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Gene transfer to the tral tegmental area dopamine neurons to glutamate after repeated nigrostriatal system by hybrid herpes simplex virus/adeno-associ- administration of cocaine or amphetamine is transient and selec- ated virus amplicon vectors discount 400 mg zovirax. Hum Gene Ther 1999;10: tively involves AMPA receptors zovirax 800 mg online. Dopamine transmission in the initiation bility of persistent adenovirus vectors in the brain: peripheral and expression of drug- and stress-induced sensitization of motor exposure to vector leads to renewed inflammation, reduced gene activity. Immune responses of opiate reward and aversion within the midbrain identified to transgene-encoded proteins limit the stability of gene expres- sion after injection of replication-defective adenovirus vectors. The role of excitatory amino acids in behavioral sensiti- ated virus vectors into the central nervous system. Molecular mechanisms of drug reinforce- systems for gene transfer. Lentiviruses as gene transfer agents for delivery to 46. Differential and persistent expres- Nature 1999;401:272–276. Repeated cocaine and related drugs in nucleus accumbens shell and frontal cortex. Amphetamine self-administration and relapse of cocaine-seeking behavior. J and cocaine induce drug-specific activation of the c-fos gene in Neurosci 1998;18:1848–1859. Cyclic AMP stimulates somato- opiate-induced c-fos mRNA expression patterns in the rat fore- statin gene transcription by phosphorylation of CREB at serine brain: comparisons between acute drug treatment and a drug 133. Conditioned place preference challenge in sensitized animals. AP-1 complex composed of altered Fos-like proteins in brain by 60. Dynorphin is a specific en- chronic cocaine and other chronic treatments. Neuron 1994;13: dogenous ligand of the kappa opioid receptor. Sensitization to the behavioral effects of long-term neural and behavioral plasticity. Brain Res 1999; of cocaine: modulation by dynorphin and kappa-opioid receptor 835:10–17. From motivation to action: Curr Opin Neurobiol 1999;9:305–313. SCHAFER Pharmacologic agents often biochemically interact with ditis elegans, and the fruit fly Drosophila melanogaster. These multiple receptor or channel proteins, and induce multiple organisms share a number of advantages that make them changes in cellular physiology and signal transduction. For Thus, identifying the biologically relevant targets and effec- example, both have short generation times (2 weeks for Dro- tors of a given neuroactive substance can be a challenging sophila, 3 days for C. This chapter describes how genetic analysis in sim- in large numbers in the laboratory, and are amenable to ple model organisms, primarily worms or flies, has been germline transformation. In addition, detailed genetic maps used to identify molecules that mediate drug responses in of both organisms are available, and the genome sequences the nervous system. Although Essentially all the studies described here rely on the same both organisms contain relatively simple nervous systems, general strategy. The drug of interest is tested for its ability they differ significantly in scale and level of characterization. Once behavior using techniques such as single-cell laser ablation, these genes are identified and cloned, human homologues and to thereby understand in a precise manner how the can be identified based on sequence similarity, and tested action of a particular gene product in a defined set of neu- for involvement in human drug responses. Thus, even process being studied; any gene that is not essential for life mutants with defects in basic neuronal functions such as and affects the behavioral response to a drug is in principle neurotransmitter release are often viable and fertile (5). The equally likely to be identified in a mutant hunt. Thus, this Drosophila nervous system is somewhat more complex, and approach is well suited for identifying previously unknown contains approximately 105 neurons. Consequently, it is receptors or signal transduction molecules that participate somewhat less well characterized at the cellular level than the in drug responses. By makes it perhaps better suited for investigating more com- making it possible to assess a particular protein function plex forms of behavior and learning (6). STUDIES OF DRUG MECHANISMS IN Among organisms with nervous systems, two are particu- MODEL ORGANISMS larly amenable to genetic analysis: the nematode Caenorhab- Genetic pharmacology has historically been a powerful ap- proach for neurobiological studies in C. Schafer: Division of Biology, University of California–San phila. Many studies of drug-resistant flies or worms have Diego, La Jolla, California 92093-0349.

