Establishment of multi-stage intravenous self-administration paradigms in mice.

Genetically tractable animal models provide needed strategies to resolve the biological basis of drug addiction. Intravenous self-administration (IVSA) is the gold standard for modeling psychostimulant and opioid addiction in animals, but technical limitations have precluded the widespread use of IVSA in mice. Here, we describe IVSA paradigms for mice that capture the multi-stage nature of the disorder and permit predictive modeling. In these paradigms, C57BL/6J mice with long-standing indwelling jugular catheters engaged in cocaine- or remifentanil-associated lever responding that was fixed ratio-dependent, dose-dependent, extinguished by withholding the drug, and reinstated by the presentation of drug-paired cues. The application of multivariate analysis suggested that drug taking in both paradigms was a function of two latent variables we termed incentive motivation and discriminative control. Machine learning revealed that vulnerability to drug seeking and relapse were predicted by a mouse's a priori response to novelty, sensitivity to drug-induced locomotion, and drug-taking behavior. The application of these behavioral and statistical-analysis approaches to genetically-engineered mice will facilitate the identification of neural circuits driving addiction susceptibility and relapse and focused therapeutic development.

Integrating buccal and occlusal dental microwear with isotope analyses for a complete paleodietary reconstruction of Holocene populations from Hungary.

Dietary reconstruction is used to make inferences about the subsistence strategies of ancient human populations, but it may also serve as a proxy to characterise their diverse cultural and technological manifestations. Dental microwear and stable isotope analyses have been shown to be successful techniques for paleodietary reconstruction of ancient populations but, despite yielding complementary dietary information, these techniques have rarely been combined within the same study. Here we present for the first time a comprehensive approach to interpreting ancient lifeways through the results of buccal and occlusal microwear, and δ13C and δ15N isotope analyses applied to the same individuals of prehistoric populations of Hungary from the Middle Neolithic to the Late Bronze Age periods. This study aimed to (a) assess if the combination of techniques yields a more precise assessment of past dietary and subsistence practices, and (b) contribute to our understanding of the dietary patterns of the prehistoric Hungarian populations. Overall, no correlations between microwear and δ13C and δ15N isotope variables were observed, except for a relationship between nitrogen and the vertical and horizontal index. However, we found that diachronic differences are influenced by the variation within the period. Particularly, we found differences in microwear and isotope variables between Middle Neolithic sites, indicating that there were different dietary practices among those populations. Additionally, microwear results suggest no changes in the abrasiveness of the diet, neither food processing methods, despite higher C4 plant resource consumption shown by carbon isotopic signal. Thus, we demonstrate that the integration of dental microwear and carbon and nitrogen stable isotope methodologies can provide complementary information for making inferences about paleodietary habits.

ß-Arrestin-Biased Allosteric Modulator of NTSR1 Selectively Attenuates Addictive Behaviors.

Small molecule neurotensin receptor 1 (NTSR1) agonists have been pursued for more than 40 years as potential therapeutics for psychiatric disorders, including drug addiction. Clinical development of NTSR1 agonists has, however, been precluded by their severe side effects. NTSR1, a G protein-coupled receptor (GPCR), signals through the canonical activation of G proteins and engages ß-arrestins to mediate distinct cellular signaling events. Here, we characterize the allosteric NTSR1 modulator SBI-553. This small molecule not only acts as a ß-arrestin-biased agonist but also extends profound ß-arrestin bias to the endogenous ligand by selectively antagonizing G protein signaling. SBI-553 shows efficacy in animal models of psychostimulant abuse, including cocaine self-administration, without the side effects characteristic of balanced NTSR1 agonism. These findings indicate that NTSR1 G protein and ß-arrestin activation produce discrete and separable physiological effects, thus providing a strategy to develop safer GPCR-targeting therapeutics with more directed pharmacological action.

Synthesis and biological evaluation of novel N-substituted nipecotic acid derivatives with a cis-alkene spacer as GABA uptake inhibitors.

