Multitasking with neurotensin in the central nervous system.

The 13-amino acid peptide neurotensin (NT) was discovered over 30 years ago and has been implicated in a wide variety of neurotransmitter and endocrine functions. This review focuses on four areas where there has been substantial recent progress in understanding NT signaling and several functions of the endogenous peptide. The first area concerns the functional activation of the high-affinity NT receptor, NTR-1, including the delineation of the NT binding pocket and receptor domains involved in functional coupling to intracellular signaling pathways. The development of NT receptor antagonists and the application of genetic and molecular genetic approaches have accelerated progress in understanding NT function in several areas, including the involvement of NT in antipsychotic drug actions, psychostimulant sensitization and the modulation of pain, and these are reviewed in that order. There is now substantial evidence indicating that NT is required for certain antipsychotic drug actions and that the peptide plays a key role in stress-induced analgesia.

Endogenous neurotensin facilitates visceral nociception and is required for stress-induced antinociception in mice and rats.

Central neurotensin (NT) administration can both facilitate and inhibit somatic and visceral nociception, depending on the dose and administration site. NT microinjection in the rostroventral medulla facilitates nociception at low doses, while NT antagonist microinjection can markedly attenuate nociception, supporting the hypothesis that endogenous NT facilitates nociception. However, higher doses of NT produce a mu-opioid receptor-independent analgesia, similar to that resulting from various intense stressors. Furthermore, intense stress results in increased NT expression in several hypothalamic nuclei that have been implicated in stress-induced antinociception (SIAN); however, there is little direct evidence that endogenous NT is required for SIAN. We have investigated the role of endogenous NT in both basal visceral nociception and SIAN using both NT knockout mice and pharmacological approaches in rats. Visceral nociception was monitored by measuring visceromotor responses during colorectal distension both prior to and following water avoidance stress. Visceral nociception was significantly attenuated in both NT knockout mice and rats pre-treated with the NT antagonist SR 48692. Disruption of NT signaling also blocked SIAN, revealing a novel stress-induced hyperalgesic response that was significantly greater in female than in male rats. NT was also required for acetic acid-induced hyperalgesia. These results indicate that endogenous NT normally facilitates visceral pain responses, is required for irritant-induced hyperalgesia, and plays a critical role in SIAN.

Endogenous neurotensin facilitates enterohepatic bile acid circulation by enhancing intestinal uptake in rats.

Initial studies on the digestive hormone neurotensin (NT) showing that intestinal NT mRNA expression and blood levels were altered in rats fed chow containing bile acid (BA) and the BA chelator cholestyramine led us to investigate the role of NT in the enterohepatic circulation of BA. In fasted, anesthetized rats with common bile ducts cannulated for bile collection, intravenous NT infusion (10 pmol. kg(-1). min(-1)) enhanced BA output relative to control over 3 h in animals administered donor bile into the duodenum (30 microl/min). This suggested that the effect of NT was on the return of BA from the intestine to the liver, which is rate determining in the normal process. In rats prepared as described above and administered [(3)H]taurocholate ([(3)H]TC; 5 mM, 1 ml) duodenally, NT infusion (3-10 pmol x kg(-1) x min(-1)) increased the [(3)H]TC recovery rate in bile approximately twofold, whereas sulfated CCK-8 (12-50 pmol x kg(-1) x min(-1)) had no effect. To investigate the roles of endogenous NT and CCK, we administered [(3)H]TC into the rat duodenum or lower jejunum and tested the effect of the NT antagonist SR-48692 (2 nmol x kg(-1) x min(-1)) or CCK-A antagonist lorglumide (100 nmol x kg(-1) x min(-1)). SR-48692 reduced the [(3)H]TC recovery rate by congruent with 50% and congruent with 24% in the duodenum and jejunum, respectively, whereas lorglumide had no effect. These results suggest that NT or a similar peptide is an endogenous regulator of enterohepatic BA cycling, which acts by enhancing BA uptake in the intestine.