The two subsequent surveys followed the same approach trusted zovirax 400 mg. Practices checked that the original sampled patients were still alive and suitable to participate discount 400mg zovirax amex, and sent them follow-up questionnaires. NWIS then replaced each patient lost to follow-up by another patient of the same age, sex and risk level. We undertook primary analysis by treatment allocated. This issue may be freely reproduced for the purposes of private research and study and extracts (or indeed, the full report) may be included in professional journals 25 provided that suitable acknowledgement is made and the reproduction is not associated with any form of advertising. Applications for commercial reproduction should be addressed to: NIHR Journals Library, National Institute for Health Research, Evaluation, Trials and Studies Coordinating Centre, Alpha House, University of Southampton Science Park, Southampton SO16 7NS, UK. METHODS TABLE 11 Overview of data sources employed in the study, matched to study objectives Objective Data source Sample Collection time 1. Measure effects on Anonymised routine All patients from participating practices Baseline service usage, particularly linked data (including emergency admissions to PRISM data) 6 months hospital 18 months Questionnaire data: Random sample of patients from Baseline CSRI participating practices (n = 800 at each time point) 6 months 18 months 2. Assess the effect of Questionnaire data: Random sample of patients from Baseline PRISM on quality of life SF-12, QCM participating practices (n = 800 at each time and patient satisfaction point) 6 months 18 months 3. Assess the technical PRISM data PRISM risk data for patients at participating Baseline performance of PRISM practices 6 months 18 months Anonymised routine Routine health data Baseline linked data 6 months 18 months 4. Estimate costs of Anonymised routine All patients from participating practices Baseline PRISM implementation linked data (including and its effects PRISM data) 6 months 18 months Questionnaire data: Random sample of patients from Baseline SF-12 was used to participating practices (n = 800 at each time derive SF-6D score point) 6 months 18 months Structured interviews PRISM users from all participating practices 18 months (n = up to 40) 5. Describe processes of Focus groups GPs, practice nurses and managers from Baseline change associated with participating practices (n = 4); local health PRISM services managers and community staff managers (n = 1) Interviews GPs from participating practices who were unable to attend focus groups (n = 12) Health board managers from sites not participating in main study (n = 6); policy-makers and NHS managers (n = 5) Interviews PRISM users from half of all participating After 3 months practices, purposively sampled (n = 16 following PRISM practices) going live (mid-trial point) and at the end Questionnaire PRISM users from remaining half of all of the intervention participating practices (n = 16 practices) (end of the trial) Focus group Local health services managers and 18 months community staff managers (n = 1) Interviews ABM UHB health service managers (n = 3) CSRI, Client Service Receipt Inventory; QCM, Quality of Care Monitor; SF-6D, Short Form questionnaire-6 Dimensions. Adapted from Hutchings HA, Evans BA, Fitzsimmons D, Harrison J, Heaven M, Huxley P, et al. Predictive risk stratification model: a progressive cluster-randomised trial in chronic conditions management (PRISMATIC) research protocol. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons. Predictive risk stratification model: a progressive cluster-randomised trial in chronic conditions management (PRISMATIC) research protocol. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons. Outcomes were analysed using an appropriate generalised linear mixed model; specifically, we used linear models for measurement outcomes (assuming normality, also assessed via residual diagnostics), in which study practice was considered as a random factor; logistic regression for binary (yes/no) outcomes; negative binomial regression models for counts; we also analysed SF-12 and QCM scores for participants returning questionnaires using a repeated measures linear model. Modelling progressed by eliminating, in turn and starting with the least significant, all covariates and factors found to be not statistically significant (that is, with a coefficient with a p-value > 0. Raw and adjusted comparisons between groups were made, with some indication of the extent of the intracluster correlation coefficient (ICC) in variables between participants at the same study practice, and details of statistically significant factors and covariates. The adjusted comparisons reflected the nature of the variable under consideration: we present an odds ratio (OR) for logistic regression models for binary variables; an incidence or event ratio, Λ from negative binomial models for count variables, and an additive group effect (Δ, in the same units as the dependent variable) for linear models for continuous variables. To test the effect of the positive skewness on the statistical results, data were log-transformed and the generalised linear model re-run as described above. Within each study practice, PRISM risk group 1 comprises those participants with the lowest 80% of scores in that practice. PRISM risk group 2 comprises those participants with the next 15% of scores, and PRISM risk group 3 comprises those participants with the next 4. PRISM risk groups for the complete sample are formed by combining the practice-level groups. This issue may be freely reproduced for the purposes of private research and study and extracts (or indeed, the full report) may be included in professional journals 27 provided that suitable acknowledgement is made and the reproduction is not associated with any form of advertising. Applications for commercial reproduction should be addressed to: NIHR Journals Library, National Institute for Health Research, Evaluation, Trials and Studies Coordinating Centre, Alpha House, University of Southampton Science Park, Southampton SO16 7NS, UK. METHODS consistent with the interpretation that the PRISM risk score is a probability of an emergency admission in 12 months, sought to analyse data on PRISM scores and emergency admissions in a 1-year period. Health economics Health economic analysis We undertook an evaluation of the costs from the perspective of the UK NHS. Costs were assigned to the resources utilised by each patient in the 32 trial practices. These consisted of the PRISM implementation costs, primary care costs and secondary care costs (including ED attendances, emergency admissions, outpatient visits, and elective and emergency inpatient stays). Unit costs (pounds sterling, cost year 2014) were derived from published information. Cost differences between the two study phases were determined and used in conjunction with differences in the primary outcome (emergency admissions) to undertake a cost-effectiveness analysis.

