Three times as many lamina I neurons project to the periaqueductal gray than to the thalamus: a retrograde tracing study in the cat Leonora J. Mouton*, Gert Holstege Department of Anatomy and Embryology, Faculty of Medical Sciences, Rijksuniversiteit Groningen, Oostersingel 69, 9713 EZ Groningen, The Netherlands Received 23 July 1998; received in revised form 2 September 1998; accepted 2 September 1998 Abstract The number and distribution of lamina I neurons projecting to the periaqueductal gray (PAG) were examined by a retrograde tracing study in the cat. WGA-HRP injections in the intermediate and caudal PAG resulted in as much as 1600 labeled lamina I neurons throughout the length of the spinal cord, counted in a 1:4 series of sections. The lamina I-PAG projection was predominantly contralateral and most labeled lamina I neurons were found in the enlargements. Comparing these results with the number of lamina I-thalamic neurons leads to the conclusion that in the cat about three times as many lamina I neurons project to the PAG than to the thalamus. Considering this, one can conclude that the spino-PAG system is a virtually neglected area in pain research.  1998 Elsevier Science Ireland Ltd. All rights reserved Keywords: Lamina I; Spinomesencephalic; Periaqueductal gray; Spinothalamic; Nociception; Cat Lamina I of the spinal cord plays an essential role in conveying information about pain and temperature. The great majority of lamina I neurons receive Ad and C fiber input and are nociceptive specific [16]. The spinothalamic tract (STT) is often considered as the main ascending spinal system for pain information. STT neurons are located both in the dorsal and ventral horn of all spinal segments, but, except for the upper cervical cord, about half of the STT neurons are located in lamina I [1,3]. Another area involved in pain regulation and that receives projections from lamina I is the mesencephalic periaque- ductal gray (PAG). Earlier retrograde tracing studies in rat [4,6–8,11], rabbit [10], cat [5,14] and monkey [2,9,15,17] showed the presence of lamina I-PAG cells. However, none of these reports precisely describes the total lamina I-PAG system. Therefore, the present study examined the lamina I- PAG system throughout the cord, with the aim to estimate the total number of lamina I-PAG neurons and to give a description of their segmental distribution. In four cases (2155, 2159, 2367 and 2385) large injec- tions of approximately 100 nl wheat germ agglutinin-con- jugated horseradish peroxidase (WGA-HRP; 2.5%) was placed in the PAG. In one control case (2338), WGA- HRP was injected in the deep tectal layers laterally to the PAG. The surgical procedures, pre- and postoperative care, handling and housing of the animals were in accordance with protocols approved by the Committee of Animal Experiments of the Faculty of Medicine of the University of Groningen. The animals were initially anesthetized with ketamin (Nimatek; 0.1 ml/kg i.m.) and xylazine (Sedamun; 0.1 ml/kg i.m.), after which they were kept anesthetized by ventilation with a mixture of O 2 ,N 2 O and halothane. During surgery, heart rate and body temperature were monitored. Following a survival time of 3 days the animals were initi- ally anesthetized with the ketamin and xylazine i.m., fol- lowed by an overdose of 6% pentobarbital sodium (Nem- butal; i.p). The cats were perfused transcardially with 2 l of heparinized saline at 37°C, directly followed by 2 l of 0.1 M phosphate buffer, containing 4% sucrose, 1% paraformalde- hyde and 2% glutaraldehyde. After perfusion, the brain and spinal cord were removed, post-fixed for 2 h and stored overnight in 20% sucrose in phosphate buffer at 4°C. Sub- sequently, the brainstem and the complete (C1-Co2) spinal Neuroscience Letters 255 (1998) 107–110 0304-3940/98/$19.00  1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3940(98)00723-X * Corresponding author. Tel.: +31 50 3632460; fax: +31 50 3632461; e-mail: l.j.mouton@med.rug.nl