Neurotensin-deficient mice show altered responses to antipsychotic drugs.

The peptide transmitter neurotensin (NT) exerts diverse neurochemical effects that resemble those seen after acute administration of antipsychotic drugs (APDs). These drugs also induce NT expression in the striatum; this and other convergent findings have led to the suggestion that NT may mediate some APD effects. Here, we demonstrate that the ability of the typical APD haloperidol to induce Fos expression in the dorsolateral striatum is markedly attenuated in NT-null mutant mice. The induction of Fos and NT in the dorsolateral striatum in response to typical, but not atypical, APDs has led to the hypothesis that the increased expression of these proteins is mechanistically related to the production of extrapyramidal side effects (EPS). However, we found that catalepsy, which is thought to reflect the EPS of typical APDs, is unaffected in NT-null mutant mice, suggesting that NT does not contribute to the generation of EPS. We conclude that NT is required for haloperidol-elicited activation of a specific population of striatal neurons but not haloperidol-induced catalepsy. These results are consistent with the hypothesis that endogenous NT mediates a specific subset of APD actions.

The neurotensin antagonist SR 48692 attenuates haloperidol-induced striatal Fos expression in the rat.

Neurotensin interacts with central dopamine systems and has been suggested to exert antipsychotic drug-like actions. Antipsychotic drugs such as haloperidol induce striatal immediate-early gene expression. In order to study neurotensin's role in antipsychotic drug actions, rats were pretreated with the neurotensin antagonist SR 48692 and then injected with haloperidol. SR 48692 dose-dependently decreased haloperidol-elicited immediate-early gene expression in the dorsolateral and central striatum but not other striatal areas. SR 48692 reduced Fos expression in the striatal patch (striosome) and matrix compartments, with a significantly greater effect in the patch. These data suggest that neurotensin may play a role in the actions of haloperidol. In view of proposed functional roles of the striatal patch and matrix, we suggest that neurotensin may be important in the therapeutic rather than side effects of antipsychotic drugs.

Sequences required for induction of neurotensin receptor gene expression during neuronal differentiation of N1E-115 neuroblastoma cells.

The promoter region of the mouse high affinity neurotensin receptor (Ntr-1) gene was characterized, and sequences required for expression in neuroblastoma cell lines that express high affinity NT-binding sites were characterized. Me(2)SO-induced neuronal differentiation of N1E-115 neuroblastoma cells increased both the expression of the endogenous Ntr-1 gene and reporter genes driven by NTR-1 promoter sequences by 3-4-fold. Deletion analysis revealed that an 83-base pair promoter region containing the transcriptional start site is required for Me(2)SO activation. Detailed mutational analysis of this region revealed that a CACCC box and the central region of a large GC-rich palindrome are the crucial cis-regulatory elements required for Me(2)SO induction. The CACCC box is bound by at least one factor that is induced upon Me(2)SO treatment of N1E-115 cells. The Me(2)SO effect was found to be both selective and cell type-restricted. Basal expression in the neuroblastoma cell lines required a distinct set of sequences, including an Sp1-like sequence, and a sequence resembling an NGFI-A-binding site; however, a more distal 5' sequence was found to repress basal activity in N1E-115 cells. These results provide evidence that Ntr-1 gene regulation involves both positive and negative regulatory elements located in the 5'-flanking region and that Ntr-1 gene activation involves the coordinate activation or induction of several factors, including a CACCC box binding complex.

Neurotensin receptor expression in prostate cancer cell line and growth effect of NT at physiological concentrations.

