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Significant necrosis order cialis jelly 20mg without a prescription erectile dysfunction treatment psychological, often accompanied by inflammation and gliosis order 20mg cialis jelly visa erectile dysfunction meme, was identified at injection sites with some genetically engineered HSV-1 vectors used for in vivo studies (see purchase cialis jelly discount zma impotence, e. However, the achievement of a more favorable ratio of vector to helper, and the virtual elimination of wild-type virus in the vector preparations (10) has greatly reduced the cytotoxicity of present-day de- fective HSV-1 amplicon vectors (see, e. An additional improvement to the packaging procedure, the banding of the virus on a sucrose step gradient, followed by a high-speed centrifugation to pellet the virus, has reduced FIGURE 20. Different vector constructions for coexpressing further the cytotoxicity of the virus preparations. Glutamate receptor GluR1 and green fluorescent protein (GFP)are used as examples. A troubling problem that has not yet been resolved for any viral vector used in the brain, except perhaps for the An improvement in gene transfer methods in general has lentiviral vector (see below), is that of persistence of expres- been the incorporation of the gene for the green fluorescent sion. Numerous investigators have had the experiences re- protein (GFP) into many vectors. In the example shown, the objec- estingly, superinfection with helper virus 5dl1. Apparently, GFP) by putting an internal ribosome entry site (IRES) transactivating factors provided by the helper virus reacti- between the two genes, which are then transcribed from the vated transcription of the transgene. The IRES enables independent translation Two recent developments suggest that the problem of of the two coding sequences even though they are present persistence of expression is not insoluble. In theory, the two fragment of the tyrosine hydroxylase promoter to drive re- coding sequences should be expressed at similar levels, but porter gene expression in an HSV-1 amplicon vector re- in practice the translation of one of the two coding se- sulted in prolonged gene expression in vivo (16), suggesting quences on the mRNA often occurs at the expense of the that neuronal, unlike viral, promoters in HSV-1 vectors other. Alternatively, they can be expressed from two inde- have the potential to produce stable gene expression. Addi- pendent transcriptional units, in a bicistronic vector. The tionally, the development of hybrid amplicons that incorpo- addition of an extra transcriptional cassette to the vector rate elements that allow autonomous replication of the epi- makes it larger and more unwieldy to use; and the level of some (17) or that incorporate adeno-associated virus (AAV) transcription from one promoter is independent of the level elements for genomic integration of the amplicon (18–20) of transcription from the other, so that the transgenes may have resulted in vectors that support long-term gene expres- be expressed at very different levels. Third, the GFP can be sion both in vitro and in vivo. This is a trickier construction, since the can be concentrated to very high titers ( 1010/mL). How- two coding sequences must be placed in frame with each ever, the use of adenovirus vectors continues to be restricted other. However, the added benefit of being able to track by the robust host immune response that they elicit (21, the subcellular location of the transgene product makes this 22). Foreign TRANSFER INTO NEURONS genes are cloned into easy-to-manipulate amplicon vectors that can be packaged directly into viral particles as head- Despite the problems that remain with the HSV-1 vector, to-tail repeats in the presence of the helper virus, with no it is a gene delivery system that has come of age. In addition, intermediate recombination step required. This enables numerous alternative and increasingly user-friendly means rapid construction of a large number of recombinant vectors of gene transfer into the brain are now available (Table simultaneously, and is particularly useful for those who are 20. Adenovirus vectors, like HSV-1 vectors, infect post- doing mutation analysis, and who wish to work with multi- mitotic cells and can enter a broad range of mammalian ple genes. Such ease of cloning is not possible with the cell types. They have the additional advantages that they genomic HSV and adenovirus vectors. Direct in vivo transfer of genes into the brain has been achieved using not only herpes virus and adenovirus vectors, but also adeno-associated virus vectors (see refs. COMPARISON OF VIRUSES USED TO