To discover new, potent, and selective inhibitors for the murine gamma-aminobutyric acid transporter 4 (mGAT4), the structure-activity relationship (SAR) study of a new cis-alkene analog family based on DDPM-1457 [(S)-2], which previously showed promising inhibitory potency at and subtype selectivity for mGAT4, was conducted. To uncover the importance of the differences between the trans- and the cis-alkene moiety in the spacer, the present publication describes the synthesis of the new compounds via catalytic hydrogenation with Lindlar's catalyst. The biological results collected by the SAR study revealed that analog rac-7j characterized by a four-instead of a three-carbon atom spacer with a cis double bond applying to the majority of the studied compounds displays a surprisingly high potency at mGAT1 (pIC50 = 6.00 ±â€¯0.04) and at the same time a reasonable potency at mGAT4 (pIC50 = 4.82).

Encoding the ß-Arrestin Trafficking Fate of Ghrelin Receptor GHSR1a: C-Tail-Independent Molecular Determinants in GPCRs.

G-protein-coupled receptors (GPCRs) can bias signaling through distinct biochemical pathways that originate from G-protein/receptor and ß-arrestin/receptor complexes. Receptor conformations supporting ß-arrestin engagement depend on multiple receptor determinants. Using ghrelin receptor GHR1a, we demonstrate by bioluminescence resonance energy transfer and fluorescence microscopy a critical role for its second intracellular loop 2 (ICL2) domain in stabilizing ß-arrestin/GHSR1a core interactions and determining receptor trafficking fate. We validate our findings in ICL2 gain- and loss-of-function experiments assessing ß-arrestin and ubiquitin-dependent internalization of the CC chemokine receptor, CCR1. Like all CC and CXC subfamily chemokine receptors, CCR1 lacks a critical proline residue found in the ICL2 consensus domain of rhodopsin-family GPCRs. Our study indicates that ICL2, C-tail determinants, and the orthosteric binding pocket that regulates ß-arrestin/receptor complex stability are sufficient to encode a broad repertoire of the trafficking fates observed for rhodopsin-family GPCRs, suggesting they provide the essential elements for regulating a large fraction of ß-arrestin signaling bias.

Synthesis and biological evaluation of novel N-substituted nipecotic acid derivatives with a trans-alkene spacer as potent GABA uptake inhibitors.

Our study presents the synthesis and structure-activity relationship (SAR) of novel N-substituted nipecotic acid derivatives closely related to DDPM-1457 [(S)-2a], a chemically stable analog of (S)-SNAP-5114 (1), in the pursuit of finding new and potent mGAT4 selective inhibitors. Iminium ion chemistry served as key step for the preparation of the desired, new N-substituted nipecotic acid derivatives containing a variety of different heterocycles attached to the nipecotic acid moiety via a trans-alkene spacer. The target compounds were characterized with regard to their potency at and subtype selectivity for the GABA transporters mGAT1-mGAT4.

Synthesis and biological evaluation of novel N-substituted nipecotic acid derivatives with an alkyne spacer as GABA uptake inhibitors.

In this study, we present the synthesis and structure-activity relationships (SAR) of novel N-substituted nipecotic acid derivatives closely related to (S)-SNAP-5114 (2) in the pursuit of finding new and potent mGAT4 selective inhibitors. By the use of iminium ion chemistry, a series of new N-substituted nipecotic acid derivatives containing a variety of heterocycles, and an alkyne spacer were synthesized. Biological evaluation of the prepared compounds showed, how the inhibitory potency and subtype selectivity for the murine GABA transporters (mGATs) were influenced by the performed modifications.

GIT1 regulates synaptic structural plasticity underlying learning.

