Neurotensin type 2 receptor is involved in fear memory in mice

Neurotensin receptor subtype 2 (Ntsr2) is a levocabastine-sensitive neurotensin receptor expressed diffusely throughout the mouse brain. Previously, we found that Ntsr2-deficient mice have an abnormality in the processing of thermal nociception. In this study, to examine the involvement of Ntsr2 in mouse behavior, we performed a fear-conditioning test in Ntsr2-deficient mice. In the contextual fear-conditioning test, the freezing response was significantly reduced in Ntsr2-deficient mice compared with that of wild-type mice. This reduction was observed from 1 h to 3 weeks after conditioning, and neither shock sensitivity nor locomotor activity was altered in Ntsr2-deficient mice. In addition, we found that Ntsr2 mRNA was predominantly expressed in cultured astrocytes and weakly expressed in cultured neurons derived from mouse brain. The combination of in situ hybridization and immunohistochemistry showed that Ntsr2 mRNA was dominantly expressed in glial fibrillary acidic protein positive cells in many brain regions including the hypothalamus, while Ntsr2 gene was co-expressed with neuron-specific microtubule associated protein-2 in limited numbers of cells. These results suggest that Ntsr2 in astrocytes and neurons may have unique function like a modulation of fear memory in the mouse brain.     

Changes in CNS response to neurotensin accompany the postpartum period in mice

Neurotensin (NT) is a highly conserved neuropeptide in mammals. Recent studies suggest that altered NT neurotransmission in postpartum females could promote the emergence of some maternal behaviors, including offspring protection. Here we evaluated how virgin and postpartum brains from mice selected for high maternal defense differ in response to NT. Virgin and postpartum mice were injected with either vehicle or 0.1 μg NT icv and brains were evaluated for c-Fos immunoreactivity, an indirect marker of neuronal activity. Using ANOVA analysis, common significant responses to NT were found in both female groups in four brain regions, including supraoptic nucleus, ventromedial nucleus, bed nucleus of stria terminalis dorsal, and a subregion of lateral septum (LS). For postpartum mice, only one additional region showed a significant response to NT relative to vehicle, whereas for virgin mice seven unique brain regions showed a significant c-Fos response: nucleus accumbens shell, paraventricular nucleus, central amygdala, and substantia nigra. Using a principal components analysis of c-Fos, we identified regions within each group with highly correlated activity. As expected, virgin and postpartum mice (vehicle conditions) showed different activity hubs and in the postpartum group the hubs matched regions linked to maternal care. The response to injected NT was different in the maternal and virgin groups with maternal mice showing a stronger coordinated activity in periaqueductal gray whereas virgin mice showed a stronger septal and amygdala linking of activity. Together, these results indicate neuronal responses of virgin and postpartum mice to NT and highlight pathways by which NT can alter maternal responses.     

Neurotensin and pain modulation

Neurotensin (NT) can produce a profound analgesia or enhance pain responses, depending on the circumstances. Recent evidence suggests that this may be due to a dose-dependent recruitment of distinct populations of pain modulatory neurons. NT knockout mice display defects in both basal nociceptive responses and stress-induced analgesia. Stress-induced antinociception is absent in these mice and instead stress induces a hyperalgesic response, suggesting that NT plays a key role in the stress-induced suppression of pain. Cold water swim stress results in increased NT mRNA expression in hypothalamic regions known to project to periaqueductal gray, a key region involved in pain modulation. Thus, stress-induced increases in NT signaling in pain modulatory regions may be responsible for the transition from pain facilitation to analgesia. This review focuses on recent advances that have provided insights into the role of NT in pain modulation.     

Loss of calcineurin from the medial preoptic area of primiparous rats

Western blot analyses reveal that calcineurin A (CNA), which is present in the hippocampus, basolateral amygdala, parietal cortex, and MPOA of virgin males and females, is undetectable only in the MPOA of primiparous females regardless of whether they had postpartum pup contact or not. In contrast, CNB was expressed at unchanging levels in the PC and MPOA. Similarly, G(alphao) and PKA(RI) were expressed at high levels in all of the brain regions of virgin males, virgin females, and primiparous females, supporting the concept that this loss of CNA is a specific event. Understanding how and why the expression of CNA, the sole neuronal Ca2+/CaM-dependent protein phosphatase, is down-regulated specifically in the MPOA of primiparous females may yield some insight into the signal transduction events that mediate the onset of mammalian maternal behavior.     

