Fos-like immunoreactivity in locus coeruleus after classical conditioning of the rabbit’s nictitating membrane response Pascal Carrive*, E. James Kehoe, Michaela Macrae, George Paxinos School of Psychology, The University of New South Wales, Sydney, 2052 Australia Received 26 September 1996; revised version received 14 January 1997; accepted 14 January 1997 Abstract We investigated the changes in Fos-like immunoreactivity in the locus coeruleus (LC) after classical conditioning of the rabbit’s nictitating membrane. Specifically, we compared unpaired versus paired presentations of a tone conditioned stimulus (CS) and a tactile unconditioned stimulus (US; near the eye). After two training sessions, only paired presentations resulted in acquisition of a conditioned response. This was associated with comparatively less LC Fos expression than with unpaired presentations. Similar observations have been reported for the ventrolateral medulla which is a major source of afferents to LC. The present results are consistent with a role of LC in attention and learning: activity increases as the animal attends to the CS and US. When the relationship between CS and US has been established, LC activity decreases.  1997 Elsevier Science Ireland Ltd. Keywords: Locus coeruleus; Lateral paragigantocellular nucleus; Fos; Nictitating membrane; Classical conditioning; Attention; Learn- ing The essential pathways for classical conditioning of the rabbit’s nictitating membrane (NM) response have been well delineated [19]. However, pathways that are collat- eral to the essential NM pathways are of interest because they may modify activity in the pathways for the NM response and/or subserve other, related response systems (e.g. heart rate). One nucleus that may lie along such a collateral pathway is the locus coeruleus (LC). The LC is the major source of noradrenaline for the brain and has been implicated in attention and learning [2,16]. In parti- cular, the LC sends dense projections to the cochlear nuclei, spinal trigeminal nucleus, and cerebellar cortex [7,10] which lie in the essential pathways for NM condi- tioning. The present experiment was conducted to deter- mine whether neurons in the LC are activated during classical conditioning by using the expression of Fos, the protein product of the immediate early gene c-fos, as an index of neural activity. Previous researchers, using NM conditioning, have found increased Fos-like immunoreac- tivity in the spinal trigeminal nucleus, raphe nucleus, and ventrolateral medulla [6]. The experiment used 21 female, albino rabbits (Orycto- lagus cuniculus), 70–90 days old and weighing 1.5–2 kg. The training apparatus was patterned after those described by Gormezano [5]. Each rabbit was trained in a chamber containing a speaker that provided an auditory CS, which was a 350 ms, 89 dB (SPL), 1000 Hz pure tone super- imposed on an ambient noise level of 82 dB. The tactile US was a 3 mA, 100 ms, 50 Hz AC electrical current delivered to the skin posterior to the right eye. To prepare the rabbits, hair around the right eye was removed, the eye was anaesthetized with proxymetacaine hydrochloride (Opthaine), and a small loop of surgical silk (000 Dynex) was sutured into the NM. This loop was used as the point of attachment for the rotating arm of the photo- electric transducer for measuring NM movements. Three days later, the rabbits were placed in the chamber for 60 min, but no stimuli were presented. Next, there were two training sessions on 2 consecutive days. Each session con- tained 60 trials separated by a mean interval of 60 s (range 50–70 s). The specific training conditions were: control (n = 4), the rabbits were restrained in the chamber without the CS or US; CS-alone (n = 3), each session had only CS- alone trials; CS/US unpaired (n = 7), CS-alone trials were intermixed with US-alone trials; and CS-US paired Neuroscience Letters 223 (1997) 33–36 0304-3940/97/$17.00  1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940(97)13395-X * Corresponding author. Tel.: +61 2 3853755; fax: +61 2 3853641; e-mail: p.carrive@unsw.edu.au