Autonomic control network active in Aplysia during locomotion includes neurons that express splice variants of R15-neuropeptides

Splice-variant products of the R15 neuropeptide gene are differentially expressed within the CNS of Aplysia. The goal of this study was to test whether the neurons in the abdominal ganglion that express the peptides encoded by this gene are part of a common circuit. Expression of R15 peptides had been demonstrated previously in neuron R15. Using a combination of immunocytochemical and analytical methods, this study demonstrated that R15 peptides are also expressed in heart exciter neuron RB(HE), the two L9(G) gill motoneurons, and L40--a newly identified interneuron. Mass spectrometric profiling of individual neurons that exhibit R15 peptide-like immunoreactivity confirmed the mutually exclusive expression of two splice-variant forms of R15 peptides in different neurons. The L9(G) cells were found to co-express pedal peptide in addition to the R15 peptides. The R15 peptide-expressing neurons examined here were shown to be part of an autonomic control circuit that is active during fictive locomotion. Activity in this circuit contributes to implementing a central command that may help to coordinate autonomic activity with escape locomotion. Chronic extracellular nerve recording was used to determine the activity patterns of a subset of neurons of this circuit in vivo. These results demonstrate the potential utility of using shared patterns of neuropeptide expression as a guide for neural circuit identification.     

Demonstration of In Vitro Host-Guest Complex Formation and Safety of para-Sulfonatocalix[8]arene as a Delivery Vehicle for Two Antibiotic Drugs

The macrocycle para-sulfonatocalix[8]arene, sCX[8], was examined with 2 antibiotic drugs, ciprofloxacin (CIP) and isoniazid. The drugs were shown to form complexes with sCX[8] using proton nuclear magnetic resonance, thermogravimetric analysis, fluorescence spectroscopy, and molecular modeling. Both drugs form 1:1 hydrated (H₂O: 13%-14% w/w) host-guest complexes, with sCX[8] binding around the pyridine ring of isoniazid, and around the piperazine and cyclopropane rings of CIP. From proton nuclear magnetic resonance, the binding constant of isoniazid to sCX[8] was 6.8 (±0.3) × 10³ M^?1. Addition of 2 equivalents of sCX[8] to CIP resulted in a 58% decrease in fluorescence, and time-resolved fluorescence anisotropy of CIP doubles with sCX[8]. Each drug binds into the cavity of the macrocycle, with binding stabilized via combinations of hydrogen bonding, electrostatic interactions, π-π stacking, and hydrophobic effects. The safety of sCX[8] was examined in vitro with human embryonic kidney 293 cells. The IC₅₀ of sCX[8] was 559 μM, which is a minimum of 5-fold higher than the concentration that would be used in the clinic. The in vitro effect of sCX[8] on the action of CIP was examined on a panel of bacterial lines. The results showed that sCX[8] has no inherent antibiotic activity and had no negative effect on the action of CIP.     

Influence of translation efficiency of homologous viral proteins on the endogenous presentation of CD8+ T cell epitopes

A significant proportion of endogenously processed CD8(+) T cell epitopes are derived from newly synthesized proteins and rapidly degrading polypeptides (RDPs). It has been hypothesized that the generation of rapidly degrading polypeptides and CD8(+) T cell epitopes from these RDP precursors may be influenced by the efficiency of protein translation. Here we address this hypothesis by using the Epstein-Barr virus-encoded nuclear antigen 1 protein (EBNA1), with or without its internal glycine-alanine repeat sequence (EBNA1 and EBNA1DeltaGA, respectively), which display distinct differences in translation efficiency. We demonstrate that RDPs constitute a significant proportion of newly synthesized EBNA1 and EBNA1DeltaGA and that the levels of RDPs produced by each of these proteins directly correlate with the translation efficiency of either EBNA1 or EBNA1DeltaGA. As a consequence, a higher number of major histocompatibility complex-peptide complexes can be detected on the surface of cells expressing EBNA1DeltaGA, and these cells are more efficiently recognized by virus-specific cytotoxic T lymphocytes compared to the full-length EBNA1. More importantly, we also demonstrate that the endogenous processing of these CD8(+) T cell epitopes is predominantly determined by the rate at which the RDPs are generated rather than the intracellular turnover of these proteins.     

