Autoradiograms were generated by apposition of labeled sections to Hyperfilm max (Amersham), for two or five days (6- and 16-day-old rats, respectively). In situ hybridization histochemistry hybridization histochemistry of NT1 receptor mRNA was performed as described (Lpe-Lorgeoux 1999), using four oligodeoxynucleotides complementary to nucleotides 128C169, 810C843, 1152C1184, 1375C1411 of the rat NT1 receptor cDNA (Tanaka 1990). neurons from fetal rats, we showed that exposure to the neurotensin agonist JMV 449 (1 nM) decreased (?43%) the amount of NT1 receptor mRNA measured by reverse transcription-PCR, an effect that was abolished by the non-peptide NT1 receptor antagonist SR Aclacinomycin A 48692 (1 M). However, daily injection of SR Aclacinomycin A 48692 to rat pups from birth for 5, 9 or 15 days, did not change [125I]neurotensin binding in brain membrane homogenates. Moreover, postnatal blockade of neurotensin transmission did not alter the density and distribution of NT1 receptors assessed by quantitative autoradiography nor NT1 receptor mRNA expression measured by hybridization in the cerebral cortex, caudate-putamen and midbrain. These results suggest that although NT1 receptor expression can be regulated by the agonist at an early developmental stage, neurotensin is not a major factor in the establishment of the ontogenetic pattern of these receptors in the rat brain. 1989). In the central nervous system (CNS), neurotensin acts as a neurotransmitter and neuromodulator of several neuronal systems; in particular, neurotensin has been shown to play an important role in the modulation of dopamine transmission (Lambert 1995). Neurotensin is also Aclacinomycin A involved in nociception, thermoregulation and modulation of neuroendocrine systems (for reviews, see Nemeroff and Kitabgi, 1992; Rostne and Alexander, 1997). Two types of neurotensin receptors, NT1 and NT2, have been T cloned and shown to belong to the family of G protein-coupled receptors (Tanaka 1990; Vita 1993; Chalon 1996; Mazella 1996). These two receptors are distinguished by their affinity for neurotensin and their capacity to bind levocabastine, a histamine H1 receptor antagonist (Schotte 1986). The levocabastine-insensitive, high-affinity neurotensin receptor (NT1) mediates most of the physiological functions ascribed to neurotensin. In contrast, only limited data are available concerning the functional role of the levocabastine-sensitive, lower-affinity neurotensin receptor (NT2). Using an anti-sense strategy in mice, the NT2 receptor was implicated in the analgesic effects induced by neurotensin (Dubuc 1999). A third neurotensin receptor (NT3) was recently cloned from human brain (Mazella 1998); it does not belong to the superfamily of G protein-coupled receptors, corresponds to the previously cloned gp95/sortilin and Aclacinomycin A could be involved in intracellular trafficking. Previous studies around the ontogeny of NT1 receptors in the rat brain using autoradiography (Palacios 1988), binding experiments in brain homogenates (Schotte and Laduron, 1987) and hybridization (Sato 1992; Lpe-Lorgeoux 1999), reported marked regional differences in their developmental profile. Thus, the cerebral cortex exhibits a high and transient expression of NT1 receptors during the early postnatal period, which declines to reach adult levels by the third postnatal week, whereas other brain areas, including the midbrain, show a gradual increase in NT1 receptor expression from late gestation to the second week of life. Interestingly, neurotensin is also highly expressed in the brain at birth, followed by a dramatic decrease to adult levels during the second and third weeks of postnatal life (Hara 1982; Sato 1991; Bennett 1998). The role of endogenous ligands in establishing the developmental pattern of expression of their corresponding receptors has been suggested for several neurotransmitter systems, including neuropeptides (Kudlacz 1991; Hill 1994; Sircar 1996; Liu 1998). However, the possible involvement of neurotensin in the regulation of NT1 receptors during ontogeny has not been studied. In a recent series of experiments, we used the non-peptide antagonist of NT1 receptors SR 48692 to investigate the role of endogenous neurotensin in the regulation of NT1 receptors in the adult rat brain. We showed that chronic administration of SR 48692 induces an up-regulation of NT1 receptor binding sites and mRNA expression, suggesting a tonic inhibitory control of neurotensin on NT1 receptors in the mature brain (Azzi 1994, 1996; Najimi 1998). The aim of the present study was to investigate the role of neurotensin in the regulation of NT1 receptors during postnatal development in the rat brain. We first examined concomitantly the ontogeny of neurotensin content and NT1 receptor expression, since previous studies examining separately either parameter rendered difficult their comparison at a given time point. Second, we used primary cultures of embryonic neurons from rat cerebral cortex, the brain region with the highest expression of NT1 receptors during early development, to investigate whether these receptors could be regulated by the agonist at an early developmental stage. The amount of NT1 receptor mRNA transcripts in the cultures was measured by quantitative reverse transcription-polymerase chain reaction (RT-PCR). Finally, we studied the effects of blockade of neurotensinergic transmission during postnatal development in rat pups injected with the NT1 receptor antagonist SR 48692. [125I]neurotensin binding to NT1 receptors was examined using brain homogenates and autoradiography, and NT1 receptor mRNA expression was assessed by hybridization histochemistry. MATERIALS AND METHODS Animals and treatments Wistar rats bred in our.