review) and lentivirus vectors (see ref. In MANIPULATE GENE EXPRESSION IN THE BRAIN contrast to other viral vectors, adeno-associated virus vectors Advantages Disadvantages do not cause an immune response or toxicity. Interestingly, the ability of adeno-associated virus vectors to transduce Herpesvirus vectors and express transgenes is not equivalent in all regions of the Broad host and cell type range Occasional cytotoxicity brain (26,27). Only in some regions will neurons bind and Episomal (no possibility of Lack of persistence of insertional activation of host expression internalize the virus, with resultant long-term expression. Long-term expression of -galac- of foreign DNA tosidase and GFP was observed in rat neurons for at least High level of expression of 9 months following intracerebral injection of the vectors, foreign genes within hours Can be concentrated to high titers with no sign of tissue pathology or immune response (28, Helper virus–free stocks possible 29). Progress has been made in achieving biosafety with Adenovirus vectors these vectors, by eliminating viral sequences nonessential Broad mammalian host and Elicits host immune for transduction. To date, the delivery of recombi- cell type range 4. One of the most success- Lentivirus vectors ful uses of this strategy has been in the field of addiction Integrates into host chromosome Can accommodate only research. Exposure to drugs of abuse causes many changes 6–8 kb of foreign DNA in gene expression within the brain. A major challenge in Expression is persistent Low titers addiction research is to determine which of these changes Potent human pathogen have a direct influence on behavior. In the case of addiction, it is thus trols their function. High expression of GluR1 favors the possible to mimic certain aspects of the drug-exposed state formation of Ca2 -permeable (GluR1-homomeric) AMPA without ever administering the drugs themselves. Examples of this subunit contains a motif that blocks Ca2 conductance behavioral changes in addiction that may result from drug- (38). Because repeated drug exposure is known to selectively induced alterations in biology (gene expression) are compul- increase the electrophysiologic responsiveness of VTA dopa- sive drug use (drug-taking) and craving (drug-seeking). However, this question is difficult to ad- Addiction Circuitry dress using traditional methods. For example, AMPA ago- nists and antagonists cannot be used to study the relation- Much research on the neuronal circuitry involved in drug ship between GluR1 expression and sensitized drug addiction has focused on the mesolimbic dopamine (DA) responses because they affect AMPA receptor function gen- system. However, the neural It was first necessary to determine if viral-mediated eleva- events that mediate the acute rewarding effects of abused tions in GluR1 expression within the VTA would increase drugs are not understood, nor are the neuroadaptations that sensitivity to the locomotor-stimulating effects of morphine, presumably underlie the transition from occasional drug use a hallmark of behavioral sensitization (36,42). Furthermore, increased activity in rats given HSV- enon that may contribute to the addiction process (36). GluR1 was seen only in response to morphine, and was This altered sensitivity is presumably a consequence of al- not evident after saline. The transient nature of the HSV- tered gene expression, and the VTA-NAc circuitry is a logi- mediated elevations in transgene expression allowed the be- cal starting point for biobehavioral studies. Because several havioral adaptations to be correlated with the time course robust and reliable drug-induced neuroadaptations have of GluR1 expression: when morphine was given only on been discovered within this circuitry (37), it has been the days 7 and 8 after microinjection—when elevations in focus of gene transfer studies in which the behavioral signifi- GluR1 expression had dissipated—the rats that were given cance of altered gene expression has been assessed. To date, HSV-GluR1 were no longer more sensitive to the stimulant the biobehavioral significance of three specific, drug-in- effects of morphine. When some rats given HSV-GluR1 duced changes in gene expression have been studied using were tested with morphine on days 3 to 4 and on days 7 viral-mediated gene transfer—the ability of drugs (cocaine, to 8, significant increases in sensitivity to the locomotor- morphine) (a) to increase expression of the AMPA (gluta- stimulating effects of the persisted in rats given HSV- mate) receptor subunit GluR1 in VTA, (b) to alter expres- GluR1, despite the fact that GluR1 labeling in the VTA sion of GluRs in the NAc, and (c) to increase the activity had dissipated. Thus when morphine is given while GluR1 of the transcription factor CREB (cAMP response element expression in the VTA is elevated, increased sensitivity to binding protein) in the NAc. These data suggest that altered expression of GluR1 in the VTA underlies, at least in part, the development and GluRs in the VTA expression of sensitized behavioral responses to morphine. AMPA receptors are made up of various combinations of The effect of elevated GluR1 expression in the VTA on the subunits GluR1, GluR2, GluR3, and GluR4 (collec- the rewarding effects of morphine was examined using place tively called GluRs) (38,39).