The signaling scaffold protein GIT1 is expressed widely throughout the brain, but its function in vivo remains elusive. Mice lacking GIT1 have been proposed as a model for attention deficit-hyperactivity disorder, due to alterations in basal locomotor activity as well as paradoxical locomotor suppression by the psychostimulant amphetamine. Since we had previously shown that GIT1-knockout mice have normal locomotor activity, here we examined GIT1-deficient mice for ADHD-like behavior in more detail, and find neither hyperactivity nor amphetamine-induced locomotor suppression. Instead, GIT1-deficient mice exhibit profound learning and memory defects and reduced synaptic structural plasticity, consistent with an intellectual disability phenotype. We conclude that loss of GIT1 alone is insufficient to drive a robust ADHD phenotype in distinct strains of mice. In contrast, multiple learning and memory defects have been observed here and in other studies using distinct GIT1-knockout lines, consistent with a predominant intellectual disability phenotype related to altered synaptic structural plasticity.

Ghrelin receptor antagonism of hyperlocomotion in cocaine-sensitized mice requires ßarrestin-2.

The "brain-gut" peptide ghrelin, which mediates food-seeking behaviors, is recognized as a very strong endogenous modulator of dopamine (DA) signaling. Ghrelin binds the G protein-coupled receptor GHSR1a, and administration of ghrelin increases the rewarding properties of psychostimulants while ghrelin receptor antagonists decrease them. In addition, the GHSR1a signals through ßarrestin-2 to regulate actin/stress fiber rearrangement, suggesting ßarrestin-2 participation in the regulation of actin-mediated synaptic plasticity for addictive substances like cocaine. The effects of ghrelin receptor ligands on reward strongly suggest that modulation of ghrelin signaling could provide an effective strategy to ameliorate undesirable behaviors arising from addiction. To investigate this possibility, we tested the effects of ghrelin receptor antagonism in a cocaine behavioral sensitization paradigm using DA neuron-specific ßarrestin-2 KO mice. Our results show that these mice sensitize to cocaine as well as wild-type littermates. The ßarrestin-2 KO mice, however, no longer respond to the locomotor attenuating effects of the GHSR1a antagonist YIL781. The data presented here suggest that the separate stages of addictive behavior differ in their requirements for ßarrestin-2 and show that pharmacological inhibition of ßarrestin-2 function through GHSR1a antagonism is not equivalent to the loss of ßarrestin-2 function achieved by genetic ablation. These data support targeting GHSR1a signaling in addiction therapy but indicate that using signaling biased compounds that modulate ßarrestin-2 activity differentially from G protein activity may be required.

Investigation of the mechanical and chemical characteristics of nanotubular and nano-pitted anodic films on grade 2 titanium dental implant materials.

OBJECTIVE: The objective of this study was to investigate the reproducibility, mechanical integrity, surface characteristics and corrosion behavior of nanotubular (NT) titanium oxide arrays in comparison with a novel nano-pitted (NP) anodic film. METHODS: Surface treatment processes were developed to grow homogenous NT and NP anodic films on the surface of grade 2 titanium discs and dental implants. The effect of process parameters on the surface characteristics and reproducibility of the anodic films was investigated and optimized. The mechanical integrity of the NT and NP anodic films were investigated by scanning electron microscopy, surface roughness measurement, scratch resistance and screwing tests, while the chemical and physicochemical properties were investigated in corrosion tests, contact angle measurement and X-ray photoelectron spectroscopy (XPS). RESULTS AND DISCUSSION: The growth of NT anodic films was highly affected by process parameters, especially by temperature, and they were apt to corrosion and exfoliation. In contrast, the anodic growth of NP film showed high reproducibility even on the surface of 3-dimensional screw dental implants and they did not show signs of corrosion and exfoliation. The underlying reason of the difference in the tendency for exfoliation of the NT and NP anodic films is unclear; however the XPS analysis revealed fluorine dopants in a magnitude larger concentration on NT anodic film than on NP surface, which was identified as a possible causative. Concerning other surface characteristics that are supposed to affect the biological behavior of titanium implants, surface roughness values were found to be similar, whereas considerable differences were revealed in the wettability of the NT and NP anodic films. CONCLUSION: Our findings suggest that the applicability of NT anodic films on the surface of titanium bone implants may be limited because of mechanical considerations. In contrast, it is worth to consider the applicability of nano-pitted anodic films over nanotubular arrays for the enhancement of the biological properties of titanium implants.

