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V. Thorus. Samuel Merritt College.

It is interesting to aversive region in paradigms such as the open field generic 50 mg asendin amex, elevated plus maze cheapest asendin, and the elevated zero maze; thus 5-HT knock- note generic asendin 50 mg overnight delivery, however, that no changes in contextual or cue-induced 1A out mice avoid the center of an open field, the open arms conditioned freezing are observed in 5-HT1B mutant mice, of a plus maze, and the unenclosed regions of a zero maze suggesting that approach-avoidance conflicts and condi- (133–135). It is worth noting that this 'increased anxiety' tioned fear may be differentially modulated by the 5-HT pattern of results was found consistently across three differ- system. The other main behavioral effect of constitutive ent research labs, indicating its robustness and reproducibil- 5-HT1B receptor deletion is a marked increase in aggressive ity. Consistent with this profile is the finding that these behavior (89,140,141). This increase in 5-HT1B knockout mice may also provide valuable informa- stress-like responding is not accompanied by changes in tion on the neural and genetic factors associated with stress overall locomotor activity or motor and spatial coordina- and anxiety-related functioning (89,142). It should be noted that mice with null mutations of other Curiously, 5-HT1A knockout mice display increased mobil- 5-HT receptor subtypes have also been generated, but these ity in response to an acute stressor such as forced swimming animals have not been found to display as robust an anxiety- or tail suspension (133–135). Taken together, these find- related behavioral profile as the 5-HT or 5-HT knock- 1A 1B ings indicate that 5-HT1A knockout mice may represent out mice. It has been found that 5-HT receptor knockout 5A another animal endophenotype of increased anxiety. These knockout mice also do not respond ingly, a recent report indicates that this mutation alters differently from control subjects in tests of startle reactivity GABA system expression and function (136). It has been or in burying a probe that delivered a brief electric shock. Anal- ysis of brain tissue from these animals indicates that GABA profile from that of the 5-HT1A or 5-HT1B knockout mice. A receptor binding is reduced and that the expression of An initial report indicates that 5-HT6 receptor deficient 1 and subunits of the GABA receptor are decreased in the mice may exhibit increased avoidance of aversive environ- 2 A amygdala. The anxiolytic actions of benzodiazepines may in ments; although these preliminary findings are interesting, part be mediated by GABAA receptors within the amygdala; further work is needed to fully characterize the phenotype the profile of results in 5-HT1A knockout mice has led to of these mutant mice (144,145). Mice lacking either the the intriguing speculation that the anxiety-like endopheno- 5-HT2A or 5-HT2C receptors have also been created; to the type in these mice may actually in part derive from a de- best of our knowledge, the stress-related behavioral func- crease in the expression and function of the GABAA recep- tioning of these animals has yet to be reported (146,147). Chapter 62: Animal Models and Endophenotypes of Anxiety and Stress Disorders 893 Clinically Effective 5-HT System Drugs for Stress- shorter form GAD65 (149). Whereas GAD67 is thought Related Disorders to maintain basal GABA levels, GAD65 is thought to regu- late the synthesis of GABA at nerve terminals in response As mentioned above, one of the most commonly prescribed to high GABA demand (150). Given the important role and effective classes of drugs that is used in the treatment of GABA in inhibitory neurotransmission associated with of depression and anxiety is the SSRIs, which block the anxiolysis, several investigators have evaluated the behav- reuptake of 5-HT by its transporter and thereby increase ioral profile of genetically altered mice that lack the GAD65 serotoninergic transmission. Two separate groups have reported that GAD65 clinical studies including those obtained from 5-HT recep- mice display an increase in stress-like behaviors in numerous tor knockout mice, 5-HT1A agonists have been developed behavioral paradigms (151,152). The clinical utility of this class had fewer entries into and time spent in the center of an of compounds, however, remains to be determined. As these open field or the open areas of an elevated zero maze (similar transgenic approaches develop and become more refined, to an elevated plus maze), indicating that they were more they will undoubtedly aid in clarifying the roles of the many avoidant of inherently aversive areas. Similarly, these mice other 5-HT receptor subtypes in processes related to stress had lower levels of activity in the bright portion of a light- and anxiety and will aid in drug development. It should be mentioned that GAD65 / mice also displayed an elevation in the occur- The GABA System rence of spontaneous and stress-induced seizures, and that these mice had a dramatically increased mortality rate start- The primary inhibitory neurotransmitter in the CNS is ing at 4 to 5 weeks after birth (151). Thus, although the GABA; GABA-synthesizing cells are distributed throughout behavioral profile of GAD65 knockout mice is suggestive the brain (128). The actions of GABA are mediated by two of increased anxiety-like responses, it is possible that these major classes of receptors, GABAA and GABAB, both of effects are secondary to the occurrence of seizures and to which modulate the activity of ion channels. The principal the factors leading to early lethality. The usefulness of this mode of inactivation of GABA transmission is the presynap- knockout as a model for anxiety-related deficits may there- tic reuptake of GABA by its transporter protein. 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, several important differences exist be- GABA receptor by enhancing GABA-induced chloride A tween the two. 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.

