Cre-expressing neurons in visual cortex of Ntsr1-Cre GN220 mice are corticothalamic and are depolarized by acetylcholine.

The Ntsr1-Cre GN220 mouse expresses Cre-recombinase in corticothalamic (CT) neurons in neocortical layer 6. It is not known if the other major types of pyramidal neurons in this layer also express this enzyme. By electrophysiological recordings in slices and histological analysis of the uptake of retrogradely transported beads we show that Cre-positive neurons are CT and not corticocortical or corticoclaustral types. Furthermore, we show that Ntsr1-Cre-positive cells are immuno-positive for the nuclear transcription factor Forkhead box protein P2 (FoxP2). We conclude that Cre-expression is limited to a specific type of pyramidal neuron: CT. However, it appears as not all CT neurons are Cre-expressing; there are indications that the penetrance of the gene is about 90%. We demonstrate the utility of assigning a specific identity to individual neurons by determining that the CT neurons are potently modulated by acetylcholine acting on both nicotinic and muscarinic acetylcholine receptors. These results corroborate the suggested function of these neurons in regulating the gain of thalamocortical transfer of sensory information depending on attentional demand and state of arousal.

Synthesis and Characterization in Vitro and in Vivo of (l)-(Trimethylsilyl)alanine Containing Neurotensin Analogues.

The silylated amino acid (l)-(trimethylsilyl)alanine (TMSAla) was incorporated at the C-terminal end of the minimal biologically active neurotensin (NT) fragment, leading to the synthesis of new hexapeptide NT[8-13] analogues. Here, we assessed the ability of these new silylated NT compounds to bind to NTS1 and NTS2 receptors, promote regulation of multiple signaling pathways, induce inhibition of the ileal smooth muscle contractions, and affect distinct physiological variables, including blood pressure and pain sensation. Among the C-terminal modified analogues, compound 6 (JMV2007) carrying a TMSAla residue in position 13 exhibits a higher affinity toward NT receptors than the NT native peptide. We also found that compound 6 is effective in reversing carbachol-induced contraction in the isolated strip preparation assay and at inducing a drop in blood pressure. Finally, compound 6 produces potent analgesia in experimental models of acute and persistent pain.

A corticothalamic switch: controlling the thalamus with dynamic synapses.

Corticothalamic neurons provide massive input to the thalamus. This top-down projection may allow the cortex to regulate sensory processing by modulating the excitability of thalamic cells. Layer 6 corticothalamic neurons monosynaptically excite thalamocortical cells, but also indirectly inhibit them by driving inhibitory cells of the thalamic reticular nucleus. Whether corticothalamic activity generally suppresses or excites the thalamus remains unclear. Here we show that the corticothalamic influence is dynamic, with the excitatory-inhibitory balance shifting in an activity-dependent fashion. During low-frequency activity, corticothalamic effects are mainly suppressive, whereas higher-frequency activity (even a short bout of gamma frequency oscillations) converts the corticothalamic influence to enhancement. The mechanism of this switching depends on distinct forms of short-term synaptic plasticity across multiple corticothalamic circuit components. Our results reveal an activity-dependent mechanism by which corticothalamic neurons can bidirectionally switch the excitability and sensory throughput of the thalamus, possibly to meet changing behavioral demands.

Layer 6 corticothalamic neurons activate a cortical output layer, layer 5a.

Layer 6 corticothalamic neurons are thought to modulate incoming sensory information via their intracortical axons targeting the major thalamorecipient layer of the neocortex, layer 4, and via their long-range feedback projections to primary sensory thalamic nuclei. However, anatomical reconstructions of individual layer 6 corticothalamic (L6 CT) neurons include examples with axonal processes ramifying within layer 5, and the relative input of the overall population of L6 CT neurons to layers 4 and 5 is not well understood. We compared the synaptic impact of L6 CT cells on neurons in layers 4 and 5. We found that the axons of L6 CT neurons densely ramified within layer 5a in both visual and somatosensory cortices of the mouse. Optogenetic activation of corticothalamic neurons generated large EPSPs in pyramidal neurons in layer 5a. In contrast, excitatory neurons in layer 4 exhibited weak excitation or disynaptic inhibition. Fast-spiking parvalbumin-positive cells in both layer 5a and layer 4 were also strongly activated by L6 CT neurons. The overall effect of L6 CT activation was to suppress layer 4 while eliciting action potentials in layer 5a pyramidal neurons. Together, our data indicate that L6 CT neurons strongly activate an output layer of the cortex.