Investigation of the in vitro photocatalytic antibacterial activity of nanocrystalline TiO2 and coupled TiO2/Ag containing copolymer on the surface of medical grade titanium.

Antibacterial surfaces have been in the focus of research for years, driven by an unmet clinical need to manage an increasing incidence of implant-associated infections. The use of silver has become a topic of interest because of its proven broad-spectrum antibacterial activity and track record as a coating agent of soft tissue implants and catheters. However, for the time being, the translation of these technological achievements for the improvement of the antibacterial property of hard tissue titanium (Ti) implants remains unsolved. In our study, we focused on the investigation of the photocatalysis mediated antibacterial activity of silver (Ag), and Ti nanoparticles instead of their pharmacological effects. We found that the photosensitisation of commercially pure titanium discs by coating them with an acrylate-based copolymer that embeds coupled Ag/Ti nanoparticles can initiate the photocatalytic decomposition of adsorbed S. salivarius after the irradiation with an ordinary visible light source. The clinical isolate of S. salivarius was characterised with MALDI-TOF mass spectrometer, while the multiplication of the bacteria on the surface of the discs was followed-up by MTT assay. Concerning practical relevance, the infected implant surfaces can be made accessible and irradiated by dental curing units with LED and plasma arc light sources, our research suggests that photocatalytic copolymer coating films may offer a promising solution for the improvement of the antibacterial properties of dental implants.

Microinjection of RFRP-1 in the central nucleus of amygdala decreases food intake in the rat.

Several members of the RFamide peptide family are known to have role in the regulation of feeding. For example, neuropeptide FF and prolactin-releasing peptide cause anorexigenic, while 26RFa and QRFP result in orexigenic effects in rodents. I.c.v. microinjection of neuropeptide RFRP-1 significantly reduced food and water intake in chicks. However, feeding related effects of RFRP-1 have not been studied in mammals yet. The central part of amygdala (CeA) is essentially involved in the regulation of feeding and body weight. RFRP-1 positive nerve cells were detected in the rat hypothalamus and RFRP-1 immunoreactive fibers were identified in the CeA. RFRP analogs bind with relatively high affinity to the NPFF1 and NPFF2 receptors (NPFF-R). RFRP-1 has potent activity for NPFF1. Significant expression of NPFF1 was detected in the CeA. To evaluate the role of RFRP-1 in feeding regulation rats were microinjected with different doses of RFRP-1 and their food intake were quantified over a 60min period. Liquid food intake of male Wistar rats was measured after bilateral intraamygdaloid administration of RFRP-1 (25, 50 or 100ng/side, RFRP-1 dissolved in 0.15M sterile NaCl/0.4µl, respectively). The 50ng dose of RFRP-1 microinjections resulted in significant decrease of food intake. The 25 and 100ng had no effect. Action of 50ng (37.8pmol) RFRP-1 was eliminated by 20ng (41.4pmol) RF9 NPFF-R antagonist pretreatment. In open-field test 50ng RFRP-1 did not modify spontaneous locomotor activity and general behavior of animals did not change. Our results are the first reporting that RFRP-1 injected to the CeA result in a decrease of liquid food consumption. This is a receptor-linked effect because it was eliminated by a NPFF-R selective antagonist.

The role of neurotensin in passive avoidance learning in the rat central nucleus of amygdala.

