Pharmacodynamic and pharmacokinetic profiles of a neurotensin receptor type 2 (NTS2) analgesic macrocyclic analog.

The current opioid crisis highlights the urgent need to develop safe and effective pain medications. Thus, neurotensin (NT) compounds represent a promising approach, as the antinociceptive effects of NT are mediated by activation of the two G protein-coupled receptor subtypes (i.e., NTS1 and NTS2) and produce potent opioid-independent analgesia. Here, we describe the synthesis and pharmacodynamic and pharmacokinetic properties of the first constrained NTS2 macrocyclic NT(8-13) analog. The Tyr11 residue of NT(8-13) was replaced with a Trp residue to achieve NTS2 selectivity, and a rationally designed side-chain to side-chain macrocyclization reaction was applied between Lys8 and Trp11 to constrain the peptide in an active binding conformation and limit its recognition by proteolytic enzymes. The resulting macrocyclic peptide, CR-01-64, exhibited high-affinity for NTS2 (Ki 7.0 nM), with a more than 125-fold selectivity over NTS1, as well as an improved plasma stability profile (t1/2 > 24 h) compared with NT (t1/2 ~ 2 min). Following intrathecal administration, CR-01-64 exerted dose-dependent and long-lasting analgesic effects in acute (ED50 = 4.6 µg/kg) and tonic (ED50 = 7.1 µg/kg) pain models as well as strong mechanical anti-allodynic effects in the CFA-induced chronic inflammatory pain model. Of particular importance, this constrained NTS2 analog exerted potent nonopioid antinociceptive effects and potentiated opioid-induced analgesia when combined with morphine. At high doses, CR-01-64 did not cause hypothermia or ileum relaxation, although it did induce mild and short-term hypotension, all of which are physiological effects associated with NTS1 activation. Overall, these results demonstrate the strong therapeutic potential of NTS2-selective analogs for the management of pain.

Identification and in vivo characterization of a brain-penetrating nanobody.

BACKGROUND: Preclinical models to determine blood to brain transport ability of therapeutics are often ambiguous. In this study a method is developed that relies on CNS target-engagement and is able to rank brain-penetrating capacities. This method led to the discovery of an anti-transferrin receptor nanobody that is able to deliver a biologically active peptide to the brain via receptor-mediated transcytosis. METHODS: Various nanobodies against the mouse transferrin receptor were fused to neurotensin and injected peripherally in mice. Neurotensin is a neuropeptide that causes hypothermia when present in the brain but is unable to reach the brain from the periphery. Continuous body temperature measurements were used as a readout for brain penetration of nanobody-neurotensin fusions after its peripheral administration. Full temperature curves were analyzed using two-way ANOVA with Dunnett multiple comparisons tests. RESULTS: One anti-transferrin receptor nanobody coupled to neurotensin elicited a drop in body temperature following intravenous injection. Epitope binning indicated that this nanobody bound a distinct transferrin receptor epitope compared to the non-crossing nanobodies. This brain-penetrating nanobody was used to characterize the in vivo hypothermia model. The hypothermic effect caused by neurotensin is dose-dependent and could be used to directly compare peripheral administration routes and various nanobodies in terms of brain exposure. CONCLUSION: This method led to the discovery of an anti-transferrin receptor nanobody that can reach the brain via receptor-mediated transcytosis after peripheral administration. This method could be used to assess novel proteins for brain-penetrating capabilities using a target-engaging readout.

Optimization and 13CH3 methionine labeling of a signaling competent neurotensin receptor 1 variant for NMR studies.

