Overexpression of Proenkephalin in the Amygdala Potentiates the Anxiolytic Effects of Benzodiazepines

Abstract

To elucidate the role of opioid peptides in control of the anxiety-like behavior and anxiety-reducing actions of benzodiazepines, a recombinant, replication-defective herpes virus (SHPE) carrying human preproenkephalin cDNA was delivered to rat amygdala. Viral infection resulted in a strong, localized transgene expression after 2-4 days which diminished after one week. Anxiety-like behavior and the anxiolytic effect of diazepam were assessed three days afterIn addition to its well known role in mediating fear and anxiety responses (Davis et al. 1994), the amygdala may also be a key neural substrate for the anxiolytic actions of benzodiazepines. Benzodiazepines (BZs) are believed to induce their anxiolytic properties by binding to GABA/benzodiazepine receptors in the brain (Haefely 1990). In addition, the amygdala has a high density of GABA/benzodiazepine receptors (Young and Kuhar 1980;Richards and Mohler 1984). Local infusion of benzodiazepine agonists into the amygdala produces anxiolytic effects which can be blocked by co-administration of GABA A or benzodiazepine antagonists (Scheel-Kruger and Petersen 1982;Petersen et al. 1985; Treit 1994, 1995). Furthermore, the anxietyreducing actions of systemic benzodiazepines can be blocked by intra-amygdala injection of GABA/benzodiazepine antagonists (Sanders and Shekhar 1995).The amygdala also has high levels of endogenous opioid peptides and opioid receptors (Loughlin et al. 1995), and opioid mechanisms have been implicated in many biological functions, including nociception, memory, and fear conditioning (Good and Westbrook 1995;McGaugh et al. 1996;Valverde et al. 1996). A partial anxiolytic action has been reported following microinjection of morphine into amygdala (File and Rodgers 1979).Although opioid agonists are not used as anxiolytics in general, recent evidence strongly indicates a role for endogenous opioid peptides in the control of stress and anxiety (Olson et al. 1996). Preproenkephalin knockout (Konig et al. 1996), and some novel -opioid agonists show anxiolytic properties (Privette and Terrian 1995). Several lines of evidence also indicate the involvement of endogenous opioid peptides in various aspects of benzodiazepine action, including their influences on food intake, locomotor activity, conflict, and anxiety-like behaviors (Millan and Duka 1981;Cooper 1983;Nowakowska and Chodera 1990). Interestingly, clinical studies suggest interactions between responses to benzodiazepines and opioids in humans (Darke et al. 1993). The anxiolytic effects of benzodiazepines can be blocked by opioid antagonists in both humans and laboratory animals (Billingsley and Kubena 1978;Koob et al. 1980;Duka et al. 1981Duka et al. , 1982Agmo et al 1995;Tsuda et al. 1996).Prior studies using herpes virus-mediated gene transfer demonstrated that overexpression of proenkephalin in amygdala produced antinociceptive effects, and illustrated the usefulness of this methodology for examining the role of neuropeptides in behavioral responses (Kang et al. 1998). To investigate if mo...

Microinjection of Naltrexone into the Central, but not the Basolateral, Amygdala Blocks the Anxiolytic Effects of Diazepam in the Plus Maze

“…This supports the concept that the effects of anxiolytic drugs are localized to, or at least regulated by, the amygdala (Pesold and Treit, 1995;Killcross et al, 1997;Kang et al, 2000;Wilson et al, 2003). It also supports the finding that systemic administration of opioid antagonists inhibits the anxiolytic properties of benzodiazepines in an anticonflict paradigm (Agmo et al, 1995;Yadin et al, 1991) and in the elevated plus maze (Agmo and Belzung, 1998).…”

“…Although the anxiolytic effects of diazepam appear to involve amygdalar processes (current study, and Kang et al, 2000;Sanders and Shekhar, 1995), microinjection of NAL into the BLA or CeA did not affect the anxiolytic properties of ethanol in the elevated plus maze. These results suggest that the anxiolytic properties of ethanol in the elevated plus maze are not regulated by opioid receptor systems within CeA or BLA nuclei of the amygdala.…”

“…or vehicle (see below) in experiments 1 and 3, or ethanol (1 g/kg, 20% v/v) or saline in experiment 2. In the first set of experiments, the nonselective opioid antagonist NAL hydrochloride (15 mg) was used to block opioid receptors in the CeA and BLA (Kang et al, 2000). This dose of NAL has been shown to affect anxiolytic drug responses when microinjected into the amygdala (Kang et al, 2000;Wilson et al, 2003), and similar doses modify behavioral responses when injected into the nucleus accumbens (Kelley et al, 1996).…”

“…This test was conducted as described in Kang et al (2000), as modified from Pellow et al (1985). The black Plexiglas-elevated plus maze consisted of two opposing open and two opposing closed arms in the shape of a cross, connected by a central square.…”

“…Microinjection of morphine into the amygdala decreases social interaction (File and Rodgers, 1979), suggesting that opioid systems in this region play a modulatory role in anxiety-like behavior. Overexpression of enkephalin in the amygdala potentiates a subeffective dose of diazepam in the elevated plus maze (Kang et al, 2000), while large injections of NAL into the amygdala block the anxiolytic properties of diazepam (Kang et al, 2000) and ethanol (Wilson et al, 2003) in the same paradigm. These previous studies used injection parameters that could not localize effects to select amygdalar nuclei.…”