Tridecapeptide neurotensin (NT) acts as a neurotransmitter and/or neuromodulator and plays a role in learning and reinforcement. The central nucleus of amygdala (CeA), which is relatively rich in NT and neurotensin-1 receptors (NTS1), participates in the regulation of memory and learning mechanisms. The aim of this study was to examine the possible effect of NT and NTS1 antagonist (ANT) on passive avoidance learning after their microinjection into the CeA of male wistar rats. NT significantly increased the latency time. Effect of NT was blocked by ANT pretreatment. ANT in itself had no effect. Our results show that in the rat CeA NT facilitates passive avoidance learning via NTS1.

Effects of neurotensin in amygdaloid spatial learning mechanisms.

Neurotensin (NT) acts as a neurotransmitter and/or neuromodulator and plays a role in learning and reward related processes. The central nucleus of amygdala (CeA) participates in the regulation of memory and learning mechanisms. In Morris water maze test, rats were microinjected with NT or neurotensin receptor-1 (NTS1) antagonist SR 48692 (ANT). NT significantly reduced the escape latency. Effect of NT was blocked by ANT pretreatment. Our results show that in the rat CeA NT facilitates spatial learning. We clarified that NTS1s are involved in this action.

The role of neurotensin in positive reinforcement in the rat central nucleus of amygdala.

In the central nervous system neurotensin (NT) acts as a neurotransmitter and neuromodulator. It was shown that NT has positive reinforcing effects after its direct microinjection into the ventral tegmental area. The central nucleus of amygdala (CeA), part of the limbic system, plays an important role in learning, memory, regulation of feeding, anxiety and emotional behavior. By means of immunohistochemical and radioimmune methods it was shown that the amygdaloid body is relatively rich in NT immunoreactive elements and NT receptors. The aim of our study was to examine the possible effects of NT on reinforcement and anxiety in the CeA. In conditioned place preference test male Wistar rats were microinjected bilaterally with 100 or 250 ng NT in volume of 0.4 microl or 35 ng neurotensin receptor 1 (NTS1) antagonist SR 48692 alone, or NTS1 antagonist 15 min before 100 ng NT treatment. Hundred or 250 ng NT significantly increased the time rats spent in the treatment quadrant. Prior treatment with the non-peptide NTS1 antagonist blocked the effects of NT. Antagonist itself did not influence the reinforcing effect. In elevated plus maze test we did not find differences among the groups as far as the anxiety index (time spent on the open arms) was concerned. Our results suggest that in the rat ACE NT has positive reinforcing effects. We clarified that NTS1s are involved in this action. It was also shown that NT does not influence anxiety behavior.

Role of intraamygdaloid acylated-ghrelin in spatial learning.

According to recently published papers acylated-ghrelin (A-Ghr) modifies memory and learning. The basolateral nucleus of amygdala (ABL) participates in the regulation of memory and learning mechanisms. Previously we verified A-Ghr responsive neurons in the ABL by electrophysiological methods. In male Wistar rats effects of bilateral intraamygdaloid microinfusion of 50 ng, 100 ng A-Ghr, 15 ng Ghr receptor antagonist d-Lys3-GHRP-6 (ANT) or ANT+50 ng A-Ghr [dissolved in 0.15M sterile saline], or vehicle in 0.4 microl volume were investigated in Morris water maze paradigm. 50 ng A-Ghr significantly reduced latency to find the platform located in one of the quadrants of the maze. Effect of 50 ng A-Ghr was blocked by ANT pretreatment. ANT alone had no effect. Our results show that place learning linked memory processes are facilitated by A-Ghr in the rat ABL. It is a specific effect, because it could be eliminated by ANT pretreatment.

Intraamygdaloid microinjection of acylated-ghrelin influences passive avoidance learning.