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However cheap cialis jelly 20mg with mastercard erectile dysfunction kidney transplant, the limited availability and ethically controversial nature of the tissue source have re- CONCLUSIONS stricted the utility of fetal grafts in this disorder order cialis jelly now erectile dysfunction pills. As an alter- native purchase generic cialis jelly pills impotence from smoking, a relatively unlimited supply of homogenous, well- Progress in molecular neurobiology has dramatically characterized viral vectors could theoretically be produced changed our understanding of psychiatric disorders. A sig- to deliver tyrosine hydroxylase, the rate-limiting enzyme in nificant proportion of these findings have been obtained dopamine synthesis. Attempts have also been made in ani- from animal experiments and postmortem human studies. Similar techniques of gene therapy have and to a lesser extent with SPECT, is ideally suited for been investigated in motor neuron degenerative diseases and such in vivo applications because of its extraordinarily high Alzheimer disease. Reporter genes whose probes can cross sensitivity and improving anatomic resolution (now about the blood–brain barrier, such as D2 receptors, can monitor 2 mm). This chapter has reviewed what is arguably the the expression of these transfected genes. On the other hand, most difficult barrier to accomplishing in vivo molecular dopamine release from the grafts could be monitored by a imaging—the development of useful and quantifiable conventional technique utilizing competition of radioligand tracers. The blood–brain barrier is a challenge to both the binding to D2 receptors, as described in the section on delivery of radiolabeled tracers and the quantification of estimation of endogenous neurotransmitter levels. However, many successful this technique of receptor displacement has been used to tracers have been developed to date. These probes have detect dopamine release in patients with embryonic nigral largely been synthesized as analogues of agents active at syn- transplants (89). Rela- imaging with small probes for relevant gene or oncogene tively little progress has been made in measuring protein products, is hampered by the development of useful intracellular signal transduction or gene expression. As described in the section on the two areas are clearly important targets for future ligand de- required properties of an in vivo tracer, it is difficult to fulfill velopment. By bridging new findings in molecular neurosci- all the requirements for a successful brain-imaging agent. Many new anticancer agents are being developed, and a significant number of these agents target signal trans- duction systems, which may also play pathophysiologic roles in psychiatric disorders (90,91). For example, Ras farnesyl- REFERENCES transferase is a target for cancer chemotherapy and poten- 1. Can receptors be imaged tially also for brain imaging. Localized 1H NMR cations, including farnesylation, Ras binds to the cell measurements of gamma-aminobutyric acid in human brain in membrane and transmits signals. Among them, a recently developed agent has tions of metabolites in the adult human brain in vivo: quantifica- a high affinity (93) and may be used as a template from tion of localized proton MR spectra. PET studies of a small molecule ligand for epidermal growth factor receptor binding competition between endogenous dopamine and the D1 11 11 has been labeled with C and has shown brain uptake (94). Rapid developments in molecular biology and the advent 5. In: of gene-targeting techniques have enabled the study of indi- Bendriem B, Townsend DW, eds. The theory and practice of 3D vidual genes in mice by means of in vitro experimental tech- PET. Recently developed animal-dedicated PET devices tion of microPET: a high-resolution lutetium oxyorthosilicate (e. J Nucl Med 1999;40: of about 2 mm and can now image these animalsin vivo 1164–1175. Performance results of a SPECT