Age-dependent memory impairment induced by co-exposure to nicotine and a synthetic cannabinoid in mice.

Co-use of marijuana and tobacco products is the second most common drug combination among adolescents. Nicotine (NIC) and cannabinoid use during adolescence induce similar detrimental changes, raising the hypothesis that simultaneous exposure could result in even more severe outcomes. Thus, we investigated whether the co-exposure to NIC and the synthetic cannabinoid WIN 55,212-2 (WIN) in adolescent mice causes behavioral outcomes different from those observed after exposure to a single drug. Male Swiss mice were exposed twice daily to NIC, WIN, or NIC + WIN during adolescence (PND28-47) or adulthood (PND70-89). Drug combination led to a greater reduction in weight gain in adolescent mice, while NIC-induced weight loss was observed in adults. During administration, NIC provoked hypothermia, and WIN produced hyperlocomotion in adolescent and adult mice. Animals exposed to NIC + WIN presented a profile of changes similar to those exposed to NIC. After drug exposure, changes in locomotion, thigmotaxis, social preference, prepulse inhibition, and working and recognition memory were evaluated. Adolescent but not adult mice exposed to NIC showed withdrawal-related hyperlocomotion unaffected by WIN co-administration. An age-specific impairment in object recognition memory was induced only by drug co-exposure during adolescence, which resolved spontaneously before reaching early adulthood. A transient decrease in hippocampal α7 nAChR subunit and CB1 receptor mRNA levels was induced by NIC exposure, which may be involved but is not enough to explain the memory impairment. Our work confirms the potential of NIC and cannabinoids association to aggravate some of the individual drug effects during critical neurodevelopmental periods.

NMDA Receptor Activation and Ca2+/PKC Signaling in Nicotine-Induced GABA Transport Shift in Embryonic Chick Retina.

Nicotinic receptors are present in the retina of different vertebrates, and in the chick retina, it is present during early development throughout to post-hatching. These receptors are activated by nicotine, an alkaloid with addictive and neurotransmitter release modulation properties, such as GABA signaling. Here we evaluated the mechanisms of nicotine signaling in the avian retina during the development of neuron-glia cells at a stage where synapses are peaking. Nicotine almost halved [3H]-GABA uptake, reducing it by 45% whilst increasing more than two-fold [3H]-GABA release in E12 embryonic chick retinas. Additionally, nicotine mediated a 33% increase in [3H]-D-aspartate release. MK-801 50 µM blocked 66% of nicotine-induced [3H]-GABA release and Gö 6983 100 nM prevented the nicotine-induced reduction in [3H]-GABA uptake by rescuing 40% of this neurotransmitter uptake, implicating NMDAR and PKC (respectively) in the nicotinic responses. In addition, NO-711 prevented [3H]-GABA uptake and release induced by nicotine. Furthermore, the relevance of calcium influx for PKC activation was evidenced through fura-2 imaging. We conclude that the shift of GABA transport mediated by nicotine promotes GABA release by inducing transporter reversal via nicotine-induced EAA release through EAATs, or by a direct effect of nicotine in activating nicotinic receptors permeable to calcium and promoting PKC pathway activation and shifting GAT-1 activity, both prompting calcium influx, and activation of the PKC pathway and shifting GAT-1 activity.

Design, synthesis and pharmacological evaluation of N-benzyl-piperidinyl-aryl-acylhydrazone derivatives as donepezil hybrids: Discovery of novel multi-target anti-alzheimer prototype drug candidates.

A new series of sixteen multifunctional N-benzyl-piperidine-aryl-acylhydrazones hybrid derivatives was synthesized and evaluated for multi-target activities related to Alzheimer's disease (AD). The molecular hybridization approach was based on the combination, in a single molecule, of the pharmacophoric N-benzyl-piperidine subunit of donepezil, the substituted hydroxy-piperidine fragment of the AChE inhibitor LASSBio-767, and an acylhydrazone linker, a privileged structure present in a number of synthetic aryl- and aryl-acylhydrazone derivatives with significant AChE and anti-inflammatory activities. Among them, compounds 4c, 4d, 4g and 4j presented the best AChE inhibitory activities, but only compounds 4c and 4g exhibited concurrent anti-inflammatory activity in vitro and in vivo, against amyloid beta oligomer (AßO) induced neuroinflammation. Compound 4c also showed the best in vitro and in vivo neuroprotective effects against AßO-induced neurodegeneration. In addition, compound 4c showed a similar binding mode to donepezil in both acetylated and free forms of AChE enzyme in molecular docking studies and did not show relevant toxic effects on in vitro and in vivo assays, with good predicted ADME parameters in silico. Overall, all these results highlighted compound 4c as a promising and innovative multi-target drug prototype candidate for AD treatment.

Design, synthesis, and evaluation of guanylhydrazones as potential inhibitors or reactivators of acetylcholinesterase.