Translaminar inhibitory cells recruited by layer 6 corticothalamic neurons suppress visual cortex.

In layer 6 (L6), a principal output layer of the mammalian cerebral cortex, a population of excitatory neurons defined by the NTSR1-Cre mouse line inhibit cortical responses to visual stimuli. Here we show that of the two major types of excitatory neurons existing in L6, the NTSR1-Cre line selectively targets those whose axons innervate both cortex and thalamus and not those whose axons remain within the cortex. These corticothalamic neurons mediate widespread inhibition across all cortical layers by recruiting fast-spiking inhibitory neurons whose cell body resides in deep cortical layers yet whose axons arborize throughout all layers. This study reveals a circuit by which L6 modulates cortical activity and identifies an inhibitory neuron able to regulate the strength of cortical responses throughout cortical depth.

The neuropeptide neuromedin U promotes autoantibody-mediated arthritis.

INTRODUCTION: Neuromedin U (NMU) is a neuropeptide with pro-inflammatory activity. The primary goal of this study was to determine if NMU promotes autoantibody-induced arthritis. Additional studies addressed the cellular source of NMU and sought to define the NMU receptor responsible for its pro-inflammatory effects. METHODS: Serum containing arthritogenic autoantibodies from K/BxN mice was used to induce arthritis in mice genetically lacking NMU. Parallel experiments examined whether NMU deficiency impacted the early mast-cell-dependent vascular leak response induced by these autoantibodies. Bone-marrow chimeric mice were generated to determine whether pro-inflammatory NMU is derived from hematopoietic cells or stromal cells. Mice lacking the known NMU receptors singly and in combination were used to determine susceptibility to serum-transferred arthritis and in vitro cellular responses to NMU. RESULTS: NMU-deficient mice developed less severe arthritis than control mice. Vascular leak was not affected by NMU deficiency. NMU expression by bone-marrow-derived cells mediated the pro-arthritogenic effect. Deficiency of all of the known NMU receptors, however, had no impact on arthritis severity and did not affect the ability of NMU to stimulate intracellular calcium flux. CONCLUSIONS: NMU-deficient mice are protected from developing autoantibody-induced inflammatory arthritis. NMU derived from hematopoietic cells, not neurons, promotes the development of autoantibody-induced inflammatory arthritis. This effect is mediated by a receptor other than the currently known NMU receptors.

Effects of neurotensin-2 receptor deletion on sensorimotor gating and locomotor activity.

Endogenous neurotensin (NT) has been implicated in brain processes relevant to schizophrenia as well as the therapeutic effects of antipsychotic drugs (APDs) used to treat this disorder. Converging evidence suggests that NT1 receptors mediate the antipsychotic-like effects of NT, such as prepulse inhibition (PPI) elevation. However, the role of NT2 receptors in these effects is not known. To investigate the contribution of NT2 receptors to the regulation of PPI, we measured baseline PPI and acoustic startle response (ASR), in male and female wild type (WT) and NT2 knockout (KO) mice. For comparison, we also measured locomotor activity. Baseline PPI was significantly elevated in both male (P<0.01) and female (P<0.01) NT2 KO compared to WT mice, while ASR was significantly decreased in KO mice of both genders (P<0.01). In contrast, female but not male KO mice exhibited significantly less baseline ambulations (P<0.05). These data support the regulation of baseline PPI, ASR and locomotor activity by endogenous NT acting at the NT2 receptor. Further studies investigating the role of NT2 receptors in the modulation of APD-like effects are warranted.