measurements of amphetamine-induced dopamine re- new DOI detector block for a high resolution PET–LSO research lease in nonhuman primates. Relationship of octanol/water partition coefficient and lated dopamine release competes in vivo for [123I]IBZM binding molecular weight to rat brain capillary permeability. Kinetic properties of the accumulation tein binding on in vivo activity and brain penetration of glycine/ of 3H-raclopride in the mouse brain in vivo. Imaging D2 receptor tween lipophilicity and brain extraction of C-11-labeled radio- occupancy by endogenous dopamine in humans. Regional rat brain distribution ing of amphetamine-induced dopamine release in drug-free of iodinated benzamides. Mapping cocaine binding trations: evidence from a novel positron emission tomography sites in human and baboon brain in vivo. Persistence of haloperi- mine transmission in schizophrenia: confirmation in a second dol in human brain tissue. Removal of en- of the 5-HT1A receptor radioligand, [O-methyl- C]WAY- dogenous dopamine reveals elevation of D2 receptors in schizo- 100635, in rat, monkey and humans plus evaluation of the brain 11 phrenia. Exquisite deline- ligand characteristics and task length for detection of activation. Principles of tracer kinetic modeling in changes with bolus or infusion delivery on neuroreceptor ligands. In: Phelps J Cereb Blood Flow Metab 1998;18:1196–1210. Comparison and autoradiography: principles and applications for the brain and heart. Parameter estimation in positron emission tomogra- receptor. Imaging extrastriatal emission tomography and autoradiography: principles and applica- dopamine D2 receptor occupancy by endogenous dopamine in tions for the brain and heart. Kinetic analysis of central the D2 dopamine receptor in calf brain. Mol Pharmacol 1983; [11C]-raclopride binding to D2 dopamine receptors studied with 25:10–17. Quantification of benzodi- tion by dopamine agonists. Sequestration of dopamine D2 and a single-experiment protocol. J Cereb Blood Flow Metab 1995; receptors depends on coexpression of G protein-coupled receptor 15:284–300. A quantitative model for the in vivo assessment of drug binding sites with posi- binding: evidence for accumulation in corpus striatum by ago- tron emission tomography. Phasic versus tonic dopamine release and modulation 1988;8:291–303. Cortical regulation of subcortical systems and its possi- of the rat: light and electron microscopy. Neuroscience 1995;65: ble relevance to schizophrenia. The regulation of forebrain dopa- cellular distribution of dopamine D2-like receptors: an immuno- mine transmission: relevance to the pathophysiology and psycho- cytochemical study of subtype-specific antibodies in rat and pathology of schizophrenia. Positron emission tomog- of [11C]raclopride: combined PET–microdialysis studies.

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In a randomized cheap cialis jelly 20mg without a prescription impotence 60 years old, controlled trial order genuine cialis jelly line erectile dysfunction ed treatment, compli- looking specifically at atypical agents discount cialis jelly 20mg on line erectile dysfunction fertility treatment, although it is gener- ance therapy was found to improve insight and observer- ally believed that reduced relapse rates reported with olan- rated adherence to treatment over an 18-month treatment zapine and clozapine may reflect, in part, improved compli- period (147). Patients in the compliance therapy group also ance (97,126). Compliance can be compromised by Cramer and Rosenheck (148) demonstrated that interven- psychosis, agitation, and comorbid substance abuse (137, tions that assist patients in remembering to take medica- 138). Van Putten (139) studied compliance in 85 schizo- tions, such as placing microchip schedulers on pill bottles, phrenia patients chronically treated with conventional neu- can also substantially improve compliance. Medication refusal was associ- Treatment Resistance ated with an early dysphoric response, which Van Putten attributed to subtle akathisia. Analysis of responses by 150 Estimates of the incidence of treatment resistance have var- schizophrenia patients to a 'Drug Attitude Inventory' re- ied with changes in the diagnostic classification of schizo- vealed that, based on responses to 10 items, 89% of patients phrenia and definitions of treatment response (149), which could be correctly assigned to compliant versus non- have tended to obscure potential improvements in outcome compliant categories as determined by clinician assessment associated with advances in pharmacologic and psychosocial of compliance (140). For example, Hegarty and colleagues (150) re- was a positive