BACKGROUND: Neurotensin (NT), a neuroendocrine peptide, exerts trophic effects in vivo and stimulates growth of some tumor cells in vitro. Androgen-sensitive prostate cells derived from lymph node carcinoma of the prostate (LNCaP) secrete NT and exhibit growth responses to NT. This study examines NT secretion, NT receptor and NT-growth responses in androgen-independent prostatic carcinoma (PC3) cells derived from prostate adenocarcinoma metastatic to bone. METHODS: Binding of 125I-NT to PC3 membranes was studied by filtration. NT was measured by RIA. Reverse transcriptase polymerase chain reaction (RT-PCR) was used for NT and NT receptor mRNA. Growth was measured as 3H-thymidine incorporation into DNA. RESULTS: Scatchard analyses gave two binding components (Kd1 = 40 pM and Kd2 = 300 pM) in equal amounts (15-30 x 10(3) sites/cell). The bioactive region of NT was essential and the specific, non-peptide NT antagonist, SR48692, inhibited (IC50 = 3 nM). GTP analogs, sodium ion and SH-directed alkylating agents also inhibited. Glutaraldehyde crosslinking labeled two substances (M(r) of 23 and 46 kDa). RT-PCR indicated robust expression of authentic NT receptor but little for NT precursor. NT was stable in PC3 cultures but it was not found in cells or conditioned media. Incubated with PC3 cells, NT exhibited a mitogenic effect with bell-shaped dose-response and maximum at 100 pM NT. CONCLUSIONS: PC3 cells expressed genuine NT receptors and generated growth responses to physiologic levels of NT which were blocked by SR48692. If NT contributes to the survival of prostate tumor cells upon androgen deprivation therapy, NT antagonists might be useful agents in further treatment.

Developmental expression and activities of specific fos and jun proteins are functionally related to osteoblast maturation: role of Fra-2 and Jun D during differentiation.

Developmental studies of oncogene expression implicate the Fos and Jun family of transcription factors in the regulation of bone growth and differentiation. Promoters of many developmentally regulated genes, including osteocalcin, a marker of osteoblast differentiation, contain AP-1 sites that bind Fos/Jun dimers. Here, we demonstrate that the selective expression of fos- and jun-related genes is functionally related to the stage of osteoblast growth and differentiation in vitro. During osteoblast proliferation, nuclear protein levels of all seven activating protein-1 (AP-1) members are maximal. Subsequently, during the period of extracellular matrix maturation, levels decline. In fully differentiated osteoblasts, Fra-2 and (to a lesser extent) Jun D are the principal AP-1 members detectable by Western blot analysis. AP-1 complex composition and binding activity also exhibit developmental changes. All Fos and Jun family members are involved in AP-1 complex formation in proliferating cells, whereas Fra-2 and Jun D predominate in AP-1 complexes in differentiated osteoblasts. Overexpression of Fos and Jun family members in ROS 17/2.8 cells markedly affects the expression of an osteocalcin promoter-chloramphenicol acetyltransferase construct. Coexpression of only one AP-1 pair, Fra-2 and Jun D, stimulated reporter expression, whereas coexpression of other AP-1 pairs down-regulated expression (i.e. c-jun and any Fos family member) or had no effect (i.e. Fra-1 and Jun B). Promoter deletion analyses indicate that these effects are site specific. Consequential effects of Fra-2 on osteoblast differentiation are further demonstrated by antisense studies in which osteoblast differentiation and the development of a bone tissue-like organization were suppressed. Consistent with recent findings suggesting that AP-1 complex composition can selectively regulate gene transcription, our findings demonstrate that differential expression of Fos and Jun family members could play a role in the developmental regulation of bone-specific gene expression and, as a result, may be functionally significant for osteoblast differentiation.

Synergistic activation of neurotensin/neuromedin N gene expression by c-Jun and glucocorticoids: novel effects of Fos family proteins.