Receptor occupancy of nonpeptide corticotropin-releasing factor 1 antagonist DMP696: correlation with drug exposure and anxiolytic efficacy

4-(1,3-Dimethoxyprop-2-ylamine)-2,7-dimethyl-8-(2,4-dichlorophenyl)-pyrazolo[1,5-a]-1,3,5-triazine (DMP696) is a highly selective and potent, nonpeptide corticotropin-releasing factor 1 (CRF(1)) antagonist. In this study, we measured in vivo CRF(1) receptor occupancy of DMP696 by using ex vivo ligand binding and quantitative autoradiography and explored the relationship of receptor occupancy with plasma and brain exposure and behavioral efficacy. In vitro affinity (IC(50)) of DMP696 to brain CRF(1) receptors measured using the brain section binding autoradiography in this study is similar to that assessed using homogenized cell membrane assays previously. The ex vivo binding assay was validated by demonstrating that potential underestimation of receptor occupancy with this procedure could be minimized by identifying an appropriate in vitro incubation time (40 min) based upon the dissociation kinetics of DMP696. Orally administrated DMP696 dose dependently occupied CRF(1) receptors in the brain, with ~60% occupancy at 3 mg/kg. In the defensive withdrawal test of anxiety, this dose of DMP696 produced approximately 50% reduction in the exit latency. The time course of plasma and brain drug levels paralleled that of receptor occupancy, with peak exposure at 90 min after dosing. The plasma-free concentration of DMP696 corresponding to 50% CRF(1) receptor occupancy (in vivo IC(50), 1.22 nM) was similar to the in vitro IC(50) (~1.0 nM). Brain concentrations of DMP696 were over 150-fold higher than the plasma-free levels. In conclusion, doses of DMP696 occupying over 50% brain CRF(1) receptors are consistent with doses producing anxiolytic efficacy in the defense withdrawal test of anxiety, and the IC(50) value estimated in vivo based on plasma-free drug concentrations is consistent with the in vitro IC(50) value.     

A comparison of cannabidiolic acid with other treatments for anticipatory nausea using a rat model of contextually elicited conditioned gaping

Rationale

The effectiveness of cannabidiolic acid (CBDA) was compared with other potential treatments for anticipatory nausea (AN), using a rat model of contextually elicited conditioned gaping reactions.

Objective

The potential of ondansetron (OND), Δ⁹-tetrahydrocannabinol (THC), chlordiazepoxide (CDP), CBDA, and co-administration of CBDA and tetrahydrocannabinolic acid (THCA) to reduce AN and modify locomotor activity was evaluated.

Materials and methods

Following four pairings of a novel context with lithium chloride (LiCl), the rats were given a test for AN. On the test trial, they received pretreatment injections of the following: vehicle, OND (0.1 or 1.0 mg/kg), THC (0.5 mg/kg), CBDA (0.0001, 0.001, 0.01, 0.1 mg/kg or 1.0 mg/kg), CDP (1, 5, or 10 mg/kg) or co-administration of subthreshold doses of CBDA (0.1 μg/kg), and THCA (5 μg/kg). Immediately following the AN test trial in all experiments, rats were given a 15 min locomotor activity test. Finally, the potential of CBDA (0.001, 0.01, 0.1, and 1 mg/kg) to attenuate conditioned freezing to a shock-paired tone was assessed.

Results

THC, CBDA, and CDP, but not OND, reduced contextually elicited gaping reactions. Co-administration of subthreshold doses of CBDA and THCA also suppressed AN, and this effect was blocked by pretreatment with either a cannabinoid receptor 1 (CB₁) receptor antagonist or a 5-hydroxytryptamine 1A (5-HT_1A) receptor antagonist. CDP (but not CBDA, THC or CBDA and THCA) also suppressed locomotor activity at effective doses. CBDA did not modify the expression of conditioned fear.

Conclusions

CBDA has therapeutic potential as a highly potent and selective treatment for AN without psychoactive or locomotor effects.