experience with medication—this factor ac- viewed results of 320 clinical trials and found that, since the counted for 60% of the total variance, whereas the factor introduction of modern antipsychotics in the mid-twentieth representing a negative subjective experience accounted for century, about 50% of patients were improved at follow- 12%. Factors representing attitudes and beliefs about medi- up, whereas the rate of improvement dropped to 35% in cation had minimal predictive power. Rates of response have tended to be higher in first- Whereas many clinicians expect atypical agents to achieve episode psychosis, although dropout rates have been high higher levels of compliance by virtue of reduced or absent in this population, particularly with conventional agents EPS, this view may seriously underestimate the impact of (102,107). Persistence of psychotic symptoms is more com- other side effects. Two studies have found that clinicians mon in drug trials involving chronic patients, presumably tend to misjudge the relative distress produced by different reflecting progression of the illness as well as a possible selec- medication side effects (142,143). Side effects associated tion bias favoring participation by more refractory patients. The advan- or failure to achieve premorbid levels of functioning, treat- tage of atypical agents in terms of compliance may stem ment resistance can be considered the rule rather than the less from their reduced EPS and more from their improved exception. Whether targeting cognitive deficits and impairment in in- Psychotic Symptoms sight will improve compliance remains to be seen. Antipsychotic Monotherapy Response of psychotic symptoms to conventional antipsy- Psychosocial Interventions chotics, risperidone, and olanzapine has been associated Most approaches to noncompliance involve psychoeduca- with D2 receptor occupancy in excess of 65% (18,57), al- tion, supervision, and supportive therapy in which the bene- though persistence of psychotic symptoms has been shown fits of treatment are emphasized, whereas barriers to adher- to occur despite adequate D2 blockade in a subgroup of ence and medication side effects are minimized (145). As noted, only clozapine has con- Family therapy and social skills training may also exert a sistently demonstrated efficacy for psychotic symptoms in positive impact on compliance. Cognitive behavioral ap- treatment of refractory patients; the mechanism responsible proaches have recently been applied to noncompliance by for this therapeutic advantage remains uncertain. In a sam- Kemp and colleagues (146,147), who developed 'compli- ple of 268 patients prospectively established to be neurolep- ance therapy,' a four- to six-session intervention based on tic resistant, 30% in the clozapine group met criteria for motivational interviewing techniques that targets attitudes response at 6 weeks compared to 7% treated with chlorpro- towards medication and discharge planning during acute mazine (11). Response rates as high as 60% have been re- Chapter 56: Therapeutics of Schizophrenia 785 ported after 6 months in open trials with clozapine in pa- therapy as an intervention for neuroleptic-resistant patients; tients less rigorously defined as treatment refractory (152). Given the risk mine efficacy of clozapine and other atypical agents is the of agranulocytosis, the burden of side effects, and the re- subject of debate (153,154). Marder and colleagues (155) found that priate first choice among these agents is unclear; two con- schizophrenia patients presumed to be treatment-resistant trolled studies that compared olanzapine and risperidone on the basis of having been hospitalized for 6 months or have produced divergent results, probably reflecting differ- longer at the time of study entry did not respond to haloper- ences in dosing of the two agents and the use of intent-to- idol 20 mg per day but significantly improved with risperi- treat versus completer analyses (63,163). The focus of this done 6 mg per day or 16 mg per day compared to placebo. Many clinicians response of psychotic symptoms to olanzapine (mean dose express the impression that certain patients do respond pref- 11 mg per day) than haloperidol (mean dose 10 mg per erentially to a single agent of this class. Sequential controlled day); this difference was significant in the intent-to-treat trials of the newer agents in treatment-resistant patients will analysis but not in a comparison of completers (76). In 67 schizophrenia patients with his- The practice of combination therapy is gaining widespread tories of neuroleptic resistance, risperidone 6 mg per day popularity in the absence of controlled