Department Of Neuroscience Research Articles

A study on optimal concentrations of isodiospyrin putative inhibitory actions against exonic splicing enhancers of Dystrophin gene exon 53 skipping in Duchenne Muscular Dystrophy

Event Abstract Back to Event A study on optimal concentrations of isodiospyrin putative inhibitory actions against exonic splicing enhancers of Dystrophin gene exon 53 skipping in Duchenne Muscular Dystrophy Hussain S. Alzahrani1*, Roslina Rashid2, Muzaimi Mustapha1 and Teguh H. Sasongko2 1 Universiti Sains Malaysia, Department of Neuroscience, School of Medical Sciences, Health Campus, Malaysia 2 Universiti Sains Malaysia, Human Genome Center, School of Medical Sciences, Health Campus, Malaysia Duchene muscular dystrophy (DMD) is an X-linked recessive disorder. It is characterized by rapid loss of muscular tissues due lacking gene of muscle replacement. The DMD gene is responsible for Dystrophin protein expression, which exists within a complex called Dystrophin glycol-protein complex (DGC). Exon mutations within DMD gene cause defective expression of Dystrophin. This study aimed to determine the inhibitory actions of Isodiospyrin targeting splicing factors and SR protein (Serine-arginine rich amino acids) a known topoisomerase inhibitor, which plays a critical role in splice site selection. previous studies demonstrated that Isodiospyrin has antitumor activity and inhibits topoisomerase enzyme from phosphorylating SF2/ASF splicing factor. In the current study we used plasmid of non-mutated exon 53 minigenes, transfected into non-mutated HEK-293 cell lines. Then, observed its actions on cells viability and exon splicing modification. The later was confirmed using RT-PCR, followed by exon 53 sequence software analysis. In consistence with previous studies, concentration of half maximal inhibitory effect (IC50) was 3.58µM, and the optimal inhibitory concentrations were: 1.79, 0.90, and 0.60µM. However, there was no detection of exon 53 skipping when exposing non-mutated HEK-293 cells to the optimal concentrations of Isodiospyrin compound. The results suggest that exon 53 splicing may occur without phosphorylation of SR proteins targeted by Isodiospyrin, which indicated that the splicing of exon 53 occurred independently of SR proteins targeted by the splicing inhibitor Isodiospyrin ,in non-mutated HEK-293 cells. Keywords: Gene Expression, inhibition, in vitro, Exon splicing, Duchenne muscular dystrophy Conference: 14th Meeting of the Asian-Pacific Society for Neurochemistry, Kuala Lumpur, Malaysia, 27 Aug - 30 Aug, 2016. Presentation Type: Poster Presentation Session Topic: 14th Meeting of the Asian-Pacific Society for Neurochemistry Citation: Alzahrani HS, Rashid R, Mustapha M and Sasongko TH (2016). A study on optimal concentrations of isodiospyrin putative inhibitory actions against exonic splicing enhancers of Dystrophin gene exon 53 skipping in Duchenne Muscular Dystrophy. Conference Abstract: 14th Meeting of the Asian-Pacific Society for Neurochemistry. doi: 10.3389/conf.fncel.2016.36.00120 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 04 Aug 2016; Published Online: 11 Aug 2016. * Correspondence: Mr. Hussain S Alzahrani, Universiti Sains Malaysia, Department of Neuroscience, School of Medical Sciences, Health Campus, Kota Bharu, Kelantan, 16150, Malaysia, hssz_23@hotmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Hussain S Alzahrani Roslina Rashid Muzaimi Mustapha Teguh H Sasongko Google Hussain S Alzahrani Roslina Rashid Muzaimi Mustapha Teguh H Sasongko Google Scholar Hussain S Alzahrani Roslina Rashid Muzaimi Mustapha Teguh H Sasongko PubMed Hussain S Alzahrani Roslina Rashid Muzaimi Mustapha Teguh H Sasongko Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Math abilities in Aging: relationship with Cognitive Reserve

Math abilities in Aging: relationship with Cognitive Reserve

Pragmatic Abilities in Patients with Traumatic Brain Injury

Pragmatic Abilities in Patients with Traumatic Brain Injury

Astrocytic structural changes correlate with overall running activities in the globus pallidus