In contrast buy cheap asendin 50mg on line,guanine nucleotides have the chemical function because this region is thought to be the ability to selectively decrease the affinity of agonists for their primary site of G-protein coupling and signal transduction order asendin 50 mg mastercard. Consistent with CRF receptors glycosylation sites discount asendin 50 mg line,which are analogous to those found on being coupled to a guanine nucleotide regulatory protein, the CRF receptor subtype. The genomic structure of the the binding of [125I]oCRF to pituitary,brain,and spleen 1 human CRF2 receptor gene is similar to that of the mouse homogenates is reciprocally increased by divalent cations CRF receptor described in the preceding and has 12 in- such as Mg2 and decreased by guanine nucleotides. Fur- 1 trons,the last ten of which interrupt the coding region in thermore,in expressed cell lines using a -galactosidase re- identical positions. These gene sequences,however,diverge porter system,CRF and related analogues could stimulate significantly at the 5′ end,and the chromosomal mapping the production of -galactosidase in whole cells with the of the human CRF2 gene has been localized to chromosome same pharmacologic rank order of potencies as those in a 7 p21-p15. The cloning of the CRF2 receptor subtype gave the first indication that other family members of this receptor system Pharmacologic Characteristics exist and have unique properties that could subserve func- The literature is replete with information on the pharmaco- tions that were previously undefined. As mentioned,a fun- logic and biochemical characterization of CRF receptors in damental element in the characterization of any receptor a variety of tissues and animal species. The initial observations clearly demonstrated that endocrine,and immune tissues have used the available ra- the CRF2 receptor subtype recognized the nonmammalian dioligands at the time,which were [125I]-Tyr0 oCRF,[125I]- analogues of CRF with high affinity (similar in profile to Tyr0 r/hCRF,and [125I]-Nle21-Tyr32 r/hCRF. These lig- the CRF subtype) but unlike the CRF receptor,had low 1 1 ands have all demonstrated high affinity for the CRF1 recep- affinity for the endogenous CRF ligands (r/hCRF and its tor subtype and lower affinity for the CRF2 subtypes (as analogues) (19). Thus,the available radioligands used in the described in the following). Thus,the discovery of the CRF2 initial studies of CRF receptors were not useful in providing receptor subtype and its isoforms has not confused the ear- information about this subtype. Recently,[125I]-Tyr0 sauvagine,a novel radioligand recently described (34). Using one of the high-affinity non- for the CRF2 receptor,has been described that binds to mammalian analogues of CRF (sauvagine),a radiolabel was both receptor subtypes with equal affinity and has become developed,and its binding specificity and selectivity deter- a useful tool in the study of CRF receptors (34). The specific signal for the labeling of the human hensive understanding of this system and its role in both CRFa receptors was greater than 85% over the entire con- normal and pathologic physiology. The ra- Autoradiographic Localization of CRF dioligand bound in a reversible,time- and protein-depen- Receptor Subtypes dent manner,reaching equilibrium within 60 minutes with the binding being stable for at least 4 hours at 22 C. Scatch- Many studies to date have described the distribution of CRF ard analyses demonstrated an affinity of about 200 pM for receptors in various tissues,including the pituitary,brain, the CRF2 receptor subtype and a maximum receptor density and spleen (29,31–33). The autoradiographic localization in the expressing cells of about 180 fmol/mg protein (34). The intermediate lobe shows a more cal to the in vitro effects of the same unlabeled peptides in uniform distribution of binding sites characteristic of the the production of cAMP in cells expressing the receptor as homogeneous population of POMC-producing cells in this described. Overall,the distribution pattern of CRF1 receptors and urotensin I that were more potent in stimulation of within the pituitary supports the functional role of CRF cAMP production were also more potent at inhibiting the as the primary physiological regulator of POMC-derived binding of [125I]sauvagine than oCRF or r/hCRF. Interest- peptide secretion from the anterior and intermediate lobes ingly,the putative antagonists for CRF receptors,D- of the pituitary. PheCRF(12-41) and -helical CRF(9-41) exhibited ap- Receptor autoradiography and binding studies in discrete proximately equal affinity for the two receptor subtypes areas of rat and primate CNS demonstrate that,in general, either in inhibiting [125I]sauvagine binding or inhibiting the highest concentration of CRF binding sites are distrib- sauvagine-stimulated cAMP production (34). These data uted in brain regions involved in cognitive function (cere- clearly indicated that although distinct pharmacologic dif- bral cortex),limbic areas involved in emotion and stress ferences exist between the two receptor subtypes of the same responses (amygdala,nucleus accumbens,and hippocam- family (in terms of their rank order profile),they still must pus),brainstem regions regulating autonomic function share some structural similarities. Further study is required (locus ceruleus and nucleus of the solitary tract),and olfac- to determine the precise common structural features of these tory bulb. In addition,there is a high density of CRF1 two family members. Although there is as yet no direct red pulp and marginal zones. The localization of [125I]oCRF evidence,this modulation of the binding of [125I]sauvagine binding sites in mouse spleen to regions known to have to the human CRF2 receptor by guanine nucleotides sug- a high concentration of macrophages