The cis-regulatory region of the neurotensin/neuromedin N (NT/N) gene integrates diverse environmental signals in the neuroendocrine PC12 cell line, resulting in remarkable synergistic regulation. An AP-1 site appears to play a pivotal role in cooperative NT/N gene activation, as mutations in this site decrease responses to all inducer combinations by at least an order of magnitude. Here we report that c-Jun acts synergistically with glucocorticoids to activate the NT/N promoter, and that Fos family proteins have novel regulatory effects on this interaction. Cotransfection of individual pCMV-AP-1 expression plasmids revealed that c-Jun most potently activates the NT/N promoter and that costimulation with dexamethasone results in a further 6- to 12-fold increase in expression. Unlike its general inhibitory effects on glucocorticoid regulation in other systems, c-Fos potentiated activation by glucocorticoids when coexpressed with c-Jun, and Fos B had a similar, but more limited, positive effect. In contrast, Fra-1 reversed the direction of glucocorticoid regulation, and Fra-2 abolished synergism. AP-1, cAMP response element, and glucocorticoid response element motifs are required for full cooperative activation by either c-Jun or c-Jun/c-Fos and glucocorticoids. These results indicate that NT/N promoter activation involves synergistic interactions between specific AP-1 complexes and ligand-activated glucocorticoid receptor, and similar mechanisms may regulate NT/N gene expression in central neurons.

Characterization of promoter elements required for cell-specific expression of the neurotensin/neuromedin N gene in a human endocrine cell line.

Expression of the gene encoding neurotensin/neuromedin N (NT/N) is mostly limited to the brain and specialized enteroendocrine cells (N cells) of the distal small intestine. We have analyzed the NT/N DNA sequences upstream of the RNA start site that direct cell-specific expression using a novel human endocrine cell line, BON, that resembles intestinal N cells in several important aspects, including NT/N precursor protein processing, ratios of different NT/N mRNA forms, and high levels of constitutive expression of the NT/N gene. Transient transfection assays with plasmids with progressive 5' deletions of the rat NT/N promoter identified the proximal 216 bp of 5' flanking sequences as essential for high-level constitutive NT/N expression in BON cells. In addition, a detailed mutational analysis defined multiple regions within the proximal 216 bp that contribute to cell-specific NT/N expression. These elements include a proximal cyclic AMP response element (CRE)/AP-1-like motif (TGACATCA) that binds c-Jun, JunD, CRE-binding (CREB), and ATF proteins, a near-consensus glucocorticoid response element, and a distal consensus AP-1 site that binds c-Fos, Fra-1, and JunD. In addition, elements contained within two 21-bp imperfect direct repeats play an important role in NT/N expression in BON cells and may bind novel factors that act as positive regulators of NT/N expression. DNase I footprinting and gel shift analyses demonstrate that the sites identified by mutational analysis, and at least one additional site, specifically bind BON cell nuclear proteins in vitro. We speculate that a complex pattern of regulation requiring interaction between a proximal CRE/AP-1-like motif and other upstream control elements play an important role in the high-level constitutive expression of NT/N in the human endocrine cell line BON. In addition, the BON cell line provides a unique model to further characterize the factors regulating cell-specific NT/N expression and to better understand the mechanisms responsible for the terminal differentiation of the N-cell lineage in the gut.

Synergistic induction of neurotensin gene transcription in PC12 cells parallels changes in AP-1 activity.

A consensus AP-1 site in the promoter of the rat neurotensin/neuromedin N (NT/N) gene is a critical regulatory element required for synergistic regulation by combinations of nerve growth factor (NGF), lithium, glucocorticoids, and adenylate cyclase activators. A rapid RNase protection assay was developed to examine the kinetics of NT/N gene activation and to determine whether activation requires newly synthesized proteins. Either NGF or lithium in combination with dexamethasone and forskolin transiently activated NT/N gene expression, but with distinct kinetics. Protein synthesis was not required for activation when NGF was used as the permissive inducer, but was required for the rapid down-regulation of the response. In contrast, lithium responses were attenuated in the absence of protein synthesis, consistent with a requirement for newly synthesized AP-1 complexes in activation. In all cases, increases in NT/N gene expression closely paralleled increases in AP-1 binding activity. Lithium in combination with other inducers caused delayed increases in both AP-1 binding activity and c-jun, c-fos and fra-1 gene expression. These results indicate that NGF and lithium exert their effects on NT/N gene expression through distinct pathways. The lithium pathway is active in neuronally-differentiated PC12 cells and could potentially be involved in the regulation of NT/N gene expression in the nervous system.

