Neurotrophins 3 and 4 enhance non-rapid eye movement sleep in rabbits Tetsuya Kushikata a , Takeshi Kubota a , Jidong Fang b , James M. Krueger c, * a Department of Anesthesiology, University of Hirosaki School of Medicine, Hirosaki 036-8506, Japan b Department of Psychiatry, College of Medicine, The Pennsylvania State University, Hershey, PA 17033, USA c Department of Veterinary and Comparative Anatomy, Pharmacology and Physiology, Washington State University, Pullman, WA 99164-6520, USA Received 14 March 2003; received in revised form 30 April 2003; accepted 5 May 2003 Abstract We determined if neurotrophins 3 and 4 (NT-3, NT-4) would promote sleep in rabbits. Two doses of NT-3 (50 and 500 ng) and three doses of NT-4 (50, 500 and 2000 ng) were intracerebroventricularly injected at dark onset. Additionally, 500 ng of each NT were injected during the light period. The electroencephalogram (EEG), brain temperature, and motor activity were recorded for 23 h following NT injection. NT- 3 (500 ng dose) and NT-4 (500 and 2000 ng doses) injected at dark onset increased the time spent in non-rapid eye movement sleep. After the 2000 ng dose of NT-4, EEG power was reduced. q 2003 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Neurotrophin; Rapid eye movement sleep; Slow wave sleep; Fever; Cytokine; Non-rapid eye movement sleep Several growth factors are implicated in sleep regulation (reviewed in Ref. [11]); the list includes endocrines such as growth hormone (GH), GH-releasing hormone (GHRH), prolactin (PRL) and insulin, and autocrines and paracrines such as interleukin-1b (IL-1b), tumor necrosis factor-a (TNFa), acidic fibroblast growth factor (aFGF), neurotro- phin 1 (NT-1: also called nerve growth factor), and neurotrophin 2 (NT-2: also called brain-derived neuro- trophic factor). All of these substances have the capacity to enhance either non-rapid eye movement sleep (NREMS) (insulin, IL-1b, TNFa, aFGF) or rapid eye movement sleep (REMS) (GH, PRL) or both (GHRH, NT-1, NT-2) [11]. Inhibition of GH, IL-1, TNF, and GHRH inhibits spon- taneous sleep and sleep rebound after sleep deprivation in the case of GHRH, IL-1 and TNF. These and much additional data strongly suggest that these growth factors are involved in physiological sleep regulation [11]. Neurotro- phins 3 and 4 (NT-3, NT-4) promote the growth and survival of CNS neurons [14]. Their receptors are widely distributed in the brain; NT-3 has partial affinity for the trkB neurotrophin receptor [1] and NT-4 has a high affinity to trkB [4]. Since NT-2 also has high affinity to trkB and it is somnogenic [10], we thought it likely that NT-3 and -4 might promote sleep. Previously it was reported that microinjection of human recombinant NT-3 into the rostro-dorsal pons of cats transiently enhanced REMS but not NREMS for about 1 h [18]. We report here that NT-3 and NT-4 enhance NREMS in rabbits. Recombinant human NT-3 and NT-4 were purchased from Peprotech (Rocky Hill, NJ) and dissolved in pyrogen- free isotonic NaCl (PFS; Abbott Laboratories, N. Chicago, IL). Injection volumes were 25 ml. Male New Zealand white rabbits (4.5–5.5 kg) were surgically implanted with electroencephalogram (EEG) electrodes, a brain thermistor, and a lateral intracerebroventricular (i.c.v.) cannula using ketamine-xylazine (35 and 5 mg/kg) anesthesia as pre- viously described [10]. The rabbits were habituated to the sleep-recording chambers for 1 day before control record- ings began. The animals were kept on a 12/12 h light/dark cycle (lights on at 06:00 h) at 21 ^ 1 8C and EEG, brain temperature (T br ) and motor activity were recorded [10]. The EEG, T br and motor activity were filtered, amplified, digitized, and saved as previously described [9]. On-line Fourier analyses of EEG data were performed. The vigilance states of wakefulness (W), NREMS, and REMS were visually identified in 10 s epochs using criteria previously reported [10]. Time spent in each vigilance state was calculated for 3 h intervals (Fig. 1) and for the entire recording period (Table 1). The EEG power density values were summed in the delta (0.5–4.0 Hz) band and 0304-3940/03/$ - see front matter q 2003 Elsevier Science Ireland Ltd. All rights reserved. doi:10.1016/S0304-3940(03)00564-0 Neuroscience Letters 346 (2003) 161–164 www.elsevier.com/locate/neulet * Corresponding author. Tel.: þ 1-509-335-6624; fax: þ1-509-335-4650. E-mail address: krueger@vetmed.wsu.edu (J.M. Krueger).