The group of investigators led by T. Cannon and A. Silva has recently described an interesting mouse model of inducible expression of the fragment of DISC1 (DISC1-cc) that is deleted in the affected members of the Scottish family. DISC1-cc expression is regulated by the CAMKII promoter, which is active in forebrain neurons. DISC1-cc spans residues 671-852, which is the portion of DISC1 crucial for binding to NUDEL and Lis1 (Morris et al., 2003; Ozeki et al., 2003; Kamiya et al., 2006). The DISC1-cc protein was fused to a HA-tagged mutant (G521R) estrogen receptor LBD (Kida et al., 2002). In this inducible system, the transgenic protein is degraded after sequestration by heat-shock chaperone proteins. When tamoxifen is administered to transgenic mice, it binds the LBD, the fusion protein complex, which includes DISC1-cc, undergoes a conformational switch such that the transgenic protein dissociates from the chaperone proteins and becomes functional. After a quick metabolism of tamoxifen, the transgenic protein acquires nonfunctional state again (Li et al., 2007).
Consistent with previous reports (Morris et al., 2003; Ozeki et al., 2003; Brandon et al., 2004; Kamiya et al., 2006), DISC1-cc was found to bind to Nudel and Lis1 6 h after induction, but not 2 days after induction. Similar to the study by Pletnikov et al., binding of DISC1-cc to Nudel and Lis1 decreased endogenous DISC1 protein in DISC1/ Nudel complexes (dominant-negative effects), indicating that this inducible transgenic system is useful for elucidating the neurobehavioral effects of disruption of endogenous DISC1 function during selective periods of brain development. Expression of the DISC1-cc protein was detected in the cortex, hippocampus, striatum, and cerebellum of the transgenic mice. Transgenic mice appeared normal and displayed no gross abnormalities. Locomotor activity in open field and anxiety-related responses in elevated plus maze were comparable between transgenic and control mice.
By regulating expression of DISC1-cc, the neurobehavioral effects of the dominant-negative DISC1-cc construct were compared between mice that expressed DISC1-cc at postnatal day 7 vs. adulthood. DISC1-cc mice were tested in a spatial working memory test [the delayed non-matched to place (DNMTP) task]. The adult DISC1-cc transgenic mice with induction at postnatal day 7 showed decreased percentage of correct DNMTP
choices compared with adult WT mice treated with tamoxifen, adult DISC1-cc transgenic or adult WT mice treated with vehicle. Importantly, no differences between DISC1-cc and WT mice treated with tamoxifen were found when this compound was injected in adulthood. The results clearly showed that disruption of DISC1 function early in development but not adulthood was responsible for deficient spatial working memory.
Another example of the developmental effects of DISC1-cc included depression-like behavior in the forced swimming test. The transgenic mice treated with the tamoxifen at day 7 had shorter latencies to floating compared control groups. In contrast, induction of expression during adulthood had no effects in this test. Thus, similar to the cognitive effects, disruption of endogenous mouse DISC1 early in development but not adulthood produced depressive-like phenotype. The effects of DISC1-cc on social behaviors were assessed in a three-chambered apparatus, which allows for evaluation of sociability and preference for social novelty (Moy et al., 2004). Induction of expression of DISC1-cc at day 7 altered the normal pattern of social activity so that transgenic mice spent the similar amounts of time with a live mouse and an inanimate object, whereas control mice demonstrated a clear preference for the live partner. Thