Research report Concurrent nociceptive stimulation impairs the anxiolytic effect of midazolam injected into the periaqueductal gray in mice Joyce Mendes-Gomes a,b , Ricardo Luiz Nunes-de-Souza a, * a Lab. Farmacologia, Faculdade de Cie ˆncias Farmace ˆuticas-UNESP, Rod. Araraquara-Jau, Km 01, 14801-902 Araraquara, SP, Brasil b Programa de Po ´ s-Graduac ¸a ˜o em Psicobiologia, FFCLRP-USP, Av. Bandeirantes, 3900, 14040-901 Ribeira ˜o Preto, SP, Brasil Accepted 13 April 2005 Available online 11 May 2005 Abstract This study investigated whether the opportunity to avoid or escape the open arms of an elevated plus-maze (EPM) affects the antinociceptive response observed when mice are subjected to open arm confinement. Furthermore, in order to better characterize the relationship between emotion and antinociception in the EPM, we examined the behavioral effects of midazolam injection into the midbrain periaqueductal gray matter (PAG). As our main aim was to evaluate the relevance of different levels of approach –avoid conflict (i.e. the presence of open and closed arms) to maze-induced antinociception, mice were exposed to one of three types of EPM—a standard EPM (sEPM), an open EPM (oEPM: four open arms) or, as a control condition, an enclosed EPM (eEPM: four enclosed arms). Nociception was assessed using the formalin test. Twenty minutes after formalin injection (50 Al, 2.5% formalin) into the dorsal right hind paw, mice received an intra-PAG injection of saline or midazolam (10– 20 nmol). Five minutes later, they were individually exposed to one of the mazes for 10 min (25 –35 min after formalin injection). Videotapes of the test sessions were scored for a variety of behavioral measures including time spent licking the formalin-injected paw. To examine whether the effects of midazolam on anxiety-like behavior may have been influenced by concurrent nociceptive stimulation (i.e. formalin pretreatment), naive mice were submitted to a similar procedure to that described above for the sEPM test but without formalin pretreatment. Results showed that mice exposed to the oEPM spent significantly less time licking the injected paw compared to groups exposed to either the sEPM or eEPM. Although exposure to the sEPM induced anxiety-like behaviors (i.e. open arm avoidance), it did not result in antinociception. Intra-PAG infusions of midazolam failed to block oEPM-induced antinociception or to alter sEPM-induced anxiety in mice that had received formalin injection. However, under normal test conditions (i.e. in the absence of formalin-induced nociceptive stimulation), intra-PAG midazolam produced clear anti-anxiety effects in mice exposed to the sEPM. Findings are discussed in terms of different emotional states induced by the oEPM and sEPM and the influence of concurrent nociceptive stimulation on the anti-anxiety effect of intra-PAG midazolam. D 2005 Elsevier B.V. All rights reserved. Theme: Neurotransmitters, modulators, transporters, and receptors Topic: Behavioral pharmacology Keywords: Anxiety; Fear; Different EPM; Antinociception; Midazolam; Periaqueductal gray; Mice 1. Introduction When animals are confronted with threatening situations, they typically display a set of behavioral (e.g., fight, flight, freezing, vocalization) and neurovegetative (e.g., tachycar- dia, hypertension, defecation) responses characterized as the fear reaction. In general, these responses are accompanied by antinociception [20,33]. The search for animal models that permit evaluation of the influence of emotionality on nociception has led to the use of the elevated plus maze (EPM) test [8,21,41]. The EPM [31] or elevated X maze [17] was originally validated to study anxiety-like behavior 0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2005.04.026 * Corresponding author. E-mail address: souzarn@fcfar.uesp.br (R.L. Nunes-de-Souza). Brain Research 1047 (2005) 97 – 104 www.elsevier.com/locate/brainres