Enhanced expression of neurotensin/neuromedin N mRNA and products of NT/NMN precursor processing in neonatal rats.

Intestinal levels of immunoreactive neurotensin (iNT) and neuromedin N (iNMN), as well as mRNA for the NT/NMN precursor, were elevated during the suckling period in rats. While transient expression of NT/NMN was observed at 1-5 days of age in the proximal small intestine and colon, NT/NMN levels in the ileum increased to peak at 10-20 days of age and then decreased to adult levels. The levels of these peptides were not elevated in the central nervous system and pituitary over this time period. Chromatographic analyses of jejunoileal extracts indicated that large molecular forms of iNT and iNMN were present, constituting approximately 1.3% of total iNT and approximately 56% of total iNMN, respectively. Treatment of the large forms with pepsin, which is known to generate the fully immunoreactive peptides, NT(3-13), NT(4-13), and NMN, increased immunoreactivity tenfold (iNT) and 1.2-fold (iNMN). Thus, large forms actually constituted approximately 13% (iNT) and approximately 60% (iNMN). Based upon its physicochemical properties, large molecular iNMN was tentatively identified as a 125 residue peptide with NMN at its C-terminus [i.e., rat prepro-NT/NMN(23-147)]. The properties of large molecular iNT were most similar to those predicted for the entire precursor [i.e., rat prepro-NT/NMN(23-169)]. These results indicate a) that enhanced expression of NT/NMN occurs in a tissue-specific manner in rats during the suckling period; b) that the pattern of precursor processing in intestine yields primarily NT and a large molecular form of NMN.

Induction of the neurotensin (NT) gene in PC12 cells gives rise to NT precursor (approximately 88%), NT(3-13)-like peptide (approximately 10%), and NT (approximately 2%).

Neurotensin (NT) is coexpressed with catecholamines in sympathetic neurons and adrenal chromaffin cells. A pheochromocytoma PC12 cell line can also be induced to express the NT gene and produce immunoreactive NT. In the present study, NT mRNA was quantified under various hormonal conditions and NT precursor synthesis rates were determined by pulse labeling and immunoprecipitation. In addition, NT precursor and NT-related products were measured using RIA and were characterized using HPLC and Sephadex chromatography. Neurotensin mRNA, NT precursor synthesis, and NT precursor/product levels were correlated. Surprisingly, NT appeared to be a minor product, both in cells and media: NT precursor (approximately 88%), NT(3-13)-like peptide (approximately 10%), and NT (approximately 2%). Neurotensin added to cultures was not converted to NT(3-13). Treatment of cells with 60 mM KCl or various secretagogues induced Ca(2+)-dependent release of NT precursor, NT(3-13), and NT in proportion to their cellular contents. These results suggest a) that NT precursor processing in induced PC12 cells was much slower than NT precursor synthesis, b) that NT(3-13) was a major product and NT a minor one, and c) that NT precursor and its products were stored within secretory vesicles.

Higher level organization of individual gene transcription and RNA splicing.

Visualization of fibronectin and neurotensin messenger RNAs within mammalian interphase nuclei was achieved by fluorescence hybridization with genomic, complementary DNA, and intron-specific probes. Unspliced transcripts accumulated in one or two sites per nucleus. Fibronectin RNA frequently accumulated in elongated tracks that overlapped and extended well beyond the site of transcription. Splicing appears to occur directly within this RNA track, as evidenced by an unambiguous spatial separation of intron-containing and spliced transcripts. Excised introns for neurotensin RNA appear free to diffuse. The transcription and processing site of the fibronectin gene localized to the nuclear interior and was associated with larger transcript domains in over 88 percent of the cells. These results support a view of nuclear function closely integrated with structure.

Cloning of human neurotensin/neuromedin N genomic sequences and expression in the ventral mesencephalon of schizophrenics and age/sex matched controls.

