transfers to the shuttle box test situation and interferes with escape. Both fear, as assessed by freezing, and escape were measured in Sprague-Dawley rats 24 hr after exposure to inescapable shock. Lesions of the basolateral region and central nucleus of the amygdala eliminated the fear that transfers to the shuttle box after inescapable shock, as well as the fear conditioned in the shuttle box by the shuttle box shocks. However, the amygdala lesions did not reduce the escape learning deficit produced by inescapable shock. In contrast, dorsal raphe nucleus lesions did not reduce the fear that transfers to the shuttle box after inescapable shock, but eliminated the enhanced fear conditioning in the shuttle box as well as the escape deficit. The implications of these results for the role of fear and anxiety in mediating inescapable shock effects are discussed.

Blockade of the hypothalamic-pituitary-adrenal response to stress by intraventricular injection of dexamethasone: a method for studying the stress-induced peripheral effects of glucocorticoids.

Interest in the mechanisms whereby stressors can influence behavior and physiological functioning has involved the use of a variety of methods to prevent the stress-induced release of glucocorticoids, an important and commonly studied stress hormone. We examined the effect of intracerebral ventricular dexamethasone (ICV DEX) on the stress-induced release of adrenocorticotropic hormone (ACTH), corticosterone, plasma epinephrine (E), and plasma norepinephrine (NE). Male Sprague-Dawley rats were stereotaxically implanted with third ventricle ICV cannulae, administered DEX or vehicle, and exposed to 100 1.6-mA tail shocks. Stress hormones were assessed from blood taken during and after the cessation the shock. We report an ICV DEX injection protocol (10 microgram given four times) that results in blocking the stress-induced release of ACTH and corticosterone, and attenuating the stress-induced release of plasma E and NE. We hypothesize that ICV DEX reduces hypothalamic corticotropin releasing hormone (CRH) synthesis and/or release. This method would be especially useful for those studying the effect of pituitary-adrenal hormones on steroid sensitive peripheral targets, such as the immune system.