The effect of chronic antipsychotic drug on hypothalamic expression of neural nitric oxide synthase and dopamine D2 receptor in the male rat

Antipsychotic-induced sexual dysfunction is a common and serious clinical side effect. It has been demonstrated that both neuronal nitric oxide (nNOS) and dopamine D2 receptor (DRD2) in the medial preoptic area (MPOA) and the paraventricular nucleus (PVN) of the hypothalamus have important roles in the regulation of sexual behaviour. We investigated the influences of 21 days' antipsychotic drug administration on expression of nNOS and DRD2 in the rat hypothalamus. Haloperidol (0.5 mg/kg/day i.p.) significantly decreased nNOS integrated optical density in a sub-nucleus of the MPOA, medial preoptic nucleus (MPN), and decreased the nNOS integrated optical density and cell density in another sub-nucleus of the MPOA, anterodorsal preoptic nucleus (ADP). Risperidone (0.25 mg/kg) inhibited the nNOS integrated optical density in the ADP. nNOS mRNA and protein in the MPOA but not the PVN was also significantly decreased by haloperidol. Haloperidol and risperidone increased DRD2 mRNA and protein expression in both the MPOA and the PVN. Quetiapine (20 mg/kg/day i.p.) did not influence the expression of nNOS and DRD2 in either the MPOA or the PVN. These findings indicate that hypothalamic nNOS and DRD2 are affected to different extents by chronic administration of risperidone and haloperidol, but are unaffected by quetiapine. These central effects might play a role in sexual dysfunction induced by certain antipsychotic drugs.     

Neurotensin receptor 1 is expressed in gastrointestinal stromal tumors but not in interstitial cells of Cajal

Gastrointestinal stromal tumors (GIST) are thought to derive from the interstitial cells of Cajal (ICC) or an ICC precursor. Oncogenic mutations of the KIT or PDGFRA receptor tyrosine kinases are present in the majority of GIST, leading to ligand-independent activation of the intracellular signal transduction pathways. We previously investigated the gene expression profile in the murine Kit(K641E) GIST model and identified Ntsr1 mRNA, encoding the Neurotensin receptor 1, amongst the upregulated genes. Here we characterized Ntsr1 mRNA and protein expression in the murine Kit(K641E) GIST model and in tissue microarrays of human GIST. Ntsr1 mRNA upregulation in Kit(K641E) animals was confirmed by quantitative PCR. Ntsr1 immunoreactivity was not detected in the Kit positive ICC of WT mice, but was present in the Kit positive hyperplasia of Kit(K641E) mice. In the normal human gut, NTSR1 immunoreactivity was detected in myenteric neurons but not in KIT positive ICC. Two independent tissue microarrays, including a total of 97 GIST, revealed NTSR1 immunoreactivity in all specimens, including the KIT negative GIST with PDGFRA mutation. NTSR1 immunoreactivity exhibited nuclear, cytoplasmic or mixed patterns, which might relate to variable levels of NTSR1 activation. As studies using radio-labeled NTSR1 ligand analogues for whole body tumor imaging and for targeted therapeutic interventions have already been reported, this study opens new perspectives for similar approaches in GIST.     

Regional Distribution of Neurotensin in Human Brain

Abstract: Neurotensin (NT) is an endogenous neuropeptide that is active in many preclinical screening tests for neuroleptic drugs. Using a radioimmunoassay, we have studied the regional distribution of NT in postmortem human brain and in cerebrospinal fluid. Highest levels were present in the hypothalamus, substantia nigra, and limbic areas, whereas much lower amounts were found in the cortex and striatum. The chromatographic properties of hypothalamic immunoreactivity on ion-exchange and high pressure liquid chromatography were similar to those of the synthetic tridecapeptide. We conclude that NT is present in human brain with a distribution resembling that seen in other species, such as rat and monkey.     