A human genomic clone encompassing exons 1-3 of the neurotensin/neuromedin N gene was identified using a canine neurotensin complementary DNA probe. Sequence comparisons revealed that the 120-amino acid portion of the precursor sequence encoded by exons 1-3 is 89% identical to previously determined cow and dog sequences and that the proximal 250 bp of 5' flanking sequences are strikingly conserved between rat and human. The 5' flanking sequence contains cis-regulatory sites required for the induction of neurotensin/neuromedin N gene expression in PC12 cells, including AP1 sites and two cyclic adenosine-5'-monophosphate response elements. Oligonucleotide probes based on the human sequence were used to examine the distribution of neurotensin/neuromedin N messenger RNA in the ventral mesencephalon of schizophrenics and age- and sex-matched controls. Neurotensin/neuromedin N messenger RNA was observed in ventral mesencephalic cells some of which also contained melanin pigment or tyrosine hydroxylase messenger RNA. Neurons expressing neurotensin/neuromedin N messenger RNA were observed in the ventral mesencephalon of both schizophrenic and non-schizophrenic humans.

Differential effects of haloperidol and clozapine on neurotensin gene transcription in rat neostriatum.

A single dose of typical neuroleptic, haloperidol, has been demonstrated to increase the expression of neurotensin/neuromedin N (NT/N) mRNA in the dorsolateral striatum within 1 hr of its administration (Merchant et al., 1991). The present study further investigated neuroleptic-induced regulation of NT/N gene transcription. Levels of NT/N mRNA were examined at various times following a single dose of haloperidol (1 mg/kg, i.p.) or the atypical antipsychotic clozapine (20, 30, or 40 mg/kg, i.p.) by in situ hybridization histochemistry and quantitative solution hybridization. In the dorsolateral striatum, the two drugs had strikingly different effects; haloperidol rapidly (within 30 min) increased the expression of mature NT/N mRNA while virtually no increase was observed in response to nontoxic doses of clozapine at any of the time points examined. Following haloperidol, maximal induction occurred at 7 hr, at which time NT/N mRNA levels were an order of magnitude higher than basal levels. By 20 hr after haloperidol, there was a significant decline in striatal NT/N mRNA levels. In situ hybridization analysis using an intron-derived probe revealed that haloperidol-induced increases in mature NT/N mRNA levels in the striatum were preceded by a transient increase in intron-containing NT/N gene transcripts. These data strongly indicate that acute haloperidol treatment results in transient transcriptional activation of NT/N gene, although a concomitant effect on the stability of NT/N primary transcripts cannot be ruled out. In contrast to their differential effects in the dorsolateral striatum, a single dose of both haloperidol and clozapine induced a small but significant increase in NT/N mRNA expression in the shell sector of the nucleus accumbens. These results raise the possibility that NT neurons in the nucleus accumbens may, at least in part, mediate the antipsychotic effects of classical neuroleptics, whereas NT cells in the dorsolateral region of the striatum may be involved in mediating other effects of typical neuroleptics such as extrapyramidal motor symptoms.

A protein kinase inhibitor, staurosporine, mimics nerve growth factor induction of neurotensin/neuromedin N gene expression.

Nerve growth factor (NGF) cooperates with glucocorticoids, activators of adenylate cyclase, and lithium to induce the expression of teh gene encoding the neuropeptides neurotensin and neuromedin N (NT/N gene) in PC 12 pheochromocytoma cells. High level expression requires simultaneous treatment with three or all four inducers. To examine the mechanism underlying this complex synergism, we have examined the effects of protein kinase inhibitors and other agents which influence intracellular signal transduction on NT/N gene expression. Two structurally similar bacterial alkaloids, staurosporine and K-252a, inhibit several protein kinases in vitro, including protein kinase C and cyclic nucleotide-dependent kinases. K-252a has been reported to specifically inhibit the effects of NGF on PC12 pheochromocytoma cells. Surprisingly, staurosporine in combination with other inducers markedly potentiated NT/N gene expression. In contrast, K-252a had no effect on NT/N gene expression when added simultaneously with other inducers. Expression of the NT/N gene was also potentiated by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate, which directly activates protein kinase C, and by bradykinin, which stimulates phosphatidylinositol turnover in PC12 cells, and these effects were not blocked by staurosporine. Staurosporine was generally more effective in stimulating NT/N gene expression when used in inducer combinations that did not include NGF. These results, taken together with recent evidence that staurosporine is also able to induce neurite outgrowth from PC12 cells, suggest that the effects of staurosporine and NGF may converge, in part, on a common intracellular target.