suggests that CRF gests that this receptor exists in two affinity states for ago- receptors are present on resident splenic macrophages. The nists coupled through a guanine nucleotide binding protein absence of specific [125I]oCRF-binding sites in the periarter- to its second messenger system. Unfortunately to date,the iole and peripheral follicular white pulp regions of the spleen only ligands available for the biochemical study of these suggests that neither T nor B lymphocytes have specific receptors have been agonists,making it very difficult to high-affinity CRF receptors comparable to those localized examine the proportions and affinities of high- and low- in the marginal zone and red pulp areas of the spleen or in affinity states of these receptors. CRF2 receptors has allowed a detailed examination of the The high affinity of the nonmammalian CRF analogues regional and cellular distribution of CRF receptor subtype for this subtype has raised the possibility that other endoge- mRNA expression utilizing both RNAse protection assays nous mammalian ligands exist that have high affinity and and in situ hybridization histochemistry. A comparison of selectivity for this receptor subtype. As described,the recent the distribution of CRF1 and CRF2 mRNA and receptor discovery of urocortin (36),although not selective for the protein defined by ligand autoradiography is demonstrated CRF2 subtype,has provided the first evidence for one such in adjacent horizontal sections of rat brain (Fig. With the increase in the complexity of the CRF that of the CRF1 and exhibits a distinct subcortical pattern. For example, the lateral septum,by virtue of widespread reciprocal con- nections throughout the brain,is implicated in a variety of physiologic processes. These range from higher cognitive functions such as learning and memory to autonomic regu- lation,including food and water intake (38). In addition, the septum plays a central role in classical limbic circuitry and thus is important in a variety of emotional conditions, including fear and aggression. Thus,the lack of CRF1 recep- tor expression in these nuclei suggests that CRF2 receptors may solely mediate the postsynaptic actions of CRF inputs to this region and strongly suggests a role for CRF2 receptors in modulating limbic circuitry at the level of septal activity. In addition,the selective expression of CRF2 receptor mRNA within hypothalamic nuclei indicates that the anxio- genic and anorexic actions of CRF in these nuclei may likely be CRF2 receptor-mediated. In contrast,within the pitui- tary,there is a predominance of CRF1 receptor expression with little or no CRF2 expression in either the intermediate and anterior lobes,indicating that it is the CRF1 receptor that is primarily responsible for CRF regulation of the HPA axis. In addition to the differences in distribution between the CRF1 and CRF2 receptor subtypes,there exists a distinct pattern of distribution between the CRF2 isoforms (CRF2 and CRF2 ) as well. The CRF2 isoform is primarily ex- pressed within the CNS,whereas the CRF2 form is found both centrally and peripherally. Digitized, color-coded images of CRF1 (Panel A) and CRF form is the predominant one,whereas the CRF CRF (Panel B) receptor mRNA expression and receptor autoradi- 2 2 2 ography in adjacent horizontal sections of rat brain. The highest form is localized primarily to non-neuronal structures,the levels of mRNA expression are coded in red, whereas the lowest choroid plexus of the ventricular system,and cerebral arteri- concentrations are coded in blue. The identification of the CRF form in cere- of receptors labeled with either [125I]oCRF (CRF only; Panel C)or 2 1 [125I]sauvagine (CRF and CRF ; Panel D) are coded in red. There bral arterioles suggests a mechanism through which CRF 1 2 was a good correspondence between the message for a particular may directly modulate cerebral blood flow. Peripherally,the receptor subtype and its protein localization; the pharmacologic highest detectable levels of mRNA were found in heart and selectivity was retained for the two radioligands. Taken together, the results of these studies demonstrating a distinct heterogeneous distribution pattern of CRF receptor subtypes in brain and peripheral tissues, strongly suggest that these receptor subtypes subserve very erally corresponded to the previously reported distribution specific physiological roles in CRF related function both of [125I]oCRF binding sites (Fig. Using the radioligand [125I]sauvagine described, Receptors CRF2 receptors could be localized to areas of high CRF2 Radioligand binding studies have demonstrated that CRF message. In addition,because [125I]sauvagine has equal af- receptors in the brain-endocrine-immune axis are coupled finity for both receptor subtypes (34),the autoradiography to a guanine nucleotide regulatory protein. In all of these revealed the localization of both the CRF1 and CRF2 recep- tissues,the primary second messenger system involved in tor subtypes,demonstrating the utility of this novel radioli- transducing the actions of CRF is stimulation of cAMP Chapter 7: Corticotropin-Releasing Factor 99 production (29,31–33,40). CRF initiates a cascade of enzy- cDNA and Amino Acid Sequences matic reactions in the pituitary gland beginning with the The CRF-BP was first isolated and purified to near homoge- receptor-mediated stimulation of adenylate cyclase,which neity for sequencing and generation of oligonucleotide ultimately regulates POMC-peptide secretion and possibly probes (47). Screening a human liver cDNA library using synthesis. POMC-derived peptide secretion mediated by the probes generated from the original amino acid sequence activation of adenylate cyclase in the anterior and neuroin- revealed a full-length cDNA containing a 1.

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