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Substance P and the NK1 receptor have a widespread distribution in the brain and are found in brain regions that regulate emotion (e discount zovirax 400mg amex. NKB is produced from a distinct precursor protein also found in close association with major catecholamine- encoded by preprotachykinin B (4) 200 mg zovirax visa. Subsequently, specific binding sites labeled also expressed in varying ratios in the CNS and spinal cord by Bolton Hunter substance P, NKA, and eledoisin were (17,18) and in the rodent (but not human) brain, and NK3 identified in the CNS (6), a finding suggesting that at least receptors and mRNA have also been demonstrated in var- three receptors mediated the actions of tachykinins. This ious regions, including the substantia nigra, raphe nuclei, was confirmed by cloning of three distinct functional cDNA and locus ceruleus (19–21). However, the endogenous neurokinins approximately 50% of ascending dorsal raphe neurons in exhibit a high degree of cross-reactivity with these tachy- the primate brain (22,23). These findings provide further illustra- tions of the marked species differences in the neuroanatomy, SPECIES DIFFERENCES IN THE and possibly physiology, of neurokinin systems. The func- DISTRIBUTION OF NEUROKININS AND tional significance of substance P and 5-HT coexpression THEIR RECEPTORS IN THE NERVOUS in the human brain is not known, but it suggests that both SYSTEM neurotransmitters may be coreleased in certain brain regions receiving terminal innervation. The substance P–preferring NK1 receptor has attracted Other evidence suggests that substance P and NKB may most interest as a CNS drug target because it is the predomi- also modulate ascending norepinephrine systems. NK1 re- nant tachykinin receptor expressed in the human brain, ceptors (25) have been shown to be expressed on tyrosine whereas NK2 and NK3 receptor expression is extremely low hydroxylase–positive cell bodies in the rat locus ceruleus, or absent (10–12). Therefore, it appears that the central and both substance P and senktide (a selective NK3 receptor actions of all tachykinins may be mediated predominantly agonist) excite the firing of locus ceruleus neurons in rats through the NK1 receptor in humans. For example, in the rat and TACHYKININ RECEPTOR PHARMACOLOGY guinea pig brain, both NK1 and NK3 receptors are expressed (10), findings suggesting that the CNS functions mediated Preclinical studies with NK1 receptor antagonists have also by NK1 receptors in the human brain may be subserved by been complicated by species variants in NK1 receptor phar- NK1 and/or NK3 receptors in rodents. Compounds such as CP-96,345 were pear to be absent in the adult mammalian brain of all species found have high (nM) affinity for the NK1 receptor ex- examined (10). For these reasons, interpretation of the ef- pressed in human, gerbil, rabbit, guinea pig, cat, and mon- fects of selective tachykinin receptor antagonists in preclini- key brain, but they had considerably lower affinity for the cal assays requires great caution. If such compounds either mouse and rat NK1 receptor. Subsequent mutation analysis succeed or fail to exhibit activity in rodent assays for psychi- revealed that subtle differences in the amino acid sequence atric and neurologic disorders, this may merely reflect differ- between the human