Pharmacological dissociation of moderate and high contextual fear as assessed by freezing behavior and fear-potentiated startle Julia Maria Santos, Ana Carolina Ga ´rgaro, Amanda Ribeiro Oliveira, Sueli Masson, Marcus Lira Branda ˜o * Laborato ´rio de Psicobiologia, Faculdade de Filosofia, Cie ˆncias e Letras de Ribeira ˜o Preto, Universidade de Sa ˜o Paulo, Av. Bandeirantes, 3900, Ribeira ˜o Preto, SP 14040-901, Brazil Received 31 July 2004; received in revised form 15 October 2004; accepted 26 October 2004 Abstract The amplitude of the whole-body acoustic startle response is reliably enhanced when elicited in the presence of foreground signals, such as light, previously paired with footshocks. It has been shown that this enhancement is evident by moderate fear levels, but is less affected by high fear levels. Potentiation of the acoustic startle reflex has also been reported in the presence of background cues previously associated with footshocks. However, the effects of anxiolytic drugs on different levels of fear elicited by moderate and intense contextual fear conditioning associated with startle reflex have not been examined yet. To approach this issue, we examined the effects of the anxiolytic, midazolam, on two intensities of contextual fear; freezing behavior and the startle response to loud noise. First, we compared the magnitude of the freezing behavior and the startle amplitude during the testing sessions in groups of rats submitted to fear conditioning using 0.3 and 0.6 mA as unconditioned stimuli (10 stimuli of 1 s each, intertrial interval from 60 to 180 s). Afterwards, the effects of midazolam (0.5 and 1.0 mg/kg) were assessed in these two conditions. Rats showed a potentiated startle reflex and a significant freezing behavior to moderate fear conditioning, which were both attenuated by midazolam. Higher levels of fear conditioning caused more intense freezing behavior without enhancing the startle reflex. Whereas midazolam reduced this freezing response, the startle response was unaffected. These results are indicative that anxiolytic-sensitive freezing and fear-potentiated startle are triggered by moderate contextual fear conditioning, while contextual conditioning with the use of high footshocks causes a distinct pattern of behavioral responses, which is only partially affected by midazolam. Due to the differential sensitivity to midazolam of these two patterns of startle responses generated as a function of the intensity of contextual fear conditioning, it is proposed that they represent moderate and intense aversive states that may be related to anxiety or panic/ phobic conditions, respectively. D 2004 Elsevier B.V. and ECNP. All rights reserved. Keywords: Startle reflex; Freezing; Contextual fear; Midazolam; Fear-potentiated startle 1. Introduction A sudden and unexpected burst of noise triggers a whole- body response, which consists of a skeletal muscle contraction throughout the body known as the acoustic startle reflex (Brown et al., 1951). When the startle-inducing noise is elicited in the presence of foreground signals [e.g., a neutral stimulus (such as a light) that does not induce a startle response by its own but has been previously paired to footshocks], the startle response is enhanced. This phenom- enon has been named fear potentiation of the startle reflex (FPS) (Brown et al., 1951; Campeau and Davis, 1992; Davis, 1992). The increase in the startle reflex in the presence of the conditioned stimuli in comparison to the startle response to noise alone is taken as an index of anxiety. The method has been considered to be a valid and reliable tool for measuring anxiety, on the basis of extensive investigation analyzing several of its behavioral, physio- logical, and pharmacological aspects. Indeed, considerable 0924-977X/$ - see front matter D 2004 Elsevier B.V. and ECNP. All rights reserved. doi:10.1016/j.euroneuro.2004.10.004 * Corresponding author. Tel.: +55 16 602 3638; fax: +55 16 633 5015. E-mail address: mbrandao@usp.br (M.L. Branda ˜o). European Neuropsychopharmacology 15 (2005) 239 – 246 www.elsevier.com/locate/euroneuro