Neonatal alcohol exposure augments voluntary ethanol intake in the absence of potentiated anxiety-like behavior induced by chronic intermittent ethanol vapor exposure.

Individuals fetally exposed to alcohol have a disproportionate risk for developing lifetime alcohol dependence, an association that may be confounded by the presence of comorbid conditions, such as anxiety. Anxiety is also observed following fetal alcohol exposure and is known to exacerbate ethanol consumption, highlighting the utility of animal models to assess this relationship. The present study evaluated the impact of third-trimester equivalent ethanol exposure on ethanol consumption and anxiety-like, marble burying behavior in adult, male C57BL/6 mice following exposure to chronic intermittent ethanol vapor, proposed to model dependence. Neonatal mice (P5-6, 2.5-3.0 g) were administered one injection of saline or ethanol (2.5 g/kg, subcutaneously [s.c.]). Pre-vapor marble burying and limited-access two-bottle choice ethanol intake (15% v/v, 2 h) were comparable in adults (8 weeks of age) across neonatal treatment groups. Five consecutive drinking sessions were repeated 72 h after each weekly ethanol vapor exposure procedure for a total of five vapor/drinking cycles. Consistent with prior research, an increase in voluntary ethanol drinking was observed in vapor-exposed, neonatal saline-treated mice throughout the study starting after the second vapor cycle compared to both air-exposed control groups. In neonatal ethanol-treated mice, this increase in ethanol intake and preference following vapor exposure was accelerated, being observed after the first vapor cycle, and observed at an augmented level compared to vapor-exposed, neonatal saline-treated mice and air controls for both neonatal conditions. Conversely, marble burying was enhanced equivalently in vapor-exposed mice from either neonatal treatment group relative to their respective air-exposed controls. These data recapitulate clinical observations of enhanced sensitivity for alcohol dependence following developmental alcohol exposure, which may reflect enhanced motivational drive rather than potentiated negative affect. The present model will facilitate the future exploration of mechanisms that underlie increased risk for alcohol use after early developmental exposure.

Design of high affinity cyclic pentapeptide ligands for kappa-opioid receptors.

Using results from our previously reported cyclic opioid peptide series and reliable models for mu-, delta-, and kappa-opioid receptors (MOR, DOR, and KOR, respectively) and their complexes with peptide ligands, we have designed and synthesized a series of cyclic pentapeptides of structure Tyr-C[D-Cys-Phe-Phe-X]-NH2, cyclized via disulfide, methylene, or ethylene dithioethers, and where X = D- or L-Cys; or D- or L-penicillamine (Pen; beta,beta-dimethylcysteine). Determination of binding affinities to MOR, DOR, and KOR revealed that members of this series with X = D- or L-Cys display KOR affinities in the low nanomolar range, demonstrating that a 'DPDPE-like' tetrapeptide scaffold is suitable not only for DOR and MOR ligands, but also for KOR ligands. The cyclic pentapeptides reported here are not, however, selective for KOR, rather they display significant selectivity and high affinity for MOR. Indeed, peptide 8, Tyr-C[D-Cys-Phe-Phe-Cys]-NH2-cyclized via a methylene dithioether, shows picomolar binding affinity for MOR ( = 16 pm) with more than 100-fold selectivity for MOR vs. DOR or KOR, and may be of interest as a high affinity, high selectivity MOR ligand. Nonetheless, the high affinity KOR peptides in this series represent excellent leads for the development of structurally related, selective KOR ligands designed to exploit structurally specific features of KOR, MOR, and DOR.

Roles of residues 3 and 4 in cyclic tetrapeptide ligand recognition by the kappa-opioid receptor.

A series of cyclic, disulfide- or dithioether-containing tetrapeptides based on previously reported potent mu- and delta-selective analogs has been explored with the aim of improving their poor affinity to the kappa-opioid receptor. Specifically targeted were modifications of tetrapeptide residues 3 and 4, as they presumably interact with residues from transmembrane helices 6 and 7 and extracellular loop 3 that differ among the three receptors. Accordingly, tetrapeptides were synthesized with Phe(3) replaced by aliphatic (Gly, Ala, Aib, Cha), basic (Lys, Arg, homo-Arg), or aromatic sides chains (Trp, Tyr, p-NH(2)Phe), and with d-Pen(4) replaced by d-Cys(4), and binding affinities to stably expressed mu-, delta-, and kappa-receptors were determined. In general, the resulting analogs failed to exhibit appreciable affinity for the kappa-receptor, with the exception of the tetrapeptide Tyr-c[d-Cys-Phe-d-Cys]-NH(2), cyclized via a disulfide bond, which demonstrated high binding affinity toward all opioid receptors (Ki(mu) = 1.26 nm, Ki(delta) = 16.1 nm, Ki(kappa) = 38.7 nm). Modeling of the kappa-receptor/ligand complex in the active state reveals that the receptor-binding pocket for residues 3 and 4 of the tetrapeptide ligands is smaller than that in the mu-receptor and requires, for optimal fit, that the tripeptide cycle of the ligand assume a higher energy conformation. The magnitude of this energy penalty depends on the nature of the fourth residue of the peptide (d-Pen or d-Cys) and correlates well with the observed kappa-receptor binding affinity.