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By E. Murat. Hardin-Simmons University.

Given that benzodiazepines and barbiturates both types of GABA receptors are widely distributed act as positive modulators of GABA transmission at the through the CNS generic claritin 10mg on-line allergy injections, several important differences exist be- GABA receptor by enhancing GABA-induced chloride A tween the two purchase 10mg claritin mastercard allergy testing when to stop antihistamines. Relevant to psychopharmacology is the find- channel opening, it is of interest to note that GAD65 / ing that traditional anxiolytics (benzodiazepines) do not mice were not sensitive to the effects of either benzodiaze- bind to GABAB receptors, but rather mediate their effects pines or barbiturates, but did respond to the direct GABA A through GABAA receptors. GABAA receptors consist of a agonist muscimol, which binds directly to the GABA site of chloride channel formed by the pentameric arrangement of the GABA receptor and increases opening of the chloride A at least 18 different protein subunits ( 1–6, 1–4, 1-3, , channel in the absence of GABA (152). This pharmacologic , , 1–3), thus allowing for considerable heterogeneity of profile is consistent with the finding that GABA synthesis the GABAA receptor isoforms (148). Typically, benzodiaze- is blocked by the GAD65 null mutation, but that GABA A pine-responsive GABAA receptors consist of , , and receptor binding is unaffected by this change. Furthermore, subunits; in addition to the benzodiazepine site, these recep- this mutation does not seem to alter the functioning of tors also contain distinct sites for the binding of GABA, GABA receptors because direct agonists stimulate the recep- barbiturates, and ethanol. These various regions act as allo- tor but indirect modulators of GABA do not. Although psychotherapeutic effects such as anxiolysis receptor subunits in the regulation of stress- and anxiety- are achieved through facilitation of GABA transmission at related behaviors, investigators have generated mutant mice this receptor, drugs that act as GABAA receptor agonists with alterations in the expression of specific GABA recep- A also produce several deleterious side effects. It was initially reported that deletion of the which differences in GABAA receptor subunit composition subunit led to a selective (94%) reduction in the expres- 2 might contribute to possible dissociations between the bene- sion of benzodiazepine sites in the CNS without alterations ficial and negative effects of these compounds is currently in the level of GABA sites or changes in the expression of being investigated. Thus, knockout A 2 mice possessed functional GABAA receptors that responded normally to GABA site ligands or barbiturates, but did not GABA System Transgenic Mice respond to benzodiazepines; these findings led to the con- The synthesis of GABA is regulated by two isoforms of the clusion that the 2subunit is not necessary for the formation enzyme glutamate decarboxylase (GAD), GAD67, and the of functional GABAA receptors, but is required to create 894 Neuropsychopharmacology: The Fifth Generation of Progress the benzodiazepine-responsive site of those receptors. Mice rated from the negative side effects of these compounds, that were homozygous for the mutation, however, did not and that the 1 subunit of the GABAA receptor is likely live past weaning in this study. In mice carrying only one to mediate some of these potentially harmful properties of copy of the functional 2 gene, a 20% reduction in benzodi- benzodiazepines. Interestingly, McKernan and colleagues azepine sites was observed, but these mice did not show (160) demonstrate that a novel benzodiazepine-site ligand overt developmental deficits. In a recent study, a detailed that binds to GABAA receptors containing 2, 3,or 5 characterization of the behavioral profile of these animals subunits but avoids receptors with the 1 subunit produces was carried out. Heterozygotes displayed a decrease in the a behavioral profile that is identical to that of the 1 subunit number of entries into and amount of time spent in the knockout mice; in normal mice, this compound decreases open arms of an elevated plus maze and the bright compart- murine anxiety-like behaviors without eliciting sedation or ment of a light-dark box. Finally, 2 heterozygotes Stress-Related Disorders were found to react to partially conditioned stimuli (only weakly paired with aversive consequences) as if they were As stated above, the most widely used GABA system-based full and potent predictors of threat; compared