data in its support significantly improved total BPRS scores compared to halo- (164). In part based on empirical experience and the dem- peridol 15 mg per day at 4 weeks, but response did not differ onstration that clozapine at optimal doses achieves relatively between groups at 8 weeks (156). In contrast, risperidone low degrees of D2 occupancy, European clinicians com- produced significantly higher response rates than haloperi- monly add low-doses of neuroleptics to clozapine in par- dol in a large, randomized open trial involving 184 schizo- tially responsive patients (165). Uncontrolled trials and case phrenia patients with a history of poor response (157). Rela- reports have described benefits associated with the addition tive response of psychotic symptoms to risperidone of risperidone (4 mg per day) (159,166) and pimozide (167) increased over time and reached a maximum improvement to clozapine in partially responsive patients. In a small, pla- compared to haloperidol at the final 12-month assessment. The same group cians perceive improved response during the cross-tapering reported that 41% of 44 patients identified as unresponsive phase of switching from one to the other. A theoretical to olanzapine in the preceding study or in an open trial rationale for this combination is less apparent, given that subsequently exhibited a response to clozapine (158). In each agent produces maximal D2 and 5-HT2 occupancy addition, open trials in which patients have been switched when appropriately dosed (57). If combined treatment with from clozapine to olanzapine or risperidone have reported olanzapine and risperidone is found in suitably controlled a high incidence of clinical deterioration, casting doubt on study designs to offer advantages over optimal monotherapy claims for therapeutic equivalence between clozapine and with either agent, such a finding would argue in favor of the second-generation agents, at least at the doses tested the existence of additional contributory receptor actions (159,160). Of interest, two controlled trials have found unique to each drug. How- ever, in one 4-week trial, the 59 participants were not Adjunctive Treatments screened for treatment resistance at baseline and, despite A diverse range of adjunctive treatments has been proposed equivalence in outcomes between groups using an LOCF for antipsychotic-resistant schizophrenia, although thera- analysis, 25% of the risperidone group dropped out owing peutic effects generally have been small or inconsistent in to lack of efficacy compared to only 5% in the clozapine controlled trials. Very little data are available from con- group (161). Lithium augmentation frequently has been cited as 786 Neuropsychopharmacology: The Fifth Generation of Progress the best-established intervention based on positive results unresponsive to ECT and a diagnosis of schizoaffective dis- from three small studies (170–172); however, two recent order did not predict a favorable response (192–195). Cases placebo-controlled studies found no benefit when well-char- describing the successful combination of ECT with cloza- acterized neuroleptic-resistant patients were treated with pine in refractory patients have also been reported, suggest- lithium (approximately 1. Recently, interest has fo- lithium may enhance response of some patients, particularly cused on the potential use of transcranial magnetic stimula- in the presence of affective symptoms or excitement (175, tion (TMS) as an alternative to ECT in schizophrenia. Carbamazepine augmentation of conventional neuro- has shown promising efficacy in depression (201–203). In leptics has been associated with modest reductions in persis- a preliminary, sham-TMS controlled crossover study in 12 tent symptoms, including tension and paranoia, in several medication-resistant schizophrenia patients, the frequency controlled trials (177–179), particularly in patients with and severity of auditory hallucinations were significantly abnormal EEGs or violence. However, induction of hepatic reduced following 12 to 16 minutes of stimulation (204). This is an intriguing in one report, resulted in clinical deterioration (181). Val- area for future research, both as a tool to explore the neural proate does not significantly affect serum concentrations circuits underlying symptoms of schizophrenia as well as a of most antipsychotic drugs, but results from two small potential treatment option in medication-resistant cases. Was- sef and colleagues (182) reported efficacy for negative symp- Psychosocial Interventions toms and global psychopathology associated with addition A particularly promising psychosocial approach to medica- of