Event Abstract Back to Event Astrocytic structural changes correlate with overall running activities in the globus pallidus Kouko Tatsumi1*, Hiroaki Okuda1, 2, Shoko M. Takemura1, Tatsuhide Tanaka1, Ayami Ishonishi1 and Akio Wanaka1 1 Nara Medical University, Department of Anatomy & Neuroscience, Faculty of Medicine, Japan 2 Kanazawa University, Department of Functional Anatomy, Graduate School of Medical Sciences, Japan Astrocytes actively modulate neurotransmission at tripartite synapses consisted of their fine processes and neuronal synapses. Morphological changes of astrocytes are primarily attributed to such processes. During lineage-tracing studies of cells expressing Olig2, we found that Olig2-lineage mature astrocytes preferentially cluster in the globus pallidus that exerts pivotal functions in the indirect pathway of the basal ganglionic circuit. Taking advantage of membrane-targeted GAP43-EGFP, which can visualize the morphology of Olig2-lineage cells in detail, we investigated how running influenced Olig2-lineage astrocyte morphology in the globus pallidus at both the light and electron microscopic levels. Runner group mice were allowed free access to a running wheel for three weeks, and Runner-Rest group mice took an enforced three-week rest after the three-week running period. GFP immunofluorescence analyses and immunoelectron microscopy revealed that astrocytic fine processes elaborated complex arborization in the Runner mice, and reverted to simple morphology in the Runner-Rest group, which is comparable to that in the Control group. The Olig2-lineage astrocytes expressed the GABA transporter GAT-3, suggesting that they participated in inhibitory GABA transmission in the globus pallidus. The fine processes of the Olig2-lineage astrocytes underwent plastic changes that correlated with overall running activities, suggesting that they actively modulate motor functions. Keywords: Astrocytes, Globus Pallidus, Motor function, in vivo, plastic changes Conference: 14th Meeting of the Asian-Pacific Society for Neurochemistry, Kuala Lumpur, Malaysia, 27 Aug - 30 Aug, 2016. Presentation Type: Poster Presentation Session Topic: 14th Meeting of the Asian-Pacific Society for Neurochemistry Citation: Tatsumi K, Okuda H, Takemura SM, Tanaka T, Ishonishi A and Wanaka A (2016). Astrocytic structural changes correlate with overall running activities in the globus pallidus. Conference Abstract: 14th Meeting of the Asian-Pacific Society for Neurochemistry. doi: 10.3389/conf.fncel.2016.36.00201 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 04 Aug 2016; Published Online: 11 Aug 2016. * Correspondence: Prof. Kouko Tatsumi, Nara Medical University, Department of Anatomy & Neuroscience, Faculty of Medicine, Kashihara, Nara, Japan, radha815@naramed-u.ac.jp Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Kouko Tatsumi Hiroaki Okuda Shoko M Takemura Tatsuhide Tanaka Ayami Ishonishi Akio Wanaka Google Kouko Tatsumi Hiroaki Okuda Shoko M Takemura Tatsuhide Tanaka Ayami Ishonishi Akio Wanaka Google Scholar Kouko Tatsumi Hiroaki Okuda Shoko M Takemura Tatsuhide Tanaka Ayami Ishonishi Akio Wanaka PubMed Kouko Tatsumi Hiroaki Okuda Shoko M Takemura Tatsuhide Tanaka Ayami Ishonishi Akio Wanaka Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Relating extrinsic connections to the intrinsic architecture of the cerebral cortex

Event Abstract Back to Event Relating extrinsic connections to the intrinsic architecture of the cerebral cortex Claus C. Hilgetag1, 2* 1 University Medical Center Hamburg-Eppendorf, Department of Computational Neuroscience, Germany 2 Boston University, Department of Health Sciences, United States The nerve fibre projections that connect different areas of the cortical sheet constitute a complex structural network for neural communication in the brain. How is the organization of this cortical connectome related to other features of brain architecture? We addressed the question by systematically investigating the relation of structural parameters of cortical areas, such as their distance, similarity in cortical cytoarchitecture as described by cortical lamination or neuronal density, as well as similarity of cortical thickness, to essential features of long-range cortico-cortical connections, such as their presence or absence as well as patterns of laminar projection origins and terminations. Across different species (primate, cat, mouse) and different cortical lobes, the essential features of cortico-cortical connections varied consistently and strongly with the cytoarchitectonic similarity of cortical areas. By contrast, such relations were not found consistently for distance or similarity of cortical thickness. As gradients in cortical cytoarchitecture also determine the specific organisation of the local micro-circuits of different areas, our findings illuminate general principles of neural wiring, linking the organization of long-range connections to intrinsic circuits of the cerebral cortex. Keywords: connectomes, Cerebral Cortex, Cytoarchitecture, connectivity, human brain, monkey, Cats, Mouse Conference: Neuroinformatics 2016, Reading, United Kingdom, 3 Sep - 4 Sep, 2016. Presentation Type: Investigator presentations Topic: General neuroinformatics Citation: Hilgetag CC (2016). Relating extrinsic connections to the intrinsic architecture of the cerebral cortex. Front. Neuroinform. Conference Abstract: Neuroinformatics 2016. doi: 10.3389/conf.fninf.2016.20.00017 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 27 Apr 2016; Published Online: 18 Jul 2016. * Correspondence: Dr. Claus C Hilgetag, University Medical Center Hamburg-Eppendorf, Department of Computational Neuroscience, Hamburg, D-20246, Germany, c.hilgetag@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Claus C Hilgetag Google Claus C Hilgetag Google Scholar Claus C Hilgetag PubMed Claus C Hilgetag Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Myelinated 3D neural culture platform as a physiological model of diabetic peripheral neuropathy

Event Abstract Back to Event Myelinated 3D neural culture platform as a physiological model of diabetic peripheral neuropathy Ashwin Sivakumar1, Reed Gioe1 and Michael Moore1, 2 1 Tulane University, Department of Biomedical Engineering, United States 2 Tulane University, Department of Neuroscience, United States Introduction: We present a microengineered 3D hydrogel platform for culture of myelinated embryonic peripheral neural tissue as shown in Figure 1. This novel in-vitro platform enables morphological and physiological metrics analogous to clinical nerve fiber density and nerve conduction tests. We demonstrate the feasibility of this model system for studying peripheral neuropathy caused by glucose-induced neurotoxicity, the most common form of neuropathy. Figure 1: Microengineered myelinated neural fiber tract 3D architecture resembling native peripheral nerve anatomy. Materials and Methods: Dorsal root ganglia (DRG) from embryonic day 15 rats were grown over a period of 4 weeks in a dual hydrogel system consisting of a methacrylated heparin gel in a polyethylene glycol micro-mold. The dual hydrogel system served to constrain the axon growth to be polarized with high density, fasciculation and myelination. The DRG’s were subject to a three-stage media regimen including ascorbic acid (AA) in the final stage to induce Schwann Cell myelination. The myelinated cultures were subjected to 60mM concentrations of the D-glucose in media over a period of 48 hours to simulate diabetic neuropathy. The control cultures were exposed to a 25mM D-glucose concentration. Electrophysiological field recordings were taken with and without the exposure of high glucose, and changes in the compound action potentials in the field recording were observed and compared to the control. Results and Discussion: Compound action potentials show little or no synaptic input, increased delay and decreased amplitude with higher glucose concentration exposure as shown in Figure 2. The higher glucose concentration is thought to trigger the sorbitol pathway and induce oxidative stress in the neurons of the DRG, leading to apoptosis and axonal degeneration. Figure 2: Average traces representing compound action potentials recorded with and without high glucose treatment. The change in amplitude and latency are also quantified as bar plots. (n=10, * = p<0.05 and ***=p<0.001) Conclusion: Microengineered myelinated neural fiber tracts have morphology, physiology and, toxicity response analogous to a peripheral nerve. The platform could be used as an in-vitro model for neuropathy studies and drug development. Funding provided by Tulane University