to wild-types, drugs for the treatment of anxiety are the benzodiazepines, which showed low levels of defensive behaviors to the par- which facilitate GABA transmission through the GABAA tially conditioned stimulus, heterozygotes displayed high receptor. As outlined in the previous section, the search for levels of conditioned freezing to the partial conditioned novel compounds that may act selectively at specific GABAA stimulus that were identical to those displayed by all animals subunits is ongoing, with the ultimate hope of discovering in response to the full conditioned stimulus. This profile ligands that produce anxiolysis but do not cause some of has been proposed to be a model for the tendency to inter- the serious side effects that are commonly associated with pret neutral situations as threatening that is seen in anxiety benzodiazepines. Taken together, the results from this extensive be- leagues (160), drugs that selectively target certain GABAA havioral profile indicate that / mice have increased receptor subunits may hold great promise for the treatment 2 neophobia and stress-like responses and may thus provide of anxiety without harmful side effects. This development a model for increased anxiety-like behaviors (154—156). The use of targeted genetic in 2 heterozygotes were blocked by the benzodiazepine alterations in identifying the roles of various GABAA sub- diazepam, suggesting that this animal model may also have units will undoubtedly aid in this effort to create 'designer good predictive validity for identifying clinically effective drugs' for the treatment of anxiety (158). It is also extremely important to mention the 1 subunit General Issues and Caveats of Transgenic transgenic mice, whose behavioral profiles have been thor- Animal Studies oughly and insightfully reviewed in recent articles (157, 158). In these mice, a single amino acid is altered (histidine As mentioned above, mice carrying certain mutations replaced by arginine at the 101 position of the peptide) in within either the CRH, the 5-HT, or the GABA system the 1 subunit of the GABAA receptor complex. It appears that these change does not produce any overt alterations in baseline genetically engineered mouse models also have some predic- responses to stress in the genetically altered mice; these ani- tive validity; the stress-like endophenotype observed in at mals behave similarly to wild-type controls in tests such least two of the aforementioned models is normalized by as the elevated plus maze and the fear-potentiated startle administration of a clinically effective antianxiety agent that paradigm, a measure of conditioned fear (159,160). Thus, acts within the system that was genetically targeted. It re- under drug-free, normal conditions, these animals do not mains to be determined, however, the extent to which these display a behavioral pattern that is consistent with an anxi- genetically altered models serve to identify potential anti- ety-like endophenotype. When these mice are treated with anxiety agents from different chemical classes. For example, conventional benzodiazepines, however, they react very dif- do benzodiazepines reduce stress-like effects of CRH over- ferently to the drug than their wild-type counterparts. The extent to which the stress-like endopheno- with the mutation in the 1 subunit display a normal reduc- type in these animals is altered by compounds that act on tion of stress-induced anxiety-like behaviors after benzodi- systems that were not directly targeted by the genetic muta- azepine treatment, but fail to display some of the more tion will aid in determining the generalizability and utility deleterious side effects associated with this class of drugs of these models as predictors of novel anxiolytic agents. These results indicate one assumes that these animals provide a model of inherent that the anxiolytic effects of benzodiazepines can be sepa- trait-like anxiety, they can serve as a powerful tool for Chapter 62: Animal Models and Endophenotypes of Anxiety and Stress Disorders 895 screening new potential anxiolytics. These models do pro- contributions to the development of stress and anxiety-like vide a sound approach to study the long-term effects of endophenotypes in animals, further information is needed congenital abnormalities in these neurotransmitter and neu- to understand the precise nature of gene–environment in- ropeptide systems. It is likely that a particular Several broad issues should be considered when inter- stressor results in alterations of gene expression in myriad preting studies utilizing genetically altered mice.