Comparison of neurotensin levels, receptors and actions in LS/Ibg and SS/Ibg mice

Neurotensin (NT), injected centrally, markedly enhances sensitivity to ethanol-induced anesthesia in SS but not in LS mice (4). Since LS and SS mice were bred selectively for differential sensitivity to ethanol, these findings suggest that neurotensinergic neuronal processes mediate some of ethanol's actions and that LS and SS mice might differ genetically in neurotensinergic systems. Indeed, in biochemical studies it was shown that LS and SS mice differ in NT-like immunoreactivity in specific brain regions, i.e., hypothalamus, and in NT receptor densities (Bmax) in frontal cortex and striatum. In other experiments LS and SS mice differed in behavioral responses to centrally administered NT. Intracerebroventricular (ICV) administration of NT produced dose-dependent changes in motor activity, hypothermia, and analgesia in both LS and SS mice. SS mice appeared to be more sensitive than LS to NT-induced analgesia but not hypothermia. Neurotensin increased or decreased locomotor activity in both SS and LS mice following intraventral tegmental area or ICV administration, respectively. The results indicate that LS and SS mice, which were selectively bred for differences in ethanol sensitivity, differ genetically in NT concentrations, receptor densities in specific brain regions, and in some receptor-mediated behavioral responses to NT.     

Neurotensin interacts with dopaminergic neurons in rat brain

Neurotensin (NT) injected intracerebroventricularly in rat increases dopamine (DA) turnover in the corpus striatum and nucleus accumbens. Significant increases in 3,4-dihydroxyphenylacetic acid (DOPAC) levels occurred within 15 minutes after injection with peak levels at 60 minutes. The effect on NT on DOPAC and homovanillic acid (HVA) accumulation was dose-dependent at 3–100 μg. NT, like haloperidol, stimulated 3,4-dihydroxyphenylalanine (DOPA) accumulation in striatal neurons, in the presence of DOPA decarboxylase inhibitor, after injection of gamma-butyrolactone (GBL). NT had a similar stimulatory effect on DOPA levels in the accumbens while haloperidol (0.25 mg·kg^?1) had no significant effect in this brain region. NT did not block the inhibitory effect of apomorphine on DOPA accumulation in both the striatum and accumbens, while haloperidol inhibited apomorphine effect in both regions. NT also failed to displace ³H-spiperone from DA receptors and the presence of NT in the binding assay did not alter the ability of DA to displace ³H-spiperone in either brain region. These experiments demonstrate that NT increases DA turnover in both the nigrostriatal and mesolimbic pathways.     

Distribution of a novel avian gonadotropin-inhibitory hormone in the quail brain

We recently identified a novel hypothalamic neuropeptide inhibiting gonadotropin release in the quail brain and termed it gonadotropin inhibitory hormone (GnIH). In this study, we investigated the localization and distribution of GnIH in both sexes of adult quails by immunohistochemistry with a specific antiserum against GnIH and in situ hybridization. Quantitative analysis demonstrated that the concentration of GnIH in the diencephalon was greater than that in the mesencephalon without sex difference. GnIH concentrations in the cerebrum and cerebellum were below the level of detectability. Clusters of GnIH-like immunoreactive (GnlH-ir) cell bodies were localized in the paraventricular nucleus (PVN) of the hypothalamus. There was no significant difference in the number of GnlH-ir cells in the PVN between males and females. By double immunostaining with antisera reacting with GnIH or avian posterior pituitary hormones (vasotocin and mesotocin), GnIH-ir cells were found to be parvocellular neurons in the ventral portion of PVN, which showed no immunoreaction with the antisera against vasotocin and mesotocin. In situ hybridization revealed the cellular localization of GnIH mRNA in the PVN. GnIH-ir nerve fibers were however widely distributed in the diencephalic and mesencephalic regions. Dense networks of immunoreactive fibers were found in the ventral paleostriatum, septal area, preoptic area, hypothalamus, and optic tectum. The most prominent fibers were seen in the median eminence of the hypothalamus and the dorsal motor nucleus of the vagus in the medulla oblongata. Thus, GnIH may participate not only in neuroendocrine functions, but also in behavioral and autonomic mechanisms.     