Electrical stimulation rescues dopaminergic degeneration in dorsal raphe nucleus and enhanced hippocampal neurogenesis of vulnerable depressive rats

Event Abstract Back to Event Electrical stimulation rescues dopaminergic degeneration in dorsal raphe nucleus and enhanced hippocampal neurogenesis of vulnerable depressive rats Sharafuddin Khairuddin1*, Wei Ling Lim2, William Wai Lun Fung1, Yasin Temel3 and Lee Wei Lim1 1 The University of Hong Kong, School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, Hong Kong (SAR) 2 Sunway University, School of Biological Sciences, Malaysia 3 Maastricht University, Departments of Neuroscience and Neurosurgery, Netherlands Electrical stimulation is a potential treatment for patients with depression. In this study, we investigate the effects of high-frequency stimulation (HFS) on various depressive-like behaviours using the stress resilience and vulnerable rat depression models. Our results demonstrated that animals with 3 weeks chronic unpredictable stress (CUS) induced exhibition of reduced sucrose consumption in 51% of animals, thus separating the resilience and vulnerable group of CUS-induced model. CUS vulnerable sham animals demonstrated anxiety-like behaviour, decreased motivation and increased immobility compared to that of the resilience group, implicating high susceptibility of vulnerable individuals to the CUS procedure. Interestingly, vmPFC HFS significantly reduced anxiety response, increased hedonia and motivation levels for food intake in the vulnerable group compared to the resilience group. HFS in ventromedial prefrontal cortex (vmPFC) and lateral habenula also showed reduced behavioural despair in both CUS vulnerable and resilience groups. In histochemistry, our results demonstrate that vmPFC HFS rescued the stress-induced dopamine neuron degeneration in the dorsal raphe nucleus, as well as increased hippocampal neurogenesis in stress vulnerable animals. In conclusion, these results suggest that vmPFC HFS effectively restores depressive-like behaviours by mechanisms of dorsal raphe dopaminergic neurons restoration and enhanced hippocampal neuroplasticity in the vulnerable CUS-induced model. Keywords: Dopamine, Hippocampus, Neurogenesis, dorsal raphe nucleus, in vivo Conference: 14th Meeting of the Asian-Pacific Society for Neurochemistry, Kuala Lumpur, Malaysia, 27 Aug - 30 Aug, 2016. Presentation Type: O01: Postgraduate Travel Awardees Oral Session 1 Topic: 14th Meeting of the Asian-Pacific Society for Neurochemistry Citation: Khairuddin S, Lim W, Fung W, Temel Y and Lim L (2016). Electrical stimulation rescues dopaminergic degeneration in dorsal raphe nucleus and enhanced hippocampal neurogenesis of vulnerable depressive rats. Conference Abstract: 14th Meeting of the Asian-Pacific Society for Neurochemistry. doi: 10.3389/conf.fncel.2016.36.00078 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 04 Aug 2016; Published Online: 11 Aug 2016. * Correspondence: Dr. Sharafuddin Khairuddin, The University of Hong Kong, School of Biomedical Sciences, Li Ka Shing Faculty of Medicine, Hong Kong, Hong Kong, Hong Kong (SAR), sharafkdin@gmail.com Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Sharafuddin Khairuddin Wei Ling Lim William Wai Lun Fung Yasin Temel Lee Wei Lim Google Sharafuddin Khairuddin Wei Ling Lim William Wai Lun Fung Yasin Temel Lee Wei Lim Google Scholar Sharafuddin Khairuddin Wei Ling Lim William Wai Lun Fung Yasin Temel Lee Wei Lim PubMed Sharafuddin Khairuddin Wei Ling Lim William Wai Lun Fung Yasin Temel Lee Wei Lim Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

A single Markov-type kinetic model reliably accounts for the macroscopic currents of all human voltage-gated sodium channel isoforms

A single Markov-type kinetic model reliably accounts for the macroscopic currents of all human voltage-gated sodium channel isoforms

Functional anatomy of cortical areas characterized by Von Economo neurons

Von Economo’s neurons (VENs) are large, bipolar or corkscrew-shaped neurons located in layers III and V of the frontoinsular and the anterior cingulate cortices. VENs are reported to be altered in pathologies such as frontotemporal dementia and autism, in which the individual’s self control is seriously compromised. We have recently reviewed the evolutionary appearance of VENs and we are currently studying their distribution in different neurodegenerative diseases. To investigate the role of VENs in the active human brain, we have explored the functional connectivity of brain areas containing VENs by analyzing resting state functional connectivity (rsFC) in 20 healthy volunteers. Our results show that cortical areas containing VENs form a network of frontoparietal functional connectivity. With the use of fuzzy clustering techniques, we find that this network comprises four sub-networks: the first network cluster resembles a “saliency detection” attentional network, which includes superior frontal cortex (Brodmann’s Area, BA 10), inferior parietal lobe, anterior insula, and dorsal anterior cingulate cortex; the second cluster, part of a “sensory-motor network”, comprises the superior temporal, precentral and postcentral areas; the third cluster consists of frontal ventromedial and ventrodorsal areas constituted by parts of the “anterior default mode network”; and the fourth cluster encompasses dorsal anterior cingulate cortex, dorsomedial prefrontal, and superior frontal (BA 10) areas, resembling the anterior part of the “dorsal attentional network”. Thus, the network that emerges from analyzing functional connectivity among areas that are known to contain VENs is primarily involved in functions of saliency detection and self-regulation. In addition, parts of this network constitute sub-networks that partially overlap with the default mode, the sensory-motor and the dorsal attentional networks.This work was supported by grants from PROGETTO TRASLAZIONALE, Department of Neuroscience, University of Torino.