Analogs of pralidoxime, which is a commercial antidote for intoxication from neurotoxic organophosphorus compounds, were designed, synthesized, characterized, and tested as potential inhibitors or reactivators of acetylcholinesterase (AChE) using the Ellman's test, nuclear magnetic resonance, and molecular modeling. These analogs include 1-methylpyridine-2-carboxaldehyde hydrazone, 1-methylpyridine-2-carboxaldehyde guanylhydrazone, and six other guanylhydrazones obtained from different benzaldehydes. The results indicate that all compounds are weak AChE reactivators but relatively good AChE inhibitors. The most effective AChE inhibitor discovered was the guanylhydrazone derived from 2,4-dinitrobenzaldehyde and was compared with tacrine, displaying similar activity to this reference material. These results indicate that guanylhydrazones as well as future similar derivatives may function as drugs for the treatment of Alzheimer's disease.

Waveform Similarity Analysis: A Simple Template Comparing Approach for Detecting and Quantifying Noisy Evoked Compound Action Potentials.

Experimental electrophysiological assessment of evoked responses from regenerating nerves is challenging due to the typical complex response of events dispersed over various latencies and poor signal-to-noise ratio. Our objective was to automate the detection of compound action potential events and derive their latencies and magnitudes using a simple cross-correlation template comparison approach. For this, we developed an algorithm called Waveform Similarity Analysis. To test the algorithm, challenging signals were generated in vivo by stimulating sural and sciatic nerves, whilst recording evoked potentials at the sciatic nerve and tibialis anterior muscle, respectively, in animals recovering from sciatic nerve transection. Our template for the algorithm was generated based on responses evoked from the intact side. We also simulated noisy signals and examined the output of the Waveform Similarity Analysis algorithm with imperfect templates. Signals were detected and quantified using Waveform Similarity Analysis, which was compared to event detection, latency and magnitude measurements of the same signals performed by a trained observer, a process we called Trained Eye Analysis. The Waveform Similarity Analysis algorithm could successfully detect and quantify simple or complex responses from nerve and muscle compound action potentials of intact or regenerated nerves. Incorrectly specifying the template outperformed Trained Eye Analysis for predicting signal amplitude, but produced consistent latency errors for the simulated signals examined. Compared to the trained eye, Waveform Similarity Analysis is automatic, objective, does not rely on the observer to identify and/or measure peaks, and can detect small clustered events even when signal-to-noise ratio is poor. Waveform Similarity Analysis provides a simple, reliable and convenient approach to quantify latencies and magnitudes of complex waveforms and therefore serves as a useful tool for studying evoked compound action potentials in neural regeneration studies.

Astrocyte transforming growth factor beta 1 promotes inhibitory synapse formation via CaM kinase II signaling.

The balance between excitatory and inhibitory synaptic inputs is critical for the control of brain function. Astrocytes play important role in the development and maintenance of neuronal circuitry. Whereas astrocytes-derived molecules involved in excitatory synapses are recognized, molecules and molecular mechanisms underlying astrocyte-induced inhibitory synapses remain unknown. Here, we identified transforming growth factor beta 1 (TGF-ß1), derived from human and murine astrocytes, as regulator of inhibitory synapse in vitro and in vivo. Conditioned media derived from human and murine astrocytes induce inhibitory synapse formation in cerebral cortex neurons, an event inhibited by pharmacologic and genetic manipulation of the TGF-ß pathway. TGF-ß1-induction of inhibitory synapse depends on glutamatergic activity and activation of CaM kinase II, which thus induces localization and cluster formation of the synaptic adhesion protein, Neuroligin 2, in inhibitory postsynaptic terminals. Additionally, intraventricular injection of TGF-ß1 enhanced inhibitory synapse number in the cerebral cortex. Our results identify TGF-ß1/CaMKII pathway as a novel molecular mechanism underlying astrocyte control of inhibitory synapse formation. We propose here that the balance between excitatory and inhibitory inputs might be provided by astrocyte signals, at least partly achieved via TGF-ß1 downstream pathways. Our work contributes to the understanding of the GABAergic synapse formation and may be of relevance to further the current knowledge on the mechanisms underlying the development of various neurological disorders, which commonly involve impairment of inhibitory synapse transmission.

Altered oxygen metabolism associated to neurogenesis of induced pluripotent stem cells derived from a schizophrenic patient.

Schizophrenia has been defined as a neurodevelopmental disease that causes changes in the process of thoughts, perceptions, and emotions, usually leading to a mental deterioration and affective blunting. Studies have shown altered cell respiration and oxidative stress response in schizophrenia; however, most of the knowledge has been acquired from postmortem brain analyses or from nonneural cells. Here we describe that neural cells, derived from induced pluripotent stem cells generated from skin fibroblasts of a schizophrenic patient, presented a twofold increase in extramitochondrial oxygen consumption as well as elevated levels of reactive oxygen species (ROS), when compared to controls. This difference in ROS levels was reverted by the mood stabilizer valproic acid. Our model shows evidence that metabolic changes occurring during neurogenesis are associated with schizophrenia, contributing to a better understanding of the development of the disease and highlighting potential targets for treatment and drug screening.