Mutually dependent response elements in the cis-regulatory region of the neurotensin/neuromedin N gene integrate environmental stimuli in PC12 cells.

The expression of the gene encoding the neuroendocrine peptides neurotensin (NT) and neuromedin N is strictly dependent on simultaneous exposure to multiple inducers in PC12 pheochromocytoma cells. NT peptide and NT/N mRNA levels are synergistically induced by combinations of NGF, dexamethasone, activators of adenylate cyclase, and lithium ion. We have used transient transfection assays to delineate the rat NT/N gene sequences necessary for this complex regulation. Progressive deletions of the 5' flanking region revealed that sequences between -216 and +56 are sufficient to confer the full spectrum of responses exhibited by the endogenous gene to a reporter gene. Detailed mutational analysis of this region indicates that it is composed of an array of inducible cis-regulatory sequences, including AP-1, cAMP response, and glucocorticoid response elements. Specific mutation of either the AP-1 site or each of two cAMP response elements indicates that they are functionally interdependent. This array of response elements serves to integrate multiple environmental stimuli into a unified transcriptional response.

Estrogen induces neurotensin/neuromedin N messenger ribonucleic acid in a preoptic nucleus essential for the preovulatory surge of luteinizing hormone in the rat.

Ovarian steroids act on unidentified neurons to trigger preovulatory secretion of GnRH. In the rat, important steroid target cells reside in the anterior medial preoptic nucleus (AMPN), a sexually dimorphic structure essential for stimulatory effects of ovarian steroids on LH secretion. The AMPN contains neurotensin (NT)-immunoreactive neurons, and immunoneutralization of NT in the preoptic region markedly attenuates steroid-induced LH surges. Using probes derived from the rat gene that encodes NT and neuromedin N (NT/N), we investigated the ability of estrogen to influence NT/N mRNA levels in the AMPN. Ovariectomized rats were treated for 14 days with sham capsules or capsules that produce supraphysiological serum levels of 17 beta-estradiol (250 +/- 20 pg/ml). As determined by in situ hybridization, estradiol markedly altered the distribution of NT/N mRNA in the medial preoptic region, causing a striking increase in NT/N mRNA abundance specifically in the AMPN and adjacent medial preoptic nucleus (MPN). In contrast, estradiol caused no obvious changes in labeling in the lateral septum, diagonal band of Broca, bed nucleus of the stria terminalis, and lateral preoptic area. The distribution of NT/N mRNA in the AMPN of normal male rats closely resembled that in ovariectomized rats, where labeled cells were rarely observed. Microdissection and S1 nuclease protection analysis were used to quantitate the effect of estradiol on NT/N mRNA levels. Supraphysiological estradiol treatment for 14 days caused a 3.4-fold increase (P less than 0.0002) in NT/N mRNA levels in the combined AMPN/MPN, whereas levels in the central amygdaloid nucleus remained constant, providing further evidence of regional specificity. Forty-eight hours of estradiol treatment, at concentrations (60 +/- 1 pg/ml) similar to those observed on the morning of proestrus, caused a 1.8-fold increase (P less than 0.001) in NT/N mRNA levels in the AMPN/MPN, indicating that the time course of NT/N mRNA induction by estrogen is compatible with events of the normal estrous cycle. Together with previous findings, our results strongly suggest that NT neurons mediate, directly or indirectly, stimulatory effects of ovarian steroids on GnRH secretion.