Impaired anorectic effect of leptin in neurotensin receptor 1-deficient mice.

Neurotensin plays a role in regulating feeding behavior. Central injection of neurotensin reduces food intake and the anorectic effect of neurotensin is mediated through neurotensin receptor 1 (Ntsr1). Ntsr1-deficient mice are characterized by mild hyperphagia and overweight without hyperleptinemia. The mechanism by which Ntsr1-deficient mice develop these metabolic abnormalities is not well understood. Leptin, secreted by adipocytes, regulates food intake by acting on hypothalamic neurons including neurotensin-producing neurons. Since the anorectic effect of leptin is blocked by neurotensin receptor antagonist, we hypothesized that the anorectic effect of leptin is mediated through Ntsr1 in the central nervous system and that decreased sensitivity to the anorectic effect of leptin contributes to metabolic perturbations in Ntsr1-deficient mice. To address this hypothesis, we examined the effect of intracerebroventricular (i.c.v.) administration of leptin on food intake in Ntsr1-deficient mice. A single i.c.v. injection of leptin caused robust reductions in food intake in wild-type mice. These effects were markedly attenuated in Ntsr1-deficient mice. These data are consistent with our hypothesis that the anorectic effect of leptin is at least partly mediated through central Ntsr1 and that the leptin-Ntsr1 signaling pathway is involved in the regulation of food intake. Our data also suggest that the lack of Ntsr1 reduces sensitivity to the anorectic action of leptin, causing hyperphagia and abnormal weight gain.

Genetic analysis of the hypothalamic neurotensin system.

This study used B x D recombinant inbred mice to detect and localize genes that control the hypothalamic neurotensin (NT) system. Abundance of transcripts that encode NT and NT receptors 1, 2, and 3 (NTR1, NTR2, and NTR3) in total hypothalamic RNA was the quantitative trait measured. Analysis of transcript abundance data revealed associations with quantitative trait loci (QTL) for NT transcript abundance (NTta) on chromosome 1, 3, 6, 7, 8, and 9; for NTR1ta on chromosome 3, 8, 12, and X; for NTR2ta on chromosome 2, 4, 9, 10, 12, 13, and 17; for NTR3ta on chromosome 1, 7, 11, and 12. NTta QTL on chromosomes 3, 7, and 8 coincide with QTL previously identified that impact NT peptide content and NTR2ta QTL on chromosome 2 and 12 coincide with genes previously associated with NTR2 receptor abundance. The NTta, NTR1ta, and NTR3ta QTL were not linked to their respective structural genes, but there is a highly significant (p<0.001) association for NTR2ta on chromosome 12 that includes the Ntsr2 structural gene. There are areas of potential shared genetic regulation between NTta and NTR3ta on chromosome 1 and 7 and for all three receptors on proximal chromosome 12. The NTta QTL on chromosome 9 includes the dopamine D2 receptor (Drd2) gene and QTL involved in responses to dopaminergic agents (Hts), antipsychotics (Hpic1) and cocaine (Cocrb8), and ethanol (Etohc3). These results further strengthen the hypothesis that the NT system is involved in mediating the actions of antipsychotic agents and drugs of abuse.

Miniaturization of whole live cell-based GPCR assays using microdispensing and detection systems.

Cell-based beta-lactamase reporter gene assays designed to measure the functional responses of G-protein-coupled receptors (GPCRs) were miniaturized to less than 2 microL total assay volume in a 3456-well microplate. Studies were done to evaluate both receptor agonists and antagonists. The pharmacology of agonists and antagonists for target GPCRs originally developed in a 96-well format was recapitulated in a 3456-well microplate format without compromising data quality or EC(50)/IC(50) precision. These assays were employed in high-throughput screening campaigns, allowing the testing of more than 150,000 compounds in 8 h. The instrumentation used and practical aspects of the assay development are discussed.

Neurotensin: peptide for the next millennium.