divalproex to haloperidol in a placebo-controlled 12-week tion-resistant psychotic symptoms is cognitive-behavioral trial in 12 schizophrenia patients hospitalized for acute exac- therapy (CBT) (205). In contrast, Ko and colleagues (183) found no of alliance formation, examination, and challenge of psy- effect when valproic acid was added to conventional neuro- chotic beliefs, and the teaching of self-monitoring and cop- leptics in six treatment-resistant patients in a placebo-con- ing skills. Four randomized trials, all performed in the trolled crossover design. Augmentation with benzodiaze- United Kingdom, demonstrated superior efficacy for CBT pines also has been advocated, in part, because of the compared to active control treatments on measures of global potential role of GABAergic agents in modulating dopa- psychopathology and positive symptoms among chronic, mine transmission, although the evidence for efficacy is not medicated patients (206–209). Short-term, acute treatment with high- mined that the between-groups effect size was.

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Indeed order cialis jelly with visa erectile dysfunction at 21, -adrenergic re- ing; higher order functions such as these are typically abol- ceptor antagonists and 2-receptor agonists are effective in ished when a situation is found threatening and defensive the treatment of certain anxiety-related symptoms in hu- behaviors are recruited (103 order cialis jelly with mastercard erectile dysfunction 2015,104) order line cialis jelly erectile dysfunction heart disease. In addition, recent preclinical evidence indicates that ing transgenic mice may represent a genetically engineered a variety of central nervous system (CNS) peptides includ- model of a murine anxiety-like endophenotype. BP knockout mice show increased stress-like behaviors. Although these peptide and neurotransmitter systems CRH-BP knockout mice displayed decreases in open arm undoubtedly play a role in stress- and anxiety-related behav- entries and open arm time in an elevated plus maze, and iors, the focus of the following section will be the CRH, showed a decrease in the number of exits from a safe box 5-HT, and GABA systems because these three systems are in a defensive withdrawal/open field paradigm (105). These perhaps the most thoroughly studied with transgenic results indicate a heightened level of neophobia in these models. Moreover, CRH-BP knockouts have reduced body provided valuable new information about the genetic regu- weight gain over several weeks (105,106), which is also syn- Chapter 62: Animal Models and Endophenotypes of Anxiety and Stress Disorders 891 tonic with increased basal CRH activity. The behavioral in the development of anxiety-related endophenotypes profile of CRH-BP knockouts is similar to that which is (123). Related Disorders Thus, as with the CRH-overexpressing mice, CRH-BP knockouts may represent another rodent anxiety-like endo- A large body of preclinical literature indicates that CRH is phenotype. In terms of the predictive validity of these a critical modulator of stress and anxiety-like behaviors in models, CRH receptor antagonists have been found to block nonhuman primates and rodents (107,124). Based on the the stress-like behavioral profile observed in these animals; ability of CRH1 receptor-selective antagonists to block one recent clinical study indicates that CRH1 receptor an- many of the behavioral effects of stress or CRH administra- tagonists may indeed prove to be effective anxiolytics or tion, these antagonists have been proposed as potentially antidepressants (108). Although certain endocrinologic responses has also revealed that the CRH system is a critical deficits are observed in CRH knockout mice, stress-related modulator of this index of anxiety-related behavior. For behavioral function in these animals remains relatively unaf- example, administration of CRH into the cerebral ventricles fected as assessed by multiple stress-related paradigms of nonhuman primates results in a constellation of behav- (109–114). This sparing of normal stress responsivity may ioral responses that closely resemble the defensive responses be due to compensatory increases in the expression of other that are exhibited upon presentation of a stressor (124). Deletion of the Consistent with the notion that increased levels of CRH are associated with increased anxiety-like responding are the CRH1 receptor gene, however, does appear to consistently recent findings that small-molecule CRH1 receptor antago- result in a putative reduction in anxiety (115–117). For nists block the expression of some behavioral, physiologic, example, CRH1 knockout