DIFFERENTIAL EXPRESSION OF NK1 AND NK3 NEUROKININ RECEPTORS IN NEURONS OF THE NUCLEUS TRACTUS SOLITARIUS AND THE DORSAL VAGAL MOTOR NUCLEUS OF THE RAT AND MOUSE I. LE BRUN, a,b A. DUFOUR, a M. CREST, a G. SZABÓ, c F. ERDELYI c AND A. BAUDE a * a Laboratoire de Neurophysiologie Cellulaire, Université de la Méditer- ranée, CNRS UMR 6150, IFR Jean-Roche, Faculté de Médecine Nord, Boulevard Pierre Dramard, 13916 Marseille Cx 20, France b Université Joseph Fourier, INSERM U 836, Site Santé, Grenoble, France c Laboratory of Molecular Biology and Genetics, Institute of Experimen- tal Medicine, Budapest, Hungary Abstract—Tachykinins (substance P, neurokinin A and neu- rokinin B) influence autonomic functions by modulating neu- ron activity in nucleus tractus solitarius (NTS) and dorsal motor nucleus of the vagus (DMV) through activation of neu- rokinin receptors NK1 and NK3. Our purpose was to identify and define by neurochemical markers, the subpopulations of NK1 and NK3 expressing neurons in NTS and DMV of rat and mouse. Because the distribution of the NK1 and NK3 expressing neurons overlaps, co-expression for both receptors was tested. By double labeling, we show that NK1 and NK3 were not co-expressed in NTS neurons. In the DMV, most of neu- rons (87%) were immunoreactive for only one of the recep- tors and 34% of NK1 neurons, 7% of NK3 neurons and 12% of NK1–NK3 neurons were cholinergic neurons. None of the neurons immunoreactive for NK1 or NK3 were positive for tyrosine hydroxylase, suggesting that catecholaminergic cells of the NTS (A2 and C2 groups) did not express neuro- kinin receptors. The presence of NK1 and NK3 was examined in GABAergic interneurons of the NTS and DMV by using GAD65-EGFP transgenic mouse. Immunoreactivity for NK1 or NK3 was found in a subpopulation of GAD65-EGFP cells. A majority (60%) of NK3 cells, but only 11% of the NK1 cells, were GAD65-EGFP cells. In conclusion, tachykinins, through differential expres- sion of neurokinin receptors, may influence the central reg- ulation of vital functions by acting on separate neuron sub- populations in NTS and DMV. Of particular interest, tachyki- nins may be involved in inhibitory mechanisms by acting directly on local GABAergic interneurons. Our results sup- port a larger contribution of NK3 compared with NK1 in me- diating inhibition in NTS and DMV. © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: tachykinin, GABA, interneuron, substance P, neurokinin A, neurokinin B. In mammals, endogenous tachykinins, substance P, neu- rokinin A, and neurokinin B act as neuromodulators in peripheral and CNS (Severini et al., 2002). The biological activity of the tachykinins depends on their interaction with three G protein– coupled receptors (NK1, NK2, and NK3) which share considerable structural homology. Each of the tachykinins is considered to have a preferred receptor. Substance P has higher affinity for NK1, while neurokinin A and neurokinin B display higher affinity for NK2 and NK3, respectively. However, all three tachykinins can bind to each of the neurokinin receptors, if present at sufficient concentration (Nakanishi, 1991; Maggi, 1995). Therefore, they can exert physiological effects through both preferred and non-preferred receptors (Harlan et al., 1989; Nakan- ishi, 1991; Hastrup and Schwartz, 1996; Wijkhuisen et al., 1999; Colin et al., 2002). Tachykinins and their receptors are involved in the central control of cardiovascular, digestive, gustatory and respiratory functions through brainstem neurons located in the nucleus tractus solitarius (NTS) and the dorsal vagal motor nucleus (DMV) (Improta and Broccardo, 1990; King et al., 1993; Otsuka and Yoshioka, 1993; Lawrence and Jarrott, 1996; Maubach and Jones, 1997; Liu et al., 1998; Mazzone and Geraghty, 2000; Severini et al., 2002; Potts et al., 2007). The NTS plays a key role in central regulation of autonomic functions as it receives primary afferents from gustatory, gastrointestinal, cardiovascular, and respi- ratory organs, particularly those supplied by the vagus nerve (Sawchenko et al., 1987; Berthoud and Neuhuber, 2000; Puizillout, 2005; Travagli et al., 2006). Apposed to the ventral face of the NTS, the DMV contains somata of the vagal efferent which innervate principally the digestive tract (Shapiro and Miselis, 1985; Blondeau et al., 2002) but also sub-diaphragmatic viscera such as the heart (Corbett et al., 2003) or lungs (Pérez Fontán and Velloff, 2001). The NTS and DMV are enriched with axon terminals containing substance P, neurokinin A and neurokinin B (Kalia et al., 1984; Baude et al., 1989; Lucas et al., 1992; Marksteiner et al., 1992; Saha et al., 1995). All of them are putatively able to activate neurokinin receptors in NTS and DMV (King et al., 1993; Maubach and Jones, 1997; Liu et al., 1998; Colin et al., 2002). Autoradiographic studies have described high specific labelings for NK1 and NK3 binding sites in NTS and DMV, while none was detected for NK2 (Saffroy et al., 2003). mRNAs coding for NK1 and NK3 receptors have been detected (Nakaya et al., 1994; Ding et al., 1996), and NK1 and NK3 receptor proteins are present in NTS and DMV neurons (Carpentier et al., 1996; Ding et al., 1996; Baude and Shigemoto, 1998). In addi- *Corresponding author. Tel: +33-4-91-69-88-52; fax: +33-4-91-69-89-77. E-mail address: agnes.baude@univmed.fr (A. Baude). Abbreviations: ChAT, choline acetyl transferase; DMV, dorsal vagal motor nucleus; NK1-/-, mice knockout for NK1; NTS, nucleus tractus solitarius; PB, phosphate buffer; PBST, phosphate buffer containing 0.9% NaCl supplemented with 0.3% Triton X-100; TH, tyrosine hy- droxylase. Neuroscience 152 (2008) 56 – 64 0306-4522/08$32.00+0.00 © 2008 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2007.12.024 56