Neurotensin is a 13-residue peptide that acts as a neuromodulator of classical neurotransmitters such as dopamine and glutamate in the mammalian central nervous system, mainly by activating the G protein-coupled receptor (GPCR), neurotensin receptor 1 (NTS1). Agonist binding to GPCRs shifts the conformational equilibrium of the transmembrane helices towards distinct, thermodynamically favorable conformations that favor effector protein interactions and promotes cell signaling. The introduction of site specific labels for NMR spectroscopy has proven useful for investigating this dynamic process, but the low expression levels and poor stability of GPCRs is a hindrance to solution NMR experiments. Several thermostabilized mutants of NTS1 have been engineered to circumvent this, with the crystal structures of four of these published. The conformational dynamics of NTS1 however, has not been thoroughly investigated with NMR. It is generally accepted that stabilized GPCRs exhibit attenuated signaling, thus we thoroughly characterized the signaling characteristics of several thermostabilized NTS1 variants to identify an optimal variant for protein NMR studies. A variant termed enNTS1 exhibited the best combination of signaling capability and stability upon solubilization with detergents. enNTS1 was subsequently labeled with 13CH3-methionine in E. coli and purified to homogeneity in the absence of bound ligands. Using solution NMR spectroscopy we observed several well dispersed 13CH3-methionine resonances, many of which exhibited chemical shift changes upon the addition of the high affinity agonist peptide, NT8-13. Thus, enNTS1 represents a novel tool for investigating ligand induced conformational changes in NTS1 to gain insights into the molecular mechanisms underlying neurotensin signaling.

Xenin-25 delays gastric emptying and reduces postprandial glucose levels in humans with and without type 2 diabetes.

Xenin-25 (Xen) is a neurotensin-related peptide secreted by a subset of glucose-dependent insulinotropic polypeptide (GIP)-producing enteroendocrine cells. In animals, Xen regulates gastrointestinal function and glucose homeostasis, typically by initiating neural relays. However, little is known about Xen action in humans. This study determines whether exogenously administered Xen modulates gastric emptying and/or insulin secretion rates (ISRs) following meal ingestion. Fasted subjects with normal (NGT) or impaired (IGT) glucose tolerance and Type 2 diabetes mellitus (T2DM; n = 10-14 per group) ingested a liquid mixed meal plus acetaminophen (ACM; to assess gastric emptying) at time zero. On separate occasions, a primed-constant intravenous infusion of vehicle or Xen at 4 (Lo-Xen) or 12 (Hi-Xen) pmol · kg(-1) · min(-1) was administered from zero until 300 min. Some subjects with NGT received 30- and 90-min Hi-Xen infusions. Plasma ACM, glucose, insulin, C-peptide, glucagon, Xen, GIP, and glucagon-like peptide-1 (GLP-1) levels were measured and ISRs calculated. Areas under the curves were compared for treatment effects. Infusion with Hi-Xen, but not Lo-Xen, similarly delayed gastric emptying and reduced postprandial glucose levels in all groups. Infusions for 90 or 300 min, but not 30 min, were equally effective. Hi-Xen reduced plasma GLP-1, but not GIP, levels without altering the insulin secretory response to glucose. Intense staining for Xen receptors was detected on PGP9.5-positive nerve fibers in the longitudinal muscle of the human stomach. Thus Xen reduces gastric emptying in humans with and without T2DM, probably via a neural relay. Moreover, endogenous GLP-1 may not be a major enhancer of insulin secretion in healthy humans under physiological conditions.

Reversal of dopamine D2 agonist-induced inhibition of ventral tegmental area neurons by Gq-linked neurotransmitters is dependent on protein kinase C, G protein-coupled receptor kinase, and dynamin.

Dopaminergic neurons of the ventral tegmental area are important components of brain pathways related to addiction. Prolonged exposure of these neurons to moderate concentrations of dopamine (DA) decreases their sensitivity to inhibition by DA, a process called DA-inhibition reversal (DIR). DIR is mediated by phospholipase C and conventional subtype of protein kinase C (cPKC) through concurrent stimulation of D2 and D1-like DA receptors, or by D2 stimulation concurrent with activation of 5-HT(2) or neurotensin receptors. In the present study, we further characterized this phenomenon by use of extracellular recordings in brain slices to examine whether DIR is linked to G protein-coupled receptor kinase-2 (GRK2) or dynamin by assessing DIR in the presence of antagonists of these enzymes. DIR was blocked by ß-ARK1 inhibitor, which inhibits GRK2, and by dynasore, which blocks dynamin. Reversal of inhibition by D2 agonist quinpirole was produced by serotonin (50 µM) and by neurotensin (5-10 nM). Serotonin-induced or neurotensin-induced reversal was blocked by ß-ARK1 inhibitor, dynasore, or cPKC antagonist 5,6,7,13-tetrahydro-13-methyl-5-oxo-12H-indolo[2,3-a]pyrrolo[3,4c]carbazole-12-propanenitrile (Gö6976). This further characterization of DIR indicates that cPKC, GRK2, and dynamin play important roles in the desensitization of D2 receptors. As drugs of abuse produce persistent increases in DA concentration in the ventral tegmental area, reduction of D2 receptor sensitivity as a result of drug abuse may be a critical factor in the processes of addiction.