Estudo etnofarmacognóstico da saracuramirá (Ampelozizyphus amazonicus Ducke), uma planta medicinal usada por comunidades quilombolas do Município de Oriximiná-PA, Brasil / Ethnopharmacology Study of Saracuramirá (Ampelozizyphus amazonicus Ducke) in the "Quilombola" communities of Oriximiná, Pará State, Brazil

Um levantamento etnobotânico realizado em comunidades quilombolas de Oriximiná, Pará, destacou a saracuramirá (SAR), Ampelozizyphus amazonicus Ducke, com vasto uso popular no tratamento da malária, como tônica e depurativa. Por este motivo, o presente trabalho objetivou realizar um estudo etnofarmacognóstico da SAR nas respectivas comunidades. Por meio de uma análise etnobotânica quantitativa, foi verificado que SAR apresentou-se dentre as 10 espécies mais versáteis pela elevada importância relativa (1,3), dentre as cinco espécies com maior importância cultural pelo elevado índice de saliência (0,311) e a espécie com maior concordância de uso principal para malária (85,7 por cento). Uma análise do índice de espuma e do índice de hemólise para SAR demonstra a presença de saponinas com elevado índice de espuma (833) e uma baixa atividade hemolítica (CH50 2,6 mg mL-1). Para realizar uma análise das agliconas das saponinas de SAR, a bebida preparada pelo método tradicional quilombola (BMT) foi hidrolisada e, após reação com diazometano, foi analisada por cromatografia gasosa. Dois sinais majoritários foram caracterizados por espectrometria de massas, um referente a um triterpeno de esqueleto damarânico, característico das saponinas da SAR, e outro referente ao éster metílico do ácido betulínico. Partindo das informações de uso popular da SAR, foi avaliada in vitro a atividade inibidora da acetilcolinesterase. Apesar de BMT não ter mostrado atividade neste ensaio, é possível supor que as indicações de uso desta planta pelos quilombolas como fortificante e contra malária podem estar relacionadas a uma possível atividade adaptógena e imunoestimulante, dada à presença das saponinas e do ácido betulínico em BMT.

In an ethnobotanical survey conducted within "Quilombola" communities of Oriximiná, Pará State, "saracuramirá" (SAR), Ampelozizyphus amazonicus Ducke, stood out as one of the most cited species with wide popular use, especially for malaria, as tonic and depurative. The aim of this paper was to carry out an ethnopharmacognostic study of SAR in these communities. Quantitative ethnobotanical analysis of the data showed that SAR stands out as one of the 10 most versatile species with a high relative importance (1,3); it remains among the five species with the greatest cultural importance, demonstrated by the high salience index (0,311), being the species with the highest major use agreement to malaria (85,7 percent). Analysis of the foam and hemolysis indexes of SAR shows the presence of saponins with high foam index (833) and low hemolytic activity (HD50 2,6 mg mL-1). In order to analyze SAR saponin aglycones, the drink was prepared by the traditional quilombola method (BMT), was hydrolyzed, and analyzed by gas chromatography after reaction with diazomethane. Two major peaks were characterized by mass spectrometry, one referring to a dammarane triterpene skeleton, characteristic from the SAR saponins, and the other identified as the methyl ester of betulinic acid. Owing to the popular usage of SAR, its in vitro acetylcholinesterase inhibitory activity was evaluated, but with negative results. However, it is possible to suggest that the indications of this plant as a tonic and for treating malaria may be related to an adaptogen and immunostimulant effect due to the presence of saponins and betulinic acid in BMT.

Binding of [3H]MK-801 in subcellular fractions of Schistosoma mansoni: evidence for interaction with nicotinic receptors.

Several studies have suggested that l-glutamate is a putative neurotransmitter in Schistosoma mansoni. Recently, we detected the presence of low-affinity binding sites for [(3)H]kainic acid in the heterogeneous (P(1)) subcellular fraction of S. mansoni. In an attempt to characterize N-methyl-d-aspartate (NMDA) receptors in this worm, we performed binding assays with [(3)H]MK-801, a NMDA non-competitive antagonist, in the P(1) fraction of adult S. mansoni. In competition experiments, MK-801 (IC(50) approximately 200 microM) and ketamine (IC(50) approximately 500 microM) exhibited a low affinity for the sites labeled with [(3)H]MK-801. Along with the lack of modulation of this binding by glutamatergic agonists and antagonists and the absence of stereoselectivity for MK-801 isomers, these results suggest that [(3)H]MK-801 could label a site different from the classical NMDA receptor in S. mansoni. Based on the evidences that MK-801 interacts with mammalian muscle and central nervous system nicotinic receptors as a low-affinity noncompetitive antagonist, we have investigated the effects of MK-801 on the nicotine-induced flaccid paralysis of the worm, in vivo. The motility of S. mansoni was quantified by image analysis through a measure of displacement of the worm's extremities. In the presence of (-)-nicotine (10-100 microM), we observed an immediate paralysis of the worms, that was inhibited by 1mM MK-801. Besides nicotine, choline (10-50mM) was also able to inhibit the worm's motility. As a conclusion, we suggest that [(3)H]MK-801 binds to nicotinic receptors, and not NMDA receptors, in subcellular fractions of S. mansoni.