Neurotensin is an endogenous tridecapeptide neurotransmitter (pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-Pro-Try-Ile-Leu-OH) that was discovered by Carraway and Leeman in bovine hypothalami in the early 1970s. Since then this peptide has been the subject of a multitude of articles detailing discoveries related to its activity, receptors, localization, synthesis, and interactions with other systems. This review article does not intend to summarize again all the history of this fascinating peptide and its receptors, since this has been done quite well by others. The reader will be directed to these other reviews, where appropriate. Instead, this review attempts to provide a summary of current knowledge about neurotensin, why it is an important peptide to study, and where the field is heading. Special emphasis is placed on the behavioral studies, particularly with reference to agonists, antagonists, and antisense studies, as well as, the interaction of neurotensin with other neurotransmitters.

Neurotensin is an antagonist of the human neurotensin NT2 receptor expressed in Chinese hamster ovary cells.

The human levocabastine-sensitive neurotensin NT2 receptor was cloned from a cortex cDNA library and stably expressed in Chinese hamster ovary (CHO) cells in order to study its binding and signalling characteristics. The receptor binds neurotensin as well as several other ligands already described for neurotensin NT1 receptor. It also binds levocabastine, a histamine H1 receptor antagonist that is not recognised by neurotensin NT1 receptor. Neurotensin binding to recombinant neurotensin NT2 receptor expressed in CHO cells does not elicit a biological response as determined by second messenger measurements. Levocabastine, and the peptides neuromedin N and xenin were also ineffective on neurotensin NT2 receptor activation. Experiments with the neurotensin NT1 receptor antagonists SR48692 and SR142948A, resulted in the unanticipated discovery that both molecules are potent agonists on neurotensin NT2 receptor. Both compounds, following binding to neurotensin NT2 receptor, enhance inositol phosphates (IP) formation with a subsequent [Ca2+]i mobilisation; induce arachidonic acid release; and stimulate mitogen-activated protein kinase (MAPK) activity. Interestingly, these activities are antagonised by neurotensin and levocabastine in a concentration-dependent manner. These activities suggest that the human neurotensin NT2 receptor may be of physiological importance and that a natural agonist for the receptor may exist.

Purification of a rat neurotensin receptor expressed in Escherichia coli.

A truncated rat neurotensin receptor (NTR), expressed in Escherichia coli with the maltose-binding protein fused to its N-terminus and the 13 amino acid Bio tag fused to its C-terminus, was purified to apparent homogeneity in two steps by use of the monomeric avidin system followed by a novel neurotensin column. This purification protocol was developed by engineering a variety of affinity tags on to the C-terminus of NTR. Surprisingly, expression levels varied considerably depending on the C-terminal tag used. Functional expression of NTR was highest (800 receptors/cell) when thioredoxin was placed between the receptor C-terminus and the tag, indicating a stabilizing effect of the thioredoxin moiety. Several affinity chromatography methods were tested for purification. NTR with the in vivo-biotinylated Bio tag was purified with the highest efficiency compared with NTR with the Strep tag or a hexa-histidine tail. Co-expression of biotin ligase improved considerably the in vivo biotinylation of the Bio tag and, therefore, the overall purification yield. Proteolysis of the NTR fusion protein was prevented by removing a protease-sensitive site discovered at the N-terminus of NTR. The ligand binding properties of the purified receptor were similar to those of the membrane-bound protein and the native receptor. The scale-up of this purification scheme, to provide sufficient protein for biophysical studies, is in progress.

Molecular cloning of a levocabastine-sensitive neurotensin binding site.

A search for sequences homologous to the neurotensin receptor cDNA in a rat hypothalamic library has identified a novel neurotensin receptor (NTR-2). The 1539 bp cDNA encodes a 416 amino acid protein and shows highest homology to the previously cloned neurotensin receptor (NTR-1) (64% homology and 43% identity). Binding and pharmacological studies demonstrate that NTR-2 expressed in COS cells recognizes neurotensin (NT) with high affinity as well as several other agonists and antagonists. However, a fundamental difference was found; unlike NTR-1, NTR-2 recognizes, with high affinity, levocabastine, a histamine H1 receptor antagonist previously shown to compete with NT for low-affinity binding sites in brain.