and the rat NK1 receptor dramatically ent roles of tachykinin receptors in rodent versus human alter antagonist binding affinity (30). Hence there is a risk of both false-positive and false- greatly hindered preclinical evaluation of high-affinity negative extrapolations from preclinical species to humans. Based on these neuroanatomic and functional PHARMACOLOGY IC FOR INHIBITION OF [125I]SP 50 studies, it was anticipated that NK1, and possibly NK2, BINDING (nM) receptor antagonists could be developed as analgesic drugs. Compound Human Gerbil Guinea Pig Rat Electrophysiologic studies on anesthetized or decerebrate animals provide evidence of potent and selective inhibition L-733060 0. Responses of dorsal horn neurons to noxious SR140333 0. NK1 receptor antago- nists have also been shown to inhibit the late-phase response commonly used preclinical species (Table 13. A few com- to formalin in gerbils (42), to inhibit carrageenan and pounds have high affinity for the rat receptor (e. SR140333), but their utility for in vivo studies may be se- Webb, S. Rupniak, unpublished observa- verely limited by poor brain penetration (31). Although tions; 43), and to attenuate peripheral neuropathy in rats these difficulties may be overcome by administering high and guinea pigs (43,44). Overall, the profile of activity of doses of NK1 receptor antagonists to rats, unspecific phar- NK1 receptor antagonists in a range of assays is comparable macologic effects are then frequently encountered, mostly to that seen with clinically used analgesic agents such as attributable to ion channel blockade. Pharmacologic differences among human, guinea cacy of these compounds in humans and are reviewed in pig, and rat NK3 receptors also exist (32). The patient populations and com- pounds examined included the following: peripheral neu- ropathy, in which CP-99,994 had no analgesic effect (47); POTENTIAL FOR USE OF TACHYKININ molar extraction, in which MK-869 was ineffective (48); RECEPTOR ANTAGONISTS TO TREAT and postherpetic neuralgia, in which MK-869 was ineffec- PSYCHIATRIC AND NEUROLOGIC tive (49). Further unpublished studies with other com- DISORDERS pounds support these conclusions. Thus, clinical studies to date indicate that NK1 receptor antagonists do not have The distribution of neurokinins in the central and periph- major potential as analgesics. The major hypotheses that are supported 10207 completely blocked both facilitation and protective by preclinical data and have been investigated in clinical nociceptive reflex responses (40), and SR48968 reduced re- trials are considered here. Numerous clinical trials have now sponses to both noxious and innocuous pressure applied to been conducted with NK1 receptor antagonists to define their therapeutic potential in psychiatric and neurologic dis- orders. In all these studies, the compounds have been ex- tremely well tolerated, with no significant side effects. PRECLINICAL EVIDENCE OF AN ANALGESIC PROFILE OF NK1 RECEPTOR are as yet no reports of clinical trials with NK2 or NK3 ANTAGONISTS receptor antagonists in patients with CNS disorders. Assay Morphine Indomethacin NK1 Antagonist Tail flick/hot plate √ Pain Paw pressure √ Writhing Radioligand-binding studies confirm the expression of Formalin paw tachykinin NK1 and NK3 (but not NK2) receptors in the Carrageenan paw dorsal horn of the spinal cord (33–35).