AAV-mediated leptin receptor installation improves energy balance and the reproductive status of obese female Koletsky rats

Leptin is a hormone secreted primarily by white adipocytes that regulates energy homeostasis and reproduction via CNS receptors. Koletsky (f/f) rats with a leptin receptor (OB-Rb) gene mutation are obese, diabetic and infertile. We employed recombinant adeno-associated viral (rAAV) vectors to transfer the human OB-Rb gene into the brains of female Koletsky rats to identify sites of leptin action in the brain. rAAV-OB-Rb was microinjected into the medial preoptic area (MPOA), the paraventricular nucleus (PVN), the ventromedial hypothalamus, the arcuate nucleus (ARC), or the dorsal vagal complex in the brainstem. Food intake and body weight were monitored bi-weekly for 55 days. Vaginal cytology was examined daily to assess estrous cyclicity. After sacrifice, uncoupling protein-1 (UCP-1) mRNA in brown adipose tissue and serum concentrations of leptin, insulin, glucose, estradiol and progesterone were measured. Expression of OB-Rb was documented by RT-PCR and site specificity of microinjection was verified by immunohistochemical detection of green fluorescent protein following a control microinjection of rAAV-GFP. OB-Rb installation in the ARC reduced food intake, however, energy expenditure, assessed by UCP-1 mRNA expression, was increased by OB-Rb installation in all sites except the PVN. When injected into the MPOA and ARC, rAAV-OB-Rb stimulated the reproductive axis as evidenced by normalization of estrous cycle length and increased luteinizing hormone releasing hormone concentrations in the hypothalamus. These studies show that long-term installation of a functional leptin receptor in the CNS is achievable using rAAV vectors and further show that leptin acts on specific sites in the brain to produce differential effects on food intake, energy expenditure and reproduction.     

Neurotensin and neurotensin receptor 1 mRNA expression in song‐control regions changes during development in male zebra finches

Learned vocalizations are important for communication in some vertebrate taxa. The neural circuitry for the learning and production of vocalizations is well known in songbirds, many of which learn songs initially during a critical period early in life. Dopamine is essential for motor learning, including song learning, and dopamine‐related measures change throughout development in song‐control regions such as HVC, the lateral magnocellular nucleus of the anterior nidopallium (LMAN), Area X, and the robust nucleus of the arcopallium (RA). In mammals, the neuropeptide neurotensin strongly interacts with dopamine signaling. This study investigated a potential role for the neurotensin system in song learning by examining how neurotensin (Nts) and neurotensin receptor 1 (Ntsr1) expression change throughout development. Nts and Ntsr1 mRNA expression was analyzed in song‐control regions of male zebra finches in four stages of the song learning process: pre‐subsong (25 days posthatch; dph), subsong (45 dph), plastic song (60 dph), and crystallized song (130 dph). Nts expression in LMAN during the subsong stage was lower compared to other time points. Ntsr1 expression was highest in HVC, Area X, and RA during the pre‐subsong stage. Opposite and complementary expression patterns for the two genes in song nuclei and across the whole brain suggest distinct roles for regions that produce and receive Nts. The expression changes at crucial time points for song development are similar to changes observed in dopamine studies and suggest Nts may be involved in the process of vocal learning. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 78: 671–686, 2018     

BiP mRNA expression is upregulated by dehydration in vasopressin neurons in the hypothalamus in mice

The immunoglobulin heavy chain binding protein (BiP) is an endoplasmic reticulum (ER) chaperone that facilitates the proper folding of newly synthesized secretory and transmembrane proteins. Here we report that BiP mRNA was expressed in the supraoptic nucleus (SON) and paraventricular nucleus (PVN) of the hypothalamus in wild-type mice under basal conditions. Dual in situ hybridization in the SON and PVN demonstrated that BiP mRNA was expressed in almost all the neurons of arginine vasopressin (AVP), an antidiuretic hormone. BiP mRNA expression levels were increased in proportion to AVP mRNA expression in the SON and PVN under dehydration. These data suggest that BiP is involved in the homeostasis of ER function in the AVP neurons in the SON and PVN.     