The cloned neurotensin receptor mediates cyclic GMP formation when coexpressed with nitric oxide synthase cDNA.

Rat neurotensin (NT) receptor (NTR) cDNA was subcloned into the pRC-CMV expression vector and transfected into 293 cells, and cellular clones that stably expressed the NTR were isolated and characterized. [3H]NT binding to membranes prepared from the NTR cDNA-transfected cells displayed specificity and saturability, with an apparent Kd of 1.25 nM and a Bmax of 43.4 pmol/mg of protein (approximately 3.5 x 10(6) binding sites/cell). NT stimulated an increase in [3H]inositol phosphate levels in the NTR-expressing cells up to 2500% of basal levels. The response was time and dose dependent, with an EC50 of 10.4 nM. NT also stimulated cAMP formation in these cells, with an EC50 of 27.0 nM. In addition, NT evoked an increase in the level of intracellular calcium. Approximately 60% of the calcium rise was attributable to the release of intracellular stores and 40% was attributable to calcium influx. Although NTR occupancy has been shown to stimulate cGMP formation in several brain preparations and cell lines, NT was unable to mediate cGMP synthesis in the NTR-expressing 293 cells. We found that 293 cells have guanylate cyclase activity but have undetectable levels of nitric oxide synthase (NOS) activity. Because it was possible that the production of nitric oxide is required as the mediator of NT-induced cGMP synthesis, we subcloned NOS cDNA into the pCEP4 expression vector and transiently expressed it in the NTR cells. We report that NT increased cGMP levels up to 375% of basal levels when NOS cDNA was coexpressed and that the increase was completely inhibited by the NOS inhibitor N omega-nitro-L-arginine. NT-induced cGMP accumulation was time and dose dependent, with an EC50 of 1.7 nM. To our knowledge, this is the first report of NT mediating cGMP formation with a cloned receptor and the first evidence that NT-induced cGMP accumulation requires the production of nitric oxide.

Expression of a rat neurotensin receptor in Escherichia coli.

With the goal of obtaining sufficient quantities of seven-helix G-protein-coupled receptors for structural analysis, we have studied the functional expression of a rat neurotensin receptor cDNA in Escherichia coli with and without a signal sequence and as a fusion with the gene coding for maltose-binding protein. The addition of an N-terminal signal peptide resulted in increased expression levels. In vitro translation at a high level revealed that the codon usage of the rat neurotensin receptor cDNA was not critical for overproduction. Expression of neurotensin receptor cDNA fused to the 3' end of the gene encoding maltose-binding protein resulted in a 40-fold increase in neurotensin-binding sites. Binding of [3H]neurotensin to intact bacteria or E. coli membranes was saturable, with a dissociation constant, KD, of 0.23 nM (Bmax. = 450 sites/bacterium or 15 pmol/mg of crude membrane protein). The binding properties of all recombinant receptors presented in this study were similar and corresponded to those of the high-affinity binding sites in rat brain. For immunological detection and future purification of neurotensin receptor, a C-terminal pentahistidine/c-myc tail was introduced. Western-blot analysis revealed the association of neurotensin receptor with E. coli membranes.

Genetic-based differences in neurotensin levels and receptors in brains of LS x SS mice.

Levels of endogenous neurotensin (NT-IR) in the LS x SS RI strains differed by 3.0-, 4.7-, 5.4-, and 6.9-fold in the ventral midbrain (VMB), hypothalamus (HY), nucleus accumbens (NA), and caudate putamen (CP), respectively. Frequency distributions and estimates of the number of genes indicate that differences in NT-IR are polygenically influenced. The NT-IR levels in NA and CP were significantly correlated, but levels in the VMB did not correlate with those in the NA or CP. Specific binding to either low (NTL)- or high (NTH)-affinity receptors as measured in the absence or presence of levocabastine differed significantly in brain regions from among LS X SS mouse strains. Results indicate a polygenic influence mediating the differences in receptor densities and suggest differences in genetic regulation of NTL and NTH receptors.