Neuroscience Gateway � Cyberinfrastructure Providing Supercomputing Resources for Large Scale Computational Neuroscience Research

Neuroscience Gateway � Cyberinfrastructure Providing Supercomputing Resources for Large Scale Computational Neuroscience Research

The Urgent Need of New Mood-Stabilizers: The Promising Results of Memantine

Giulia Serra1,2, Francesca Demontis3 and Gino Serra4* 1Clinical and Research fellow, Child Neuropsychiatry, Department of Neuroscience, I.R.C.C.S. Children Hospital Bambino Gesu, Rome, Italy. 2NESMOS Department, Sant'Andrea Hospital, Sapienza University, Rome Italy 3International Consortium for Bipolar Disorder Research, McLean Hospital, Belmont (MA), USA 4Dipartimento di Scienze Biomediche, University of Sassari, Italy

Restorative Effects of the TrkB agonist 7,8-Dihydroxyflavone on Neuronal Network Activity in an in vitro model of Huntington's disease

Event Abstract Back to Event Restorative Effects of the TrkB agonist 7,8-Dihydroxyflavone on Neuronal Network Activity in an in vitro model of Huntington's disease Alberto Capurro1*, Pablo Baliño1, Candise Baez1, Ayaka Sano2 and Ruth Luthi-Carter1 1 University of Leicester, Department of Neuroscience, Psychology and Behavior, United Kingdom 2 Hokkaido University, Japan Motivation Impaired transcription and transport of brain-derived neurotrophic factor (BDNF) have been strongly implicated in the pathogenesis of HuntingtonÕs disease (HD) [1,2]. Here we assess the possibility that the TrkB agonist 7,8-Dihydroxyflavone (7,8-DHF) [3] rescues HD-related changes in neuronal activity. Methods We compared micro-electrode array (MEA) recordings of a cellular model of HD comprised of rat P0 cortical neurons transfected with lentiviral vectors encoding an 171 aa fragment of either a normal (N171-18Q, 18Q) protein or disease-causing mutant (N171-82Q, 82Q) human Huntingtin protein [2]. The spike detection and data analyses were performed with customized R and Matlab codes run using the cluster machine of Leicester University (ÒAliceÓ). Results Population spike time histograms of selected examples of MEA recordings are shown in Fig. 1. They were examined together with their corresponding raster plots. Statistical comparisons of the three groups (18Q and 82Q with N = 7 MEAs, and 82Q+7,8-DHF with N = 4 MEAs) are depicted in Fig. 2. We confirmed previous results showing that the number and size of population bursts (PBs) decreases in 82Q cells. Moreover we show that treatment with the TrkB agonist 7,8-Dihydroxyflavone (7,8-DHF) can partially rescue the abnormal electrophysiological phenotype of 82Q cells, as has been previously shown in studies with BDNF. 82Q cells showed decreased spike counts, decreased burst numbers and burst intensities (Hz x active channels) and decreased mean correlation between the channels. 7,8-DHF treatment (50 nM) restored the spike counts and burst numbers (Fig. 2). Discussion and Conclusions This study confirmed our labÕs previous data showing that the number and size of PBs decreases in 82Q cells when compared to control (18Q) cells. Treatment with 7,8-DHF can significantly rescue the abnormal electrophysiological phenotypes of 82Q cells, showing an effect size similar to BDNF. Our results highlight the utility of MEAs as an in vitro model for studying HD, and indicate that 7,8-DHF or other TrkB agonists may have potential as HD treatments that restore synaptic connectivity in abnormal neuronal circuits. To further assess the role of connection probability and synaptic weight on the bursting behavior we will perform numerical simulations of randomly connected neural networks with excitatory and inhibitory neurons including adaptation using NEST (http://www.nest-initiative.org/). Acknowledgements The authors thank Dr Sandro Perrone for useful comments regarding data analysis. Supported by European CommissionÕs Seventh Framework Program FP7-PEOPLE-2011-IAPP project number 286403 Ð NEUROACT.

Examining the Impact of Patient-Reported Hope for Improvement and Patient Satisfaction with Clinician/Treatment on the Outcome of Major Depressive Disorder Treatment.

AimsThis analysis aims at examining if patient-reported variables such as hope for improvement and patient satisfaction with clinician/treatment could influence the outcome major depressive disorder (MDD) treatment, namely depression remission, in the Sequenced Treatment Alternatives to Relieve Depression (STAR*D) trial.Study DesignRetrospective cohort study.Place and Duration of StudyThe STAR*D study was conducted at 18 primary care and 23 psychiatric care settings in the United States from 2001–2007 and was funded by the National Institute of Mental health (NIMH). The analysis contained in this manuscript was conceptualized at the Cedars-Sinai Department of Psychiatry and Behavioral Neurosciences and performed at the UCLA School of Public Health.MethodologyUsing data from STAR*D, the current study used logistic regression and survival analyses to examine the relationship between depressive symptoms remission and two sets of self-reported factors: Hope for improvement and, Patient satisfaction with treatment/clinician.ResultsFirst, more than 90% of STAR*D patients reported having high hope for improvement (agree or strongly agree) and more than 66% endorsed high satisfaction with clinicians and more than 50% expressed high satisfaction with treatments (very or mostly satisfied). Second, hope for improvement was predictive of depression remission (p<0.05). Third, satisfaction with clinician/treatment, did not predict remission.ConclusionThis study shows the impact that patients’ subjective hope for improvement can have on predicting depression remission in contrast to satisfaction with clinician/treatment. Future studies should prospectively incorporate patients’ subjective attitudes regarding hope for improvement and satisfaction with clinicians and treatments as mediators and moderators of MDD treatment success.

