Research reportOpioid-dependent effects of inescapable shock on escape behavior and conditioned fear responding are mediated by the dorsal raphe nucleus
Introduction
Exposure to stressors over which the subject has no behavioral control produces a constellation of behavioral changes that are not observed following controllable stressors of equal duration, intensity and number. This `learned helplessness' phenomenon, initially examined as poor shuttlebox escape performance following exposure to inescapable shock [59], is now known to include other forms of poor escape responding 27, 58as well as increased anxiety [61], enhanced fear conditioning [37], an opioid form of stress-induced analgesia (SIA) 28, 41, reduced activity [13]and reduced defensive behavior 80, 81, 82.
A number of neural and neurochemical systems are involved in mediating the behavioral sequelae of uncontrollable aversive events 2, 16, 56, 77. However, 5-HT neurons in the DRN appear to play a key role. In general, manipulations designed to reduce the activity of 5-HT neurons within the DRN block the behavioral effects of IS when administered before IS. For example, 5-HT neurons in the DRN are under GABAergic inhibition 74, 76, exerted, at least in part, by GABAA receptors. Benzodiazepines act at a subunit of the GABAA receptor to facilitate GABAergic inhibition [64], and the microinjection of benzodiazepines into the region of the DRN before IS blocks consequent failure to escape and enhancement of fear conditioning assessed 24 h after IS [43]. Consistent with these findings, DRN lesions completely block the interference with escape and exaggeration of fear conditioning produced by IS [42]. Conversely, the intra-DRN administration of the benzodiazepine receptor inverse agonist DMCM, which would be expected to increase DRN 5-HT activity [35], mimics the behavioral effects of IS [44].
These findings, combined with data indicating a large release of 5-HT within the DRN during IS [47], suggest that IS produces activation of 5-HT neurons within the DRN essential to producing the behavioral effects of IS. It has been suggested that this activation leads to a sensitization of DRN 5-HT neurons, so that the test conditions used to assess learned helplessness effects (e.g. escape training, morphine administration) lead to an exaggerated release of 5-HT in projection regions of the DRN [38], the putative proximate cause of the behavioral outcomes. Consistent with this view, learned helplessness effects can be reversed by manipulations that inhibit DRN activity at the time of testing 24 h after IS. Intra-DRN benzodiazepines [43]and intra-DRN 8-OH-DPAT [46]administered before testing prevented the escape deficit and enhanced conditioned fear in rats exposed to IS 24 h earlier. Furthermore, the DRN does project to sites 54, 72likely to mediate the behaviors that result from IS exposure. For example, the DRN projects to the dorsal periaqueductal gray area (dPAG) and to the amygdala which have been shown to be involved in escape behavior [20]and conditioned fear [17], respectively. Furthermore, stressors do lead to an increase in extracellular 5-HT in these regions [32].
Although there is accumulating evidence that supports this scheme, the mechanism(s) by which IS activates the DRN are largely unknown. The present experiments were conducted to explore the possible role of opioid mediated disinhibition of the DRN in the behavioral effects of IS. Endorphin-, enkephalin- and dynorphin-containing fibers and terminals have all been demonstrated to be present in the DRN 74, 86. Opioid terminals synapse on GABA interneurons within the DRN, which normally inhibit 5-HT cells [74]. Thus the release of opioids within the DRN would disinhibit 5-HT neurons, thereby increasing their activity. As would be expected, systemic and intra-DRN administration of morphine increases 5-HT metabolism within the DRN [57]as well as 5-HT release within the DRN and its terminal regions 66, 67. Indeed, this effect of morphine on 5-HT release is GABA receptor dependent 65, 68, as would be expected given the neural organization of opioid, GABAergic and serotonergic cells in the DRN. In addition, met-enkephalin has been shown to reduce the number of inhibitory postsynaptic currents measured in DRN 5-HT neurons [30]. Thus, IS could induce the release of opioid peptides within the DRN, thereby altering 5-HT release and subsequent behavior. The effect of opioid release in the DRN would summate with other mechanisms of IS-induced DRN activation, resulting in enhanced release of 5-HT.
There is considerable evidence that stressor exposure does lead to the release of endogenous opioid peptides 1, 36, 62, 71. Behavioral and pharmacological data suggest that IS, in particular, does so. Exposure to IS, but not to escapable shock, results in a `short-term', or immediate, analgesia [28]and a `long-term' analgesia that is measured 24 h later when subjects are re-exposed to a small amount of shock [28]. Both types of analgesia are blocked by opiate antagonists administered before IS [40]and before testing [40]. In addition, the long-term analgesia is cross-tolerant with morphine [15]. Furthermore, rats exposed to IS 24 h earlier exhibit hyperactivity to morphine's analgesic effects 22, 26. Consistent with these findings, the escape learning deficit is prevented by opiate antagonist administration before IS or before escape testing 14, 24, 49, 78, 79, supporting the view that endogenous opioids mediate learned helplessness effects in general. The purpose of the present experiments was to directly assess the role of opioid interactions within the DRN in mediating learned helplessness effects.
Section snippets
Experiment 1
Forty seven male naive Sprague–Dawley rats approximately 100 days old were used in Experiment 1A and 34 were used in Experiment 1B. They were housed individually with free access to standard laboratory chow and water under a 12 h light/dark cycle. All experiments were conducted during the light part of the cycle and were approved by the University of Colorado Institutional Animal Care and Use Committee.
Experiment 2
Subjects were 28 naive rats housed as described above.
Experiment 3
Subjects were 44 naive rats housed as
Experiment 1A: effect of intra-DRN naltrexone
Previous work has shown that systemic administration of the opiate antagonists naloxone or naltrexone prevents or reverses learned helplessness behaviors including `long-term' stress-induced analgesia, exaggerated fear conditioning, and poor shuttlebox escape performance 14, 28, 37, 49, 78, 79. Experiment 1A was conducted to determine whether naltrexone would have the same effect if microinjected into the region of the DRN. Naltrexone was microinjected prior to exposure to IS or prior to
Opioid mediation of learned helplessness behavior
The present studies suggest that the poor shuttlebox escape responding and enhanced conditioned fear that are a normal consequence of IS involve mediation by an opioid mechanism in the DRN. It has been known that a) opiate antagonists administered systemically before IS or before later behavioral testing block or reduce many of the behavioral sequelae of IS 14, 28, 37, 49, 78, 79, b) IS sensitizes the organisms reactivity to opiates 22, 26, c) activation of 5-HT neurons within the DRN at the
Acknowledgements
We would like to thank Lee Silbert for his invaluable technical assistance and Brian Otero for help with Experiment 1B. This research was supported by NIMH MH50479 and RSA MH00314 to SFM and a grant to MBM from the University of Colorado Undergraduate Research Oportunity Program (UROP).
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