Neurotensin inhibits glutamate-mediated synaptic inputs onto ventral tegmental area dopamine neurons through the release of the endocannabinoid 2-AG.

Neurotensin (NT), a neuropeptide abundant in the ventral midbrain, is known to act as a key regulator of the mesolimbic dopamine (DA) system, originating in the ventral tegmental area (VTA). NT activates metabotropic receptors coupled to Gq heterotrimeric G proteins, a signaling pathway often triggering endocannabinoid (EC) production in the brain. Because ECs act as negative regulators of many glutamate synapses and have also been shown recently to gate LTP induction in the VTA, we examined the hypothesis that NT regulates glutamate-mediated synaptic inputs to VTA DA neurons. We performed whole cell patch-clamp recordings in VTA DA neurons in TH-EGFP transgenic mouse brain slices and found that NT induces a long-lasting decrease of the EPSC amplitude that was mediated by the type 1 NT receptor. An antagonist of the CB1 EC receptor blocked this decrease. This effect of NT was not dependent on intracellular calcium, but required G-protein activation and phospholipase C. Blockade of the CB1 receptor after the induction of EPSC depression reversed synaptic depression, an effect not mimicked by blocking NT receptors, thus suggesting the occurrence of prolonged EC production and release. The EC responsible for synaptic depression was identified as 2-arachidonoylglycerol, the same EC known to gate LTP induction in VTA DA neurons. However, blocking NT receptors during LTP induction did not facilitate LTP induction, suggesting that endogenously released NT is not a major source of EC production during LTP inducing stimulations.

Different interactions of prolyl oligopeptidase and neurotensin in dopaminergic function of the rat nigrostriatal and mesolimbic pathways.

Prolyl oligopeptidase (PREP) is an intracellular enzyme digesting small proline-containing peptides. Since PREP resides the same brain areas as neurotensin in the nigrostriatal and mesolimbic dopaminergic pathways, we were interested to study if there is an intracellular interaction between them. A colocalization of PREP with neurotensin and neurotensin receptor 1 (NTS1) in the rat striatum, nucleus accumbens (NAcc), substantia nigra (SN) and ventral tegmental area (VTA) was studied with immunofluorescence. From the same brain areas, the levels of dopamine and its metabolites were measured 1 h after the injection of saline, NTS1 ligands (JMV-449; 5 µg) or antagonist (SR142948; 5 µg) to the rat striatum or NAcc. We also studied whether an intraperitoneal injection of a PREP inhibitor (KYP-2047; 5 mg/kg) affects the levels of dopamine and its metabolites alone or modifies the effects of the NTS1 ligands. PREP was highly colocalized with neurotensin and NTS1 in the VTA, and with NTS1 in the SN. Colocalization was moderate or low in other brain areas. When injected to the striatum, JMV-449 had a tendency to increase dopamine (p = 0.052) and metabolite levels in the striatum and SN, whereas SR142948 did not. After the injection to the NAcc, JMV-449 but not SR142948, increased dopamine levels in the VTA and dopamine metabolite levels in the NAcc and VTA. KYP-2047 decreased the dopamine levels in the striatum, but increased dopamine metabolite levels in the NAcc and VTA. Our results suggest a novel role for PREP in the modulation of dopaminergic transmission, which may be different in nigrostriatal and mesolimbic pathways.

Neuropeptide receptor ligands as drugs for psychiatric diseases: the end of the beginning?

The search for novel drugs for treating psychiatric disorders is driven by the growing medical need to improve on the effectiveness and side-effect profile of currently available therapies. Given the wealth of preclinical data supporting the role of neuropeptides in modulating behaviour, pharmaceutical companies have been attempting to target neuropeptide receptors for over two decades. However, clinical studies with synthetic neuropeptide ligands have been unable to confirm the promise predicted by studies in animal models. Here, we analyse preclinical and clinical results for neuropeptide receptor ligands that have been studied in clinical trials for psychiatric diseases, including agents that target the receptors for tachykinins, corticotropin-releasing factor, vasopressin and neurotensin, and suggest new ways to exploit the full potential of these candidate drugs.