Microgels as vehicles for effective delivery of hydrophilic drugs to the brain

Event Abstract Back to Event Microgels as vehicles for effective delivery of hydrophilic drugs to the brain Madeline Simpson1, Justin Piazza2, Yogesh K. Katare2, Sharnpreet K. Kooner2, Niels B. Smeets1, Ram K. Mishra2 and Todd Hoare1 1 McMaster University, Department of Chemical Engineering, Canada 2 McMaster University, Department of Psychiatry and Behavioural Neuroscience, Canada Introduction: The treatment of central nervous system (CNS) disorders is challenging due to difficulties in drug transport across the blood-brain barrier (BBB). Nanoparticle-based therapies are of interest as they can be designed for specific targets, to control drug release, and lower the required dosage for therapeutic intervention. Degradable microgels (hydrogel-based nanoparticles) composed of poly(oligoethylene glycol methacrylate) (POEGMA), are promising delivery vehicles which have demonstrated protein-repellent properties[1]. In comparison to “hard” nanoparticles widely cited to cross the BBB with diameters of <150 nm[2], we show that larger elastic microgels can be transported across the BBB, which can improve drug loading and biodistribution of the hydrophilic drug 3(R)-[(2(S)-pyrrolidinylcarbonyl)amino]-2-oxo-1-pyrrolidineacetamide) (PAOPA). This drug is a positive allosteric modulator that selectively targets the dopamine D2 receptor and has been demonstrated to reduce the negative symptoms of schizophrenia in animal models[3]. Materials and Methods: POEGMA-based microgels were synthesized by precipitation polymerization of diethylene glycol methacrylate, acrylic acid (AA) and 2-hydroxyethyl disulfide dimethacrylate (degradable crosslinker) with various quantities of sodium dodecyl sulfate (SDS). The AA groups were functionalized using carbodiimide chemistry with Solanum tuberosum lectin (STL) which enables binding to the nasal epithelium for intranasal delivery. Microgel characterisation was carried out by light scattering (size), transmission electron microscopy (morphology) and conductometric titration (functional group content). PAOPA was passively loaded, with excess drug removed by centrifugation. The in vitro drug release was assessed using HPLC, and microgel cytotoxicity was determined by an MTT assay using RPMI 2650 nasal septum and SH-SY5Y dopamine 2D receptor-transduced neuronal cells. Rhodamine-labelled microgels were injected into the peritoneum of male Sprague-Dawley rats to assess the in vivo biodistribution of different microgel sizes. Blood and tissues were recovered after one hour and analyzed using fluorescence to quantify microgel biodistribution. Results and Discussion: Microgels with sizes of 99±2 nm, 152±3 nm, and 249±4 nm can be prepared with low polydipsersity (<0.1). PAOPA was loaded with an encapsulation efficiency of 30±2%, independent of size. The diffusion of PAOPA from the two smallest microgels occured over four days, with 50% of the drug still retained after 24 hours (Figure 1) while the largest microgel prolonged release to five days. The biodistribution studies with the rhodamine-labelled microgels demonstrated that the smallest microgels were the most effective at crossing the BBB; however the largest microgels were transported in significant numbers despite their larger size than the 150 nm limit cited while also accumulating less in major non-target organs (Figure 2). In vivo efficacy studies as a function of microgel size in a rat schitzophrenia model will be presented. Conclusion: POEGMA microgels are effective delivery vehicles for the transport of hydrophilic drugs across the BBB. Due to their elastic nature, larger microgels may be advantageous as the compressibility may enable the delivery of bigger, longer circulating, and more highly drug loaded particles across tight biological barriers. Canadian Institutes for Health Research (CIHR); Natural Sciences and Engineering Research Council of Canada (NSERC)

Migration and differentiation of neural stem cells in novel random and aligned nanofibrous scaffolds.

Event Abstract Back to Event Migration and differentiation of neural stem cells in novel random and aligned nanofibrous scaffolds. Hadi Hajiali1, 2, Andrea Contestabile3, Athanassia Athanassiou1 and Elisa Mele1, 4 1 Istituto Italiano di Tecnologia (IIT), Smart Materials, Nanophysics,, Italy 2 University of Genoa, DIBRIS, Italy 3 Istituto Italiano di Tecnologia (IIT), Neuroscience and Brain Technologies Department, Italy 4 Loughborough University, Department of Materials, United Kingdom Introduction: In tissue engineering, stem cell therapies have been proposed to boost the repair of injured tissues. In particular, several studies on the regeneration of the central nervous system have demonstrated the importance of combining neural stem cells (NSCs) and polymer scaffolds. In fact, expandable sources of neurons and properly engineered scaffolds, which can provide neural differentiation to specific lineage, can be instrumental for the effective repair of the neural tissue [1]. Among the different structures of scaffolds, nanofibers are highly attractive for their ability to mimic the extracellular matrix in regenerative medicine [2]. Materials and Methods: In this study, polymer scaffolds, constituted by randomly distributed or aligned nanofibers, were fabricated by electrospinning. The realized fibrous scaffolds were characterized in terms of morphology by Scanning Electron Microscope (SEM), mechanical properties, and biocompatibility. The attachment, migration and differentiation of neural stem cells were also investigated in random and aligned nanofibers. Results and Discussion: The results of SEM show that the fibers are free from beads and defects and very well aligned in the case of a specific type of scaffold. The Young’s modulus and tensile strength of aligned fibers significantly increase compared to random fibers. The scaffolds are able to promote the proliferation and differentiation of NSCs without laminin functionalization. The NSCs, seeded on the 3D scaffolds, not only attached and proliferated, but also were able to migrate and infiltrate inside the porosity of the fibrous mats. In this way, 3D cellular networks were created. Furthermore, the alignment of the fibers induced the growth of NSCs along one specific direction. In fact, the cells were able to highly elongate following the direction of fibers, creating aligned parallel patterns (Figure 1). Figure 1. Morphology of neural stem cells in the aligned fibers Conclusion: The obtained results demonstrate that the developed scaffolds are characterized by exceptional biocompatibility and that the topographical cues can be advantageously used in neural tissue engineering.