mice show increased exploration and neuroendocrine responses to stressors in rhesus mon- of the open arms on an elevated plus maze and spend more keys (126,127). The first report of an open-label clinical time in the brightly lit compartment of a dark-light transi- trial with a CRH1 antagonist was recently published, and tion box than do wild-type controls. Moreover, CRH1 revealed a significant effect of this compound in ameliorat- knockout mice appear to be immune to the anxiogenic ef- ing symptoms of depression and anxiety (108). Studies of CRH2 recep- further research is needed to firmly establish the utility of tor knockout mice, on the other hand, indicate that these CRH1 antagonists as psychotherapeutic agents and also to mice display a less consistent behavioral profile than the determine the possible side effects associated with their use, CRH1 knockout mice (118–120). Part of the behavioral these preliminary data support the notion that these com- profile of CRH2 knockouts is suggestive of increased stress- pounds represent an important new class of drugs that may like responding, but other aspects of the behavioral profile offer great promise for the treatment of illnesses associated indicate either no alteration of stress-related responding with increased anxiety and stress. The observed increases in anxiety-like behaviors in these genetically altered mice may be due to increased levels of The 5-HT System brain CRH and/or urocortin; in two of the three studies, Serotonin is a member of the monoamine family of trans- an elevation of baseline CRH or urocortin mRNA levels in mitters that also include dopamine and norepinephrine. As the CNS was seen in CRH2 knockout mice (118,119). It should be noted that 5-HT produces its effects through at least 15 different 5- acute blockade of CRH2 receptors results in a decrease in HT receptors that are differentially distributed throughout stress-induced defensive behaviors; thus the behavioral pro- the CNS; the principal mode of 5-HT inactivation is cellu- file of these animals is opposite to that of mice that are lar reuptake via terminal transporter proteins (129). Thus, the timing HT system has long been implicated in the regulation of of the gene deletion may critically influence the nature of mood states and anxiety, and selective serotonin reuptake the behavioral phenotype that ensues. Future studies utiliz- inhibitors (SSRIs) constitute a major class of antidepressants ing novel inducible-knockout technologies may help in clar- that have anxiolytic effects. As outlined below, 5-HT trans- ifying the developmental versus acute role of various genes mission also plays a critical role in the regulation of anxiety- 892 Neuropsychopharmacology: The Fifth Generation of Progress like behaviors. This proposed mechanism is consistent with the the paucity of highly selective ligands for these multiple increase in stress-related behaviors that are seen in certain target sites, several investigators have employed murine gene transgenic mice with mutations in the GABAA receptor (see targeting strategies to elucidate the roles of specific 5-HT below). Further work is necessary to determine the precise receptors in the regulation of stress and anxiety. In contrast to 5-HT1A knockout mice, mice that lack Studies of targeted gene deletions within the 5-HT system the 5-HT receptor show decreased anxiety-like behaviors 1B have revealed an important role for this system in the regula- in several tests of approach-avoidance conflicts. The knockout mice spend more time in the center of an open behavioral sequelae of disrupting 5-HT receptor gene field and more readily explore novel objects than their wild- expression have been elegantly summarized in several review type controls; this profile is opposite from that of 5-HT1A articles (89,130–132). Perhaps the best-characterized 5-HT knockout mice, and is suggestive of diminished neophobia mutant mice are the 5-HT1A and 5-HT1B receptor knock- (89,137). Consistent with this pattern of results is the find- outs. Mice with a mutation in the 5-HT1A receptor gene ing that as pups, 5-HT1B mice emit fewer ultrasonic vocali- have been found to display increased stress-like behaviors zations when separated from their mothers; separation- in multiple tests of approach-avoidance conflicts. These ani- induced vocalizations are thought to provide a measure of mals show decreased entries into and time spent in the more anxiety and distress in pups (138,139). It is interesting to aversive region in paradigms such as the open field, elevated plus maze, and the elevated zero maze; thus 5-HT knock- note, 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.