The first step was a baseline which all the practices were required to meet claritin 10 mg line allergy treatment dr oz. This was a mandatory requirement to remain a member of this CCG cheap 10mg claritin fast delivery allergy symptoms jaw pain. The second step was to standardise the local enhanced services offer. This meant that practices (in cluster form if necessary) were asked to improve their range of services so as to meet an acceptable standard. This started out as a voluntary exercise but increasingly became a requirement. The third step was a higher level of innovation in services offered. The CCG used a budget provided from the centre, which was geared towards care for the elderly, to invite bids for new enhanced services in this area. We first describe the primary care improvement programme (a pseudonym of the title actually used by this CCG) as it was presented in official terms. We then present an analysis of how the programme was received and understood by multiple agents, including some of the designers of the programme and those who were the recipients. The official picture The main initiative driven by this CCG was a service improvement programme designed to make a step change in the quality of primary care. The key declared objective was to: Reduce the level of variation in general practice and bring all practices up to the same standards of primary care. Through [primary care improvement programme] we will ensure there is universal coverage of services across our member practices and that these services are available for all patients, regardless of where they live. Case B: CCG policy document Attention focused on holistic care, integrated care, long-term conditions management and better care for the elderly and vulnerable. The constituent elements of the new model of primary care included universal coverage of some basic service standards across the whole CCG population; an overarching framework that allowed the freedom to identify creative solutions for how patients receive their care while ensuring accountability for care remains with practices; delivery of a patient-centred and integrated approach to improving primary care management of long-term conditions; and an up-skilled general practice workforce to deliver services that had previously been provided by secondary/community providers. Integration, access to mental health services and a transformation of urgent care were all elements of the total package of reform. The new plan from the top leadership duo was for a more integrated system built around general practices. This included new models of care with GPs and others working in new ways with support from secondary care, while also bringing in associated community services, community nursing and district nursing. Supporting elements included data sharing and use of the BCF to integrate social care. The problems of pressures on general practice, fragmentation and lack of a universal and equitable service provision were further reasons justifying action. In exchange for extra funding and support, the GP practices in the CCG were expected to offer care closer to home, delivering a wider range of tests and investigations in primary care settings, such as electrocardiography, spirometry and insulin initiation. These steps were expected to reduce referrals to secondary care. Each service area, such as diabetes care, chronic obstructive pulmonary disease and asthma, had an associated set of target outcomes. For example, the plan stated that 90% of patients with type 2 diabetes should be managed outside the acute trust. The plans also included a place for appraisal of practices in relation to their conformance. None of these interventions was without controversy. A number of GPs were very reluctant to accept the changes. The leadership skills of the CCG chairperson and accountable officer were very necessary. They undertook institutional work in conceptualising the required nature of the changes and in the creation of new institutional forms in place of long-standing and embedded institutional practices, which tended to emphasise and privilege professional autonomy. The programme included some elements of PPI and, as was indicated in the survey results, clinicians are sometimes leaders of this process, as in this case. Part of this was through ongoing channels, as in the cases of respiratory and mental health, but in addition there were some special stakeholder consultation events. These included talks with the Patients Council and other patient participation groups. The part played by clinicians as front communicators was seen as very important in this context.

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In one example claritin 10 mg sale allergy testing and zantac, three groups indepen- CONDITIONAL MUTATIONS dently generated lines of mice with null mutations of the 5-HT1A receptor subtype (21–23) order 10mg claritin otc allergy treatment for foods. Interestingly, although New technologies are under development for circumventing each group placed this mutation on a different genetic back- the limitations of standard gene targeting approaches by ground, all observed enhanced anxiogenic-like behaviors in creating mutations that may be induced in adult animals the mutant lines. Thus, particularly strong evidence is pro- and/or restricted to particular brain regions. Although these vided for a contribution of the 5-HT1A receptor to the strategies are not yet in widespread use, it is likely that rapid regulation of anxiety. This potential problem may Cell Type–Specific Mutation Strategies be addressed through breeding programs to place targeted When a null mutation of a gene