Antagonism of RXFP-3 receptor in the lateral hypothalamus increases alcohol self-administration in rats

Event Abstract Back to Event Antagonism of RXFP-3 receptor in the lateral hypothalamus increases alcohol self-administration in rats Christina J. Perry1, 2*, Hanna E. Kastman1, 2, Sarah S. Ch'Ng1, 2, Leigh C. Walker1, 2, Craig M. Smith1, 2, Andrew L. Gundlach1, 2 and Andrew J. Lawrence1, 2 1 The Florey Institute of Neuroscience and Mental Health, Behavioural Neuroscience, Australia 2 The University of Melbourne, Florey Department of Neuroscience and Mental Health, Australia Alcohol is the most widely abused drug worldwide, and alcohol abuse is a significant contributor to the global burden of disease. The search for novel and effective therapeutic targets to treat alcoholism is an ongoing task, especially given the high rates of relapse observed following alcohol rehabilitation. RXFP-3, the cognate receptor for the neuropeptide relaxin-3, is a potential therapeutic target. Recently we showed that RXFP3 is necessary for alcohol consumption and seeking, since central antagonism of this receptor reduced alcohol self-administration and relapse-like behaviour in alcohol preferring rats. Relaxin-3 is predominantly expressed in the brainstem, in neurons that project rostrally to the septum, hippocampus and lateral hypothalamus (LH). Of these, the lateral hypothalamus in particular is frequently implicated in alcohol consumption and relapse to alcohol-seeking. We sought, therefore, to examine the effect of RXFP3 antagonism in the LH. Alcohol-preferring P rats were trained to lever press for 10% ethanol for a minimum of 30 days. The selective antagonist R3(B1-22)R was then infused bilaterally into the LH prior to a standard self-administration session. In contrast to the effect of a central (icv) or intra-BNST infusion, rats showed increased alcohol consumption following administration of R3(B1-22)R compared to vehicle. To better understand this unexpected result we subsequently sought to characterise RXFP-3 positive neurons in the hypothalamus using a transgenic reporter mouse expressing GFP protein on RXFP-3 neurons. RXFP-3 receptors were expressed on orexin negative neurons that project to the ventral tegmental area (VTA). These findings have implications for understanding how relaxin 3 acts centrally to regulate alcohol-seeking and alcohol consumption, and on-going studies will elucidate the mechanism behind this finding, which somewhat parallels observations with the opioid receptor antagonist naltrexone. Keywords: Hypothalamus, Neuron, RXFP3, Alcohol consumption, alcohol seeking behaviour Conference: 14th Meeting of the Asian-Pacific Society for Neurochemistry, Kuala Lumpur, Malaysia, 27 Aug - 30 Aug, 2016. Presentation Type: YIC01: Young Investigator Colloquium 1 Topic: 14th Meeting of the Asian-Pacific Society for Neurochemistry Citation: Perry CJ, Kastman HE, Ch'Ng SS, Walker LC, Smith CM, Gundlach AL and Lawrence AJ (2016). Antagonism of RXFP-3 receptor in the lateral hypothalamus increases alcohol self-administration in rats. Conference Abstract: 14th Meeting of the Asian-Pacific Society for Neurochemistry. doi: 10.3389/conf.fncel.2016.36.00053 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 04 Aug 2016; Published Online: 11 Aug 2016. * Correspondence: Dr. Christina J Perry, The Florey Institute of Neuroscience and Mental Health, Behavioural Neuroscience, Melbourne, Victoria, Australia, christina.perry@mq.edu.au Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Christina J Perry Hanna E Kastman Sarah S Ch'Ng Leigh C Walker Craig M Smith Andrew L Gundlach Andrew J Lawrence Google Christina J Perry Hanna E Kastman Sarah S Ch'Ng Leigh C Walker Craig M Smith Andrew L Gundlach Andrew J Lawrence Google Scholar Christina J Perry Hanna E Kastman Sarah S Ch'Ng Leigh C Walker Craig M Smith Andrew L Gundlach Andrew J Lawrence PubMed Christina J Perry Hanna E Kastman Sarah S Ch'Ng Leigh C Walker Craig M Smith Andrew L Gundlach Andrew J Lawrence Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Ultra-short self-assembled beta-peptide hydrogels as matrices for neural tissue engineering