Discovery of highly potent and neurotensin receptor 2 selective neurotensin mimetics.

The neurotensin receptor subtype 2 (NTS2) is involved in the modulation of tonic pain sensitivity and psychiatric diseases and is, therefore, regarded as a highly attractive pharmacological target protein. Aiming to discover NTS2 selective ligands, we herein describe the identification of screening hits and the chemical synthesis of structural variants leading to the highly potent and NTS2 selective peptide-peptoid hybrids of type 3. The neurotensin mimetics 3a and 3e-g incorporating an N-(4-hydroxyphenethyl)glycine substructure exhibit single digit nanomolar affinity (K(i) = 4.3-8.8 nM) and 1900-12000 fold selectivity over the neurotensin receptor subtype 1 (NTS1). According to functional experiments, the test compounds 3a and 3e-g displayed an inhibition of constitutive mitogen-activated protein kinase (MAPK) activity exceeding 2.6-4.6 times the inverse agonist activity of the endogenous ligand neurotensin.

Conotoxins: molecular and therapeutic targets.

Marine molluscs known as cone snails produce beautiful shells and a complex array of over 50,000 venom peptides evolved for prey capture and defence. Many of these peptides selectively modulate ion channels and transporters, making them a valuable source of new ligands for studying the role these targets play in normal and disease physiology. A number of conopeptides reduce pain in animal models, and several are now in pre-clinical and clinical development for the treatment of severe pain often associated with diseases such as cancer. Less than 1% of cone snail venom peptides are pharmacologically characterised.

Synthesis and biological effects of c(Lys-Lys-Pro-Tyr-Ile-Leu-Lys-Lys-Pro-Tyr-Ile-Leu) (JMV2012), a new analogue of neurotensin that crosses the blood-brain barrier.

The central administration of neurotensin (NT) or of its C-terminal hexapeptide fragment NT(8-13), produces strong analgesic effects in tests evaluating acute pain. The use of NT-derived peptides as pharmaceutical agents to relief severe pain in patients could be of great interest. Unfortunately, peptides do not readily penetrate the blood-brain barrier. We have observed that the cyclic NT(8-13) analogue, c(Lys-Lys-Pro-Tyr-Ile-Leu-Lys-Lys-Pro-Tyr-Ile-Leu) (JMV2012, compound 1), when peripherally administered to mice produced analgesic and hypothermic effects, suggesting the peptide penetrates the blood-brain barrier and functions effectively like a drug. Moreover, dimeric compounds show increased potency compared to their corresponding monomer. We present the synthesis of the cyclic dimer compound 1 (JMV2012). In mice, compound 1 induced a profound hypothermia and a potent analgesia, even when peripherally administered. Compound 1 appears to be an ideal lead compound for the development of bioactive NT analogues as novel analgesics drugs.

Neurotensin enhances GABAergic activity in rat hippocampus CA1 region by modulating L-type calcium channels.

Neurotensin (NT) is a tridecapeptide that interacts with three NT receptors; NTS1, NTS2, and NTS3. Although NT has been reported to modulate GABAergic activity in the brain, the underlying cellular and molecular mechanisms of NT are elusive. Here, we examined the effects of NT on GABAergic transmission and the involved cellular and signaling mechanisms of NT in the hippocampus. Application of NT dose-dependently increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) recorded from CA1 pyramidal neurons with no effects on the amplitude of sIPSCs. NT did not change either the frequency or the amplitude of miniature (m)IPSCs recorded in the presence of tetrodotoxin. Triple immunofluorescent staining of recorded interneurons demonstrated the expression of NTS1 on GABAergic interneurons. NT increased the action potential firing rate but decreased the afterhyperpolarization (AHP) amplitude in identified CA1 interneurons. Application of L-type calcium channel blockers (nimodipine and nifedipine) abolished NT-induced increases in action potential firing rate and sIPSC frequency and reduction in AHP amplitude, suggesting that the effects of NT are mediated by interaction with L-type Ca(2+) channels. NT-induced increase in sIPSC frequency was blocked by application of the specific NTS1 antagonist SR48692, the phospholipase C (PLC) inhibitor U73122, the IP(3) receptor antagonist 2-APB, and the protein kinase C inhibitor GF109203X, suggesting that NT increases gamma-aminobutyric acid release via a PLC pathway. Our results provide a cellular mechanism by which NT controls GABAergic neuronal activity in hippocampus.