Antagonism of V1b receptors promotes maternal motivation to retrieve pups in the MPOA and impairs pup-directed behavior during maternal defense in the mpBNST of lactating rats

Recent studies using V1b receptor (V1bR) knockout mice or central pharmacological manipulations in lactating rats highlighted the influence of this receptor for maternal behavior. However, its role in specific brain sites known to be important for maternal behavior has not been investigated to date. In the present study, we reveal that V1bR mRNA (qPCR) and protein levels (Western blot) within either the medial preoptic area (MPOA) or the medial-posterior part of the bed nucleus of the stria terminalis (mpBNST) did not differ between virgin and lactating rats. Furthermore, we characterized the effects of V1bR blockade via bilateral injections of the receptor subtype-specific antagonist SSR149415 within the MPOA or the mpBNST on maternal behavior (maternal care under non-stress and stress conditions, maternal motivation to retrieve pups in a novel environment, maternal aggression) and anxiety-related behavior in lactating rats. Blocking V1bR within the MPOA increased pup retrieval, whereas within the mpBNST it decreased pup-directed behavior, specifically licking/grooming the pups, during the maternal defense test. In addition, immediately after termination of the maternal defense test, V1bR antagonism in both brain regions reduced nursing, particularly arched back nursing. Anxiety-related behavior was not affected by V1bR antagonism in either brain region. In conclusion our data indicate that V1bR antagonism significantly modulates different aspects of maternal behavior in a brain region-dependent manner.     

Expression of proopiomelanocortin and proenkephalin mRNA in sexually dimorphic brain regions are altered in adult male and female rats treated prenatally with morphine.

The present study demonstrates that prenatal morphine exposure on gestation days 11-18 differentially alters proopiomelanocortin (POMC) and proenkephalin (pENK) mRNA in the hypothalamus and limbic system of adult male and female rats. In adult, prenatally morphine-exposed male rats POMC mRNA levels are decreased in the arcuate nucleus of the hypothalamus (ARC), while the pENK mRNA levels are increased in the paraventricular nucleus of the hypothalamus (PVN) and in the ventrolateral subdivision of the ventromedial nucleus of the hypothalamus (VMH), specifically in the ventrolateral subdivision of the VMH. In adult, prenatally morphine-exposed female rats, POMC mRNA levels in the ARC are increased in ovariectomized (OVX) but not in OVX, estradiol benzoate- (EB) or EB- and progesterone- (P) treated females. In contrast, pENK mRNA levels are decreased in the VMH of morphine-exposed, OVX females and increased in EB-treated females. Further, prenatal morphine exposure decreases pENK mRNA in the ARC and increases it in the medial pre-optic area independently of female gonadal hormones. Finally, POMC mRNA levels are increased in the ARC of saline-exposed, EB- or EB- and P-treated females but not in OVX females. Thus, the present study suggests that prenatal morphine exposure sex and brain region specifically alters the level of POMC and pENK mRNA.     

Neurotensin increases mortality and mast cells reduce neurotensin levels in a mouse model of sepsis

Sepsis is a complex, incompletely understood and often fatal disorder, typically accompanied by hypotension, that is considered to represent a dysregulated host response to infection. Neurotensin (NT) is a 13-amino-acid peptide that, among its multiple effects, induces hypotension. We find that intraperitoneal and plasma concentrations of NT are increased in mice after severe cecal ligation and puncture (CLP), a model of sepsis, and that mice treated with a pharmacological antagonist of NT, or NT-deficient mice, show reduced mortality during severe CLP. In mice, mast cells can degrade NT and reduce NT-induced hypotension and CLP-associated mortality, and optimal expression of these effects requires mast cell expression of neurotensin receptor 1 and neurolysin. These findings show that NT contributes to sepsis-related mortality in mice during severe CLP and that mast cells can lower NT concentrations, and suggest that mast cell-dependent reduction in NT levels contributes to the ability of mast cells to enhance survival after CLP.