Event Abstract Back to Event Ultra-short self-assembled beta-peptide hydrogels as matrices for neural tissue engineering Sepideh Motamed1*, Mark Del Borgo2*, Ketav Kulkarni3*, Nathan Habila2, Kun Zhou1, David Finkelstein4, Patrick Perlmutter3*, Marie I. Aguilar2 and John S. Forsythe1* 1 Monash University, Department of Materials Science and Engineering, Australia 2 Monash University, Department of Biochemistry and Molecular Biology, Australia 3 Monash University, School of Chemistry, Australia 4 The University of Melbourne, Florey Department of Neuroscience and Mental Health, Australia Hydrogels have physical features of soft tissue and have been explored for their use in nerve regeneration and drug delivery [1]. Hydrogels based on peptide self-assembly are extremely promising candidates to provide a suitable microenvironment for cells due to their facile synthesis, simple building blocks, inherent biocompatibility and the ability to control the structural and functional properties of the end product [2]-[4]. However, applying the peptide hydrogel matrices in brain tissue engineering is faced with a number of key challenges. Peptide-based biomaterials that are used in neural tissue engineering have been based on α-amino acid peptides which can undergo rapid proteolysis and are unable to provide long term structural support. In cases where the matrix must fill and provide structural support in a large brain lesion, it may be preferable to use a non-degrading or permanent peptide matrix [5]. Herein we introduce for the first time hydrogels consisting of peptide matrix composed of only β3-amino acids as an efficient alternative for neural tissue engineering and to utilise their inherent proteolytic stability in vivo [6]. To provide the required conditions for self-assembly the N-terminus of β-tripeptide (AzKA) was capped with an acetyl group which produced a total of six axially oriented hydrogen bonding interactions [7]. A hydrophobic alkyl chain was added laterally to the peptide backbone to ensure the formation of stable hydrogel. Peptide self-assembled spontaneously to form a hydrogel upon dissolving in PBS buffer (pH 7.4) under physiological conditions at a concentration of 10mg/mL. The morphology of the formed nanofibers was further investigated by AFM and TEM which revealed a network of nanofibers with consistent diameter. The mechanical properties of the peptide hydrogel were tested via rheological studies. The hydrogel showed viscoelastic properties with storage modulus in the range of 1kPa. A balance between hydrophilic and hydrophobic domain in the peptide, allowed the hydrogel to flow under applied shear strain and to recover completely within seconds upon relaxation. To check the feasibility of the hydrogel for neural tissue engineering, the viability of SN4741, substantia nigra dopaminergic neuronal progenitor cell line, cultured on the hydrogel were assayed. The hydrogel proved to be highly biocompatible and even though it did not possess any bioactive motif, by pre-depositing protein from serum, it provided an environment for cells to adhere and proliferate with 80% cell viability in comparison to the positive control (cultured on TCPS). The facile design and synthesis of a β-peptide hydrogel can thus allow formation of controlled and variable biomaterials for different types of tissue engineering applications.

Synapsin III alterations in Parkinson's disease

s / Parkinsonism and Related Disorders 22 (2016) e149ee192 e189 Conclusions: The results indicate that the a-mannosidase activity present in the CSF is of lysosomal type and of brain origin. Furthermore, the intermediate form of a-mannosidase from plasma does not cross the bloodbrain-barrier. Thus CSF a-mannosidase activity might mirror the brain pathological changes linked to neurodegenerative disorders such as PD. P 6.058. SYNAPSIN III ALTERATIONS IN PARKINSON'S DISEASE Arianna Bellucci , Michela Zaltieri , Francesca Longhena , Gaia Faustini , Jessica Grigoletto , Gaia Favero , Stefania Castrezzati , Rita Rezzani , Marina Pizzi , Fabio Benfenati , Maria Grazia Spillantini , Cristina Missale , PierFranco Spano . Department of Molecular and Translational Medicine, University of Brescia, Brescia, Italy; Department of Clinical and Experimental Sciences, University of Brescia, Brescia, Italy; Department of Neuroscience and Brain Technologies, Italian Institute of Technology, Genoa, Italy; Department of Clinical Neuroscience, University of Cambridge, Cambridge, United Kingdom; 5 IRCCS San Camillo Hospital for Neurorehabilitation (NHS-Italy), Venice, Italy Objectives: The main neuropathological hallmarks of Parkinson's disease (PD) are loss of nigro-striatal dopamine neurons and intraneuronal Lewy Bodies (LB), proteinaceous inclusions mainly composed by a-synuclein. Recently, we found that a-synuclein interacts and cooperates with a specific member of the synapsin phosphoprotein family: synapsin III, in the regulation of dopaminergic neuron function. The aim of this study was to investigate whether synapsin III alterations may be related to a-synuclein pathology in PD. We thus investigated the occurrence of alterations of synapsin III expression and distribution in “the brain of patients affected by PD and DLB (Human brain samples were kindly provided by the UK Parkinson's disease Brain Bank). Methods: We used molecular biology and immunohistochemical techniques as well as the “in situ” proximity ligation assay (PLA) to investigate the expression levels and the distribution of synapsin III in the PD brain. Results: By immunohistochemistry, we showed a marked accumulation of synapsin III in the caudate/putamen of patients affected by PD when compared to age matched controls. In addition, many LB-like structures in the substantia nigra of PD patients were found to be immunoreactive for synapsin III, that by confocal fluorescence microscopy resulted to be accumulated in the core of LB. By co-immunoprecipitation and “in situ” PLA we found that synapsin III directly interacted with a-synuclein and that these two proteins were co-redistributed in mice and human brains. Finally, western blot analysis showed significant alterations of synapsin III levels that correlated with alteration of a-synuclein in the caudate/putamen and substantia nigra of PD patients. Conclusions: Altogether, our data support a critical involvement of synapsin III in PD pathophysiology P 6.059. UNDERSTANDING THE DIFFERENTIAL REGULATION OF GBA AND GBAP1 EXPRESSION Valeria Rimoldi , Giulia Rovaris , Letizia Straniero , Gianni Pezzoli , Stefano Goldwurm , Giulia Sold a , Rosanna Asselta , Stefano Duga . Humanitas University, Humanitas Clinical and Research Center, Rozzano, Milano, Italy; Universit a degli Studi di Milano, Humanitas Clinical and Research Center, Rozzano, Milano, Italy; 3 Parkinson Institute, Istituti Clinici di Perfezionamento, Milano, Italy Objectives: GBA downregulation was demonstrated to have a crucial role in Parkinson's Disease. We previously showed that there is a miRNA-mediated regulatory circuit linking GBA and its pseudogene (GBAP1) expression and that they are coexpressed inmost tissues with a GBA/GBAP1 ratio ranging from 2 to 200. Furthermore, we demonstrated that GBAP1 levels are downregulated by the NMD pathway. GBA and GBAP1 share a similar genomic structure characterized by a proximal (P1) and a distal (P2) promoter but there are no data on the differential regulation at the transcriptional level. Hence, wewant to study the P1 and P2 promoters of both genes. Methods: We cloned 1kb of the 4 promoters in a luciferase vector and produced serial 5'-deletion constructs of each promoter. These plasmids were transfected in different cell lines. Results: We demonstrated that: P2 promoters are stronger than P1s, for both GBA and GBAP1; in HeLa cells, as expected, GBAP1 promoters are weaker than GBA ones, while in SH-SY5Y cells, the pseudogene P2 promoter has the highest activity; the TFEB binding sites seem to be the most important regulatory elements for P1 promoters. Conclusions: The transcriptional activity of both GBAP1 promoters, as assessed by luciferase assays, was unexpectedly higher compared to the mRNA levels detected in vivo. Besides differences in epigenetic regulation not faithfully modeled in our system, NMD is likely to be the major determinant of the relative expression of GBA/GBAP1 transcripts. P 6.061. STRUCTURAL ANALYSIS OF ALPHA-SYNUCLEIN OLIGOMERS VIA ANTIBODY FINGERPRINTING Lina Nilsson , Tohidul Islam , Irina Iakovleva , Kristoffer Br€annstr€ om, Anders Olofsson . Department of Chemistry, Umea, Sweden; Department of Medical Biochemistry and Biophysics, Umea,