The brain-gut peptide acylated-ghrelin (A-Ghr) is a potent growth hormone (GH) secretagogue substance. A-Ghr is also known to influence on memory and learning processes. Its effect is mediated partly via GH secretagogue receptor (GHS-R) type 1a. The amygdaloid body (AMY) plays important role in memory and learning processes. Projections of ghrelinergic neurons were identified in the AMY, and previously we verified that A-Ghr infused into basolateral nucleus of the AMY (ABL) caused liquid food intake decrease. The aim of the present study was to examine the possible effects of A-Ghr in the ABL on learning. Male Wistar rats were examined in two-compartment passive avoidance paradigm. Animals were shocked with 0.4mA current and subsequently were microinjected bilaterally with 50 or 100 ng A-Ghr, 30 ng GHS-R antagonist d-Lys3-GHRP-6 (ANT), ANT+50 ng A-Ghr (dissolved in 0.15M sterile NaCl/0.4 microl) or vehicle into the ABL. Fifty nanogram A-Ghr significantly increased the latency time, the 100 ng and the ANT alone were ineffective. The effect of 50 ng A-Ghr was eliminated by the ANT pretreatment. Our results suggest that intraamygdaloid A-Ghr enhances learning processes and memory in aversive situations, and this effect can specifically be prevented by ANT pretreatment.

Effects of intraamygdaloid microinjections of acylated-ghrelin on liquid food intake of rats.

Ghrelin (Ghr) has two main forms in the blood: the acylated (A-Ghr) and non-acylated (NA-Ghr) Ghr. A-Ghr was discovered as a potent growth hormone (GH) secretion increasing substance acting on GH secretagouge receptor (GHS-R) type 1a. A-Ghr facilitates food intake after its i.p., i.c.v. or direct hypothalamic application. Immunohistological assays identified projections of ghrelinergic neurons to the basolateral nucleus (ABL) of the amygdala (AMY). A-Ghr injected into the hypothalamus caused c-Fos overexpression in the AMY area that has an important role in food intake and body weight regulation. In separate experiments, liquid food intake of male wistar rats was measured after bilateral intraamygdalar or bilateral i.c.v. administration of A-Ghr (25, 50, 100, 250, and 500 ng/side or 500 and 1000 ng/side, A-Ghr dissolved in 0.15 M sterile NaCl/0.4 microl or 1 microl, respectively). In the ABL, A-Ghr microinjections in the 50-250 ng dose range resulted in significant decrease of food intake. The 25 and 500 ng had no effect. Action of 50 ng (14.83 pmol) or 100 ng (30.16 pmol) A-Ghr was eliminated by 15 ng (16.13 pmol) or 30 ng (32.25 pmol) GHS-R antagonist (D-Lys3-GHRP-6) pretreatment. The administration of 30 ng D-Lys3-GHRP-6 in itself had no influence on feeding. I.c.v. applied 1000 ng A-Ghr increased liquid food intake. Our results are the first ones reporting that A-Ghr injected into the ABL resulted in a decrease of liquid food consumption, within a limited dose range. This is a receptor-linked effect because it was eliminated by a GHS-R specific antagonist.

Neuromedin C microinjected into the amygdala inhibits feeding.

Bombesin-like peptides including gastrin releasing peptide and neuromedin C are known to inhibit feeding. Bombesin receptors have been found in moderate to high densities in the amygdaloid body, which is essentially involved in the regulation of feeding and body weight. In the present experiments neuromedin C (15, 30, and 60 ng), a carboxyterminal decapeptid fragment of gastrin releasing peptide, was bilaterally microinjected into the central part of the amygdala in ad libitum fed male CFY rats. Food intake was measured every 5 min for 30 min and also 6 min following neuromedin C or vehicle microinjections. Fifteen nanograms neuromedin C significantly suppressed liquid food consumption for 5 min and 30 ng for 25 min. However, 60 ng was not effective. Neuromedin C effects were blocked by prior application of the bombesin receptor antagonist [Leu(13)-psi(CH(2)NH)-Leu(14)]-bombesin. Neuromedin C treatment increased latency to feeding, decreased food intake, decreased the time spent feeding and their ratio, the number and the duration of feeding episodes during the first 5 min, without modifying body temperature or stereotype activity. Results indicate that neuromedin C may decrease the efficiency of feeding and that activation of bombesin receptors in the central amygdala may reduce appetite.