Neurotensin receptor mechanisms and its modulation of glutamate transmission in the brain: relevance for neurodegenerative diseases and their treatment.

The extracellular accumulation of glutamate and the excessive activation of glutamate receptors, in particular N-methyl-D-aspartate (NMDA) receptors, have been postulated to contribute to the neuronal cell death associated with chronic neurodegenerative disorders such as Parkinson's disease. Findings are reviewed indicating that the tridecaptide neurotensin (NT) via activation of NT receptor subtype 1 (NTS1) promotes and reinforces endogenous glutamate signalling in discrete brain regions. The increase of striatal, nigral and cortical glutamate outflow by NT and the enhancement of NMDA receptor function by a NTS1/NMDA interaction that involves the activation of protein kinase C may favour the depolarization of NTS1 containing neurons and the entry of calcium. These results strengthen the hypothesis that NT may be involved in the amplification of glutamate-induced neurotoxicity in mesencephalic dopamine and cortical neurons. The mechanisms involved may include also antagonistic NTS1/D2 interactions in the cortico-striatal glutamate terminals and in the nigral DA cell bodies and dendrites as well as in the nigro-striatal DA terminals. The possible increase in NT levels in the basal ganglia under pathological conditions leading to the NTS1 enhancement of glutamate signalling may contribute to the neurodegeneration of the nigro-striatal dopaminergic neurons found in Parkinson's disease, especially in view of the high density of NTS1 receptors in these neurons. The use of selective NTS1 antagonists together with conventional drug treatments could provide a novel therapeutic approach for treatment of Parkinson's disease.

Receptor-receptor interactions as studied with microdialysis. Focus on NTR/D2 interactions in the basal ganglia.

Using mono and dualprobe(s) microdialysis in the basal ganglia of the freely moving rat evidence has been obtained that neurotensin (NT) in threshold concentrations can counteract the D(2) agonist (intrastriatally perfused) induced inhibition of striatal dopamine (DA) release and of pallidal GABA release from the striato-pallidal GABA pathway, effects that are blocked by a NTR(1) antagonist SR48692. These results indicate the existence of antagonistic intramembrane NTR/D(2) receptor interactions in the striatal DA terminals and in the somato-dendritic regions of the striato-pallidal GABA neurons. By the NT-induced reduction of the D(2) mediated signals at the striatal pre- and postjunctional level DA transmission is switched towards a D(1) mediated transmission leading to increased activity in the striatopallidal and striatonigral GABA pathways. The former action will contribute to the motor inhibition and catalepsy found with NT treatment and underlies the use of NT receptor antagonists as a treatment strategy for Parkinson's disease. Nigral NT by an antagonistic NTR/D(2) receptor interaction in the DA cell body and dendrites may also increase nigral DA release leading to a D(2) mediated inhibition of the nigrothalamic GABA pathway. Such an effect, will instead result in antiparkinsonian actions. Thus, increases in NT transmission will have different consequences for the motor system depending upon where in the basal ganglia the increase takes place.

Interactions between NTS2 neurotensin and opioid receptors on two nociceptive responses assessed on the hot plate test in mice.