Revisiting allostatis and allostatic load in bipolar disorder

1Prof. Dr., Dokuz Eylul University, Faculty of Medicine, Department of Psychiatry, Izmir Turkey Dokuz Eylul University, Institute of Health Sciences, Department of Neuroscience, Izmir Turkey Dokuz Eylul University, Brain Dynamics Center, Izmir Turkey Guest Editorial / Misafir Editoryal Dusunen Adam The Journal of Psychiatry and Neurological Sciences 2015;28:183-188 DOI: 10.5350/DAJPN20152803001

A New Role for Clonidine in Addictions: Catching Relapses Before They Happen.

Relapse is one of the greatest challenges facing drug abusers and their caretakers. Many addicts can successfully achieve initial abstinence through a variety of sticks and carrots, medications and incarcerations.However, uponreturn to their usual environment, users are very likely to relapse to old drug habits, even longafter the effectsof acutewithdrawal aregone. Why people return to drug use, despite the known adverse consequences, is not understood. Because relapse occurs in an uncontrolled environment, and at unpredictable times, it is extremely difficult to study. In their article in this issue, Kowalczyk and colleagues (1) present promising new data supporting the idea that stress induces relapse, and that reducing the stress response with medication can reduce lapses to drug use. Perhaps more importantly, their study describes new research approaches that can be used to understand the processes involved in, and treatments to reduce, relapse to drug use, within the natural drug-taking environment. Acute stress is thought to be a key cause of relapse to drug use (2). This idea is supported not only by clinical reports but also by extensive research in animal models of drug relapse, which also examine the neural mechanisms underlying the return to drug seeking (3). Indeed, these animal studies suggest that alpha-2 adrenoceptor agonists specifically reduce stress-induced relapse, compared with relapse induced by other factors, such as cues or small amounts of the drug. In the Kowalczyk et al. study, the authors examined the effects of clonidine, analphanoradrenergic agonist, on stressinduced relapse. Clonidine is commonly used during detoxification, but how andwhy it alters drug-seeking behavior is not known. Opioid-dependent patients who had already demonstrated a successful response to buprenorphine treatment (i.e., 5–6 weeks of abstinence) were randomly assigned to continuing 14 weeks of buprenorphine maintenance treatment either with or without adjunctive clonidine. The investigators monitored the participants’ drug use, and also obtained repeated daily reports (four times a day, at random) of participants’ mood and craving. Clonidine extended the duration of abstinence, compared with placebo, although both groups ultimately showed similar rates of relapse. The most intriguing aspect of the study was that the investigators studied the temporal relationship betweendrug useanddaily reports of stress,mood, andcravingbyproviding participants with handheld electronic devices. This innovative data collection strategy allowed the investigators to conclude that clonidine reduced the temporal association between stress and craving for opioids, supporting the idea that the drug exerted its therapeutic effect by dampening stress. Despite an enormous scientific literature derived from controlled studies of drugs of abuse, our understanding of the natural ecology of drug use, outside of the laboratory setting, is very limited. For example, we have limited knowledge of the patterns of drug use that lead to dependence, or of the environmental or psychological factors that influence specific occasions of drug use, including during periods of attempted abstinence. This lack of knowledge of the natural course of drug use constitutes an enormous gap in our understanding of the determinants of addiction. The gap can be addressed using innovative techniques such as that used in the study by Kowalczyk andcolleagues. Ecological momentary assessment, or the collection of psychological and behavioral data using electronic devices in real time, in the natural environment,was pioneered by Shiffman and colleagues (4). This technology now allows researchers to monitor the patterns and the contexts in which people use drugs. It allows investigators to determine the temporal relationships between events (e.g., mood states, stress) and stimuli (e.g., reminders of druguse) that are thought to inducedrug taking, and between events and stimuli and drug use itself. In the present study, Kowalczyk et al. used this technology to support their hypothesis that clonidine improves treatment outcome by reducing the link between acute stress and drug use. This technique has enormous potential to improve our understanding of the processes of onset, maintenance, and relapse to drug use and the effectiveness of potential treatments. In future research, a similar approach could be taken to study precipitants of relapse other than stress, or to study other problematic behaviors, such as gambling. It would be possible to study the role of drug-related cues (possibly even by adding a visual component to the monitoring) or to study how small (“priming”) doses of an abused drug increase the Despite an enormous scientific literature derived from controlled studies of drugs of abuse, our understandingof the natural ecology of drug use, outside of the laboratory setting, is very limited.