With this method cheap 200mg zovirax visa, the gene of interest is inserted into a random locus Laurence H cheap zovirax 400 mg without prescription. The techniques required for intro- 242 Neuropsychopharmacology: The Fifth Generation of Progress ducing transgenes into the mouse genome have been highly rates of integration of transgenes than other known methods refined, permitting their widespread use. After microinjection, the embryos are ment of this technique, many thousands of lines of surgically transferred into the oviduct of pseudopregnant transgenic mice have been generated, and it has been the mice. Pseudopregnant females are generated by matings most widely utilized technique of genetic manipulation in with vasectomized males. The foster mothers give birth 19 Methods of Production of Transgenic to 21 days after oviduct transfer. For genotyping, DNA is Mice typically isolated from mouse tail biopsies and screened for Techniques for producing transgenic mice involve the mi- the presence of the transgene by Southern blotting or poly- croinjection of DNA constructs into fertilized mouse eggs merase chain reaction (PCR). DNA constructs used for the generation of of the mice that develop to term possess the transgene. In transgenic mice typically consist of a gene of interest located the majority of cases, integration of the transgene occurs 3′ to promoter sequences selected to produce a desired dis- during the one-cell stage, so that the transgene is present tribution of gene expression. The maximum length of the in every cell of the transgenic mouse. Integration usually DNA sequence that may be successfully incorporated into occurs at a single random chromosomal location, and, for the mouse genome is not known, and up to 70 kilobase reasons that are not fully understood, there are usually mul- (kb) DNA fragments have been successfully integrated. The tiple copies of the transgene inserted as head-to-tail conca- transgene is linearized and purified from prokaryotic vector tamers. Mice identified to possess the integrated transgene sequences. For optimal integration efficiency, about 1 to 2 are referred to as founders. The founders are typically used picoliter (pL) of DNA at a concentration of 1 to 2 ng/ L in a breeding strategy to produce animals that are homozy- (corresponding to a few hundred molecules of a 5-kb DNA gous for the transgene insertion. Although labor intensive, direct injec- Uses of Transgenic Mice tion of DNA into the pronucleus results in much higher Because transgenic mice often possess multiple copies of the transgene, this method can be used to produce animals with increased levels of expression of particular genes, i. In addition, it can be used to express altered forms of a gene product in the distri- bution of the endogenous gene. One example is a transgenic 2 line bearing a transgene composed of the Ca /calmodulin- dependent protein kinase subunit (CaMKII ) promoter driving expression of a mutant form of CaMKII that con- ferred Ca2 -independent activation. These mice exhibited an increased stimulation threshold for the induction of syn- aptic plasticity in the hippocampus, as well as deficits in spatial memory (3,4). Studies of these animals led to an enhanced understanding of the role of CaMKII in synaptic plasticity and spatial memory acquisition. In many cases, it is desirable to express a gene with an anatomic distribution that does not mirror its native expres- sion pattern in the mouse. Such ectopic expression of a gene may be achieved using a transgenic construct in which the gene of interest is preceded by promoter elements that direct expression in an anatomic distribution characteristic of an- other gene. An example of this approach is a transgenic line in which the D1 dopamine receptor promoter was used to FIGURE 19. A: drive expression of a cholera toxin subunit (which constitu- One-celled fertilized zygotes located in the oviduct ampullae of pregnant donor mice are surgically harvested. B: DNA encoding tively activates G ) in cells that express D1 dopamine recep-s the gene of interest is microinjected directly into the pronucleus tors (5). Studies of these animals revealed that chronic over- of the zygotes. C: Injected zygotes are surgically transferred into stimulation (by constitutively activated G ) of forebrain s the oviducts of pseudopregnant female mice. D: DNA from the progeny can be analyzed by Southern blot or polymerase chain neurons expressing D1 receptors results in an abnormal be- reaction (PCR) for the presence of the transgene. For most genes, the promoter elements necessary cause the likelihood of two founders possessing the same to reproduce the native patterns of expression are not well transgene integration site is minimal. A useful approach for identifying important pro- moter elements for genes of interest involves the generation of transgenic mice in which putative promoter sequences GENE TARGETING PROCEDURES are used to direct expression of reporter genes, whose expres- sion is readily determined in brain tissue. Comparisons may A mutational approach has proved to be invaluable to inves- then be made between the pattern of reporter gene expres- tigators examining the roles of gene products in complex sion and that of the gene of interest (6–8). Recently it has become possible to apply this ap- the expression of a particular gene product. The introduction of mutations that produce more thus decreasing production of the gene product of interest subtle alterations in gene function has also been achieved. Alternatively, the function of gene products that Two major developments have made gene targeting experi- aggregate into multimeric complexes may be disrupted by ments feasible: (a) the generation of totipotent embryonic dominant-negative mutations that produce dysfunctional stem (ES) cells, and (b) the elucidation of techniques to subunits of the complex. The most prevalent approach used achieve homologous recombination in mammalian cells. Blastocysts Transgenic Mouse Phenotypes are cultured individually under conditions that permit the An important factor that frequently complicates the inter- proliferation of the inner-cell mass cells, which are those pretation of studies with transgenic mice is the difficulty cells that would normally become the fetus. These cells are that may be encountered in achieving a desired anatomic then disaggregated, and individual ES cells clones are grown. Promoter elements are Under optimal conditions, ES cells retain the ability to con- often quite large, and additional regulatory elements may tribute to all of the tissues of the developing fetus.

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