The intracerebroventricular administration of the tridecapeptide neurotensin (NT) produces strong analgesic effects in tests evaluating acute pain. We investigated whether these effects are mediated by the opioid receptors. In the hot plate test, the NT receptors agonist NT1 (N(alpha)Me-Arg-Lys-Pro-Trp-Tle-Leu), s.c. injected (0.3-3 mg/kg), increased paw licking and jump latencies. These effects were inhibited by the NTS2 antagonist levocabastine (2.5 mg/kg, i.p.) but not by the selective NTS1 antagonist SR48692 (3 mg/kg, i.p.). The opioid receptor antagonist naloxone did not modify (up to the dose of 4.5 mg/kg, s.c.) the NT1 effect on licking, but abolished the increase in the jump latency (from the dose of 1.5 mg/kg). In mice made tolerant to the analgesic effect of morphine (2 mg/kg, s.c.) by previous morphine injections (32 mg/kg, s.c., twice a day, 4 days), NT1 maintained its effect on licking, but its effect on jump latency was suppressed. Levocabastine (up to the dose of 4.5 mg/kg) failed to antagonize the effects of morphine (2 mg/kg, s.c.) on both licking and jump latencies. In mice made tolerant to the analgesic effect of NT1 (0.3 mg/kg, s.c.) by previous NT1 injections (3 mg/kg, s.c., twice a day, 4 days) morphine maintained its analgesic effects both on licking and jumping latencies. We can conclude that neurotensinergic and opioidergic transmissions are functionally independent as regards the licking response. However, in the jump response, neurotensinergic transmission seems to regulate opioidergic transmission, inducing its stimulation.

Bioactive analogs of neurotensin: focus on CNS effects.

Neurotensin (NT) is a 13-amino acid neuropeptide found in the central nervous system and in the gastrointestinal tract. It is closely associated anatomically with dopaminergic and other neurotransmitter systems, and evidence supports a role for NT agonists in the treatment of various neuropsychiatric disorders. However, NT is readily degraded by peptidases, so there is much interest in the development of stable NT agonists, that can be injected systemically, cross the blood-brain barrier (BBB), yet retains the pharmacological characteristics of native NT for therapeutic use in the treatment of diseases such as schizophrenia, Parkinson's disease and addiction.

Potent spinal analgesia elicited through stimulation of NTS2 neurotensin receptors.

Intrathecal administration of the neuropeptide neurotensin (NT) was shown previously to exert antinociceptive effects in a variety of acute spinal pain paradigms including hotplate, tail-flick, and writhing tests. In the present study, we sought to determine whether some of these antinociceptive effects might be elicited via stimulation of low-affinity NTS2 receptors. We first established, using immunoblotting and immunohistochemical techniques, that NTS2 receptors were extensively associated with putative spinal nociceptive pathways, both at the level of the dorsal root ganglia and of the superficial layers of the dorsal horn of the spinal cord. We then examined the effects of intrathecal administration of NT or selective NTS2 agonists on acute thermal pain. Both NT and NTS2 agonists, levocabastine and Boc-Arg-Arg-Pro-Tyrpsi(CH2NH)Ile-Leu-OH (JMV-431), induced dose-dependent antinociceptive responses in the tail-flick test. The effects of levocabastine and of JMV-431 were unaffected by coadministration of the NTS1-specific antagonist 2-[(1-(7-chloro-4-quinolinyl)-5-(2,6-dimethoxy-phenyl)pyrazol-3-yl)carboxylamino]tricyclo)3.3.1.1.(3.7))-decan-2-carboxylic acid (SR48692), confirming that they were NTS2 mediated. In contrast, the antinociceptive effects of NT were partly abolished by coadministration of SR48692, indicating that NTS1 and NTS2 receptors were both involved. These results suggest that NTS2 receptors play a role in the regulation of spinal nociceptive inputs and that selective NTS2 agonists may offer new avenues for the treatment of acute pain.

Design, synthesis, and evaluation of the antipsychotic potential of orally bioavailable neurotensin (8-13) analogues containing non-natural arginine and lysine residues.