results in a mutant pheno- mutations on different inbred backgrounds, and by the gen- type, limitations in the interpretation of that phenotype can eration of ES cell lines derived from other inbred strains. It is possible that the absence of a gene inherent limitations. The null mutations engineered into product in the periphery may lead to embryonic lethality, knockout mice are typically constitutive, i. For genes that are widely expressed within the CNS, Therefore, the potential for developmental perturbations is it may be difficult to identify neural circuits through which a major caveat to the interpretation of mutant phenotypes mutations produce behavioral perturbations. It may be difficult to determine whether inactivate genes in restricted subpopulations of the cells that a mutant phenotype reflects a normal adult role for the normally express them will be a valuable asset in studies to targeted gene or an indirect effect of the mutation attribut- uncover the neural mechanisms underlying neural pheno- able to perturbed development. Conversely, if significant com- veloped to exert spatial control over the pattern of expres- pensation for the loss of a gene product occurs during devel- sion of genetic changes introduced into mice. This approach opment, then the severity of the mutant phenotype may utilizes somatic cell recombination rather than germ cell (or underestimate the functional significance of the gene prod- embryonic stem cell) recombination to inactivate a gene in uct. The nature of such compensatory mechanisms and the restricted populations of cells or tissues. In this approach, extent to which they exist may be difficult to determine. It had been previously demonstrated that tion to only those cells expressing the recombinase. The widespread gene inactivation in NMDAR1 null mutants two recombinase systems that have been utilized for genetic resulted in perinatal lethality (36,37). When the mutation manipulation in mice have been the Flp-frt system from was restricted to hippocampal CA1 neurons, animals were yeast (26), and the Cre-lox system from bacteriophage P1 viable and exhibited impaired spatial learning and impaired (27–29), with the large majority of reports using this tech- plasticity at CA1 synapses (31). Thus, spatial restriction of nique utilizing the Cre-lox system. This approach, lines will speed and simplify the production of animals with then, involves generating two independent lines of mice—a restricted patterns of gene inactivation. Animals Inducible Mutation Strategies with a gene or gene region of interest flanked by loxP sites (floxed) are generated by gene targeting. Because the loxP As described above, the absence of a gene product through- sites are relatively small and placed in intronic regions, they out development complicates the interpretation of mutant do not typically interfere with normal gene transcription. Efforts are currently under way to overcome Of course, WT patterns and levels of expression need to be this limitation through the use of gene expression systems documented in these floxed mouse lines, because inadvert- that may be induced in the adult animal. Strategies are ent placement of lox sites into promoter elements or RNA under exploration for achieving this goal using a variety of splice sites could disrupt gene expression. The Cre mice are compounds, such as tetracycline, steroid receptor antago- most commonly generated by creating a transgenic line of nists, and ecdysone to induce gene expression. Although mice in which Cre expression is driven by a tissue-specific these approaches have yet to be optimized for general use, promoter. As discussed above in the section on transgenic this development is likely to be close at hand. The tetracy- mice, variability in transgenic expression patterns requires cline system has been the most utilized and best developed several lines of Cre mice that need to be generated and approach to inducible gene regulation. An alternative strat- Since the introduction of the Tet system by the Bujard egy is to use gene targeting procedures to place Cre under laboratory in 1992, many laboratories have validated the the control of an endogenous promoter (30). The advantage utility of this approach to inducible gene regulation, and of this approach is that Cre expression should closely ap- many refinements/improvements in the system have been proximate the WT expression pattern of the gene it is replac- introduced (38). A potential disadvantage is that Cre may coli, in which the transcription of tetracycline resistance disrupt expression of the gene into which it has integrated. When tetracycline is present, tetR binds is identified, it is crossed with an appropriate floxed line to the tetracycline and loses its capacity to bind to the operator commence a breeding strategy resulting in the generation sequences (tetO) located within the promoter of the tetracy- of animals with a restricted pattern of gene inactivation (Fig. The first example of this approach was the DNA binding and activation specificity of the tetR. The inactivation of the glutamate receptor subunit NMDAR1 desired regulatable gene of interest is placed under tetO plus in CA1 pyramidal neurons of the hippocampus, with a minimal promoter (Pmin), that contains the basic promoter expression in other brain areas mostly intact (31).

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This ious regions purchase claritin 10 mg fast delivery allergy katy tx, including the substantia nigra purchase claritin 10mg free shipping allergy shots worth it, 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.

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