Neurotensin (NT) and its active fragment NT(8-13) elicit behavioral responses typical of clinically used antipsychotic drugs when administered directly to the brain. However, limited peptide stability and oral bioavailability have prevented these compounds from being developed as relevant pharmaceuticals. Recently, our laboratory designed and studied a first-generation NT(8-13) derivative, KK13, that elicited key pharmacokinetic and behavioral responses typical of clinically used antipsychotic drugs when administered to rats parenterally. This compound was the basis for the rational design of a series of second-generation NT(8-13) analogues (KH1-KH30) studied in this paper. Initial screening of these analogues for CNS activity by monitoring hypothermia induction after peripheral administration defined several compounds (KH11, KH24, KH26, and KH28-KH30) that warranted further investigation. Each compound maintained binding affinity for NTR(1), however, only KH24, KH26, and KH28 (as well as KK13) elicited significant hypothermic responses after oral administration. Of these, KH28 demonstrated an oral activity 3-fold greater than any other analogue; hence it was further characterized in a series of rat behavioral assays. KH28 attenuated d-amphetamine induced hyperlocomotion, a hallmark of current clinically effective antipsychotic drugs, after both IP and oral administration. In addition, tolerance to the compound did not develop after repeated daily dosing, as measured by hypothermic induction as well as attenuation of d-amphetamine induced hyperlocomotion. Finally, KH28 did not produce catalepsy, a deleterious side-effect elicited by classical antipsychotic drugs. KH28 is considered to be an ideal compound for further development as a potential novel antipsychotic.

Activation of central neurotensin receptors reinstates cocaine seeking in the rat: modulation by a D1/D5, but not D2/D3, receptor antagonist.

RATIONALE: Neurotensin (NT) has been implicated in some of the behavioral effects of psychostimulants. Thus, there is reason to think that NT may play a role in the reinstatement of cocaine seeking, and that it may do so via an interaction with dopamine (DA). OBJECTIVES: To assess (1) whether NT and an NT analog, D-TYR[11]NT, induce reinstatement of cocaine seeking; (2) whether the effects of NT receptor activation on reinstatement can be modulated by D1/D5 or D2/D3 antagonists; (3) the specificity of the effects of NT receptor activation on the reinstatement of cocaine seeking. METHODS: In Experiment 1, rats were initially trained to self-administer cocaine. Following a subsequent period of extinction training, they were tested for the reinstatement of cocaine seeking by NT or D-TYR[11]NT (15, 30 microg i.c.v.). In Experiment 2, rats were pretreated with the D1/D5 antagonist, SCH 23390 (0.05, 0.10 mg/kg i.p.) or the D2/D3 antagonist, raclopride (0.25, 0.50 mg/kg i.p.), prior to testing for reinstatement by D-TYR[11]NT (15 microg i.c.v.). In Experiment 3, rats that had been trained to self-administer sucrose pellets were tested for the reinstatement of sucrose seeking by D-TYR[11]NT (15, 30 microg i.c.v.). RESULTS: (1) Both NT and D-TYR[11]NT produced robust reinstatement of cocaine seeking; (2) the effect of the analog was attenuated by pretreatment with the D1/D5, but not D2/D3, receptor antagonist; (3) the analog did not induce the reinstatement of sucrose seeking. CONCLUSIONS: The findings suggest that an interaction between NT and DA may contribute to the neurobiology of reinstatement in animals with a history of cocaine self-administration.

Use of TH-EGFP transgenic mice as a source of identified dopaminergic neurons for physiological studies in postnatal cell culture.

The physiological and pharmacological properties of dopaminergic neurons in the brain are of major interest. Although much has been learned from cell culture studies, the physiological properties of these neurons remain difficult to study in such models because they are usually in minority and are difficult to distinguish from other non-dopaminergic neurons. Here we have taken advantage of a recently engineered transgenic mouse model expressing enhanced green fluorescence protein (EGFP) under the control of the tyrosine hydroxylase promoter to establish a more effective dopaminergic neuron cell culture model. We first evaluated the specificity of the EGFP expression. Although ectopic expression of EGFP was found in cultures derived from postnatal day 0 pups, this decreased over time in culture such that after 2 weeks, approximately 70% of EGFP-expressing neurons were dopaminergic. We next sought to validate this dopaminergic neuron culture model. We evaluated whether EGFP-expressing dopaminergic neurons displayed some of the well-established properties of dopaminergic neurons. Autoreceptor stimulation inhibited the activity of dopaminergic neurons while neurotensin receptor activation produced the opposite effect. Confocal imaging of the synaptic vesicle optical tracer FM4-64 in EGFP-expressing dopaminergic neurons demonstrated the feasibility of high resolution monitoring of the activity of single terminals established by these neurons. Together, this work provides evidence that primary cultures of postnatal TH-EGFP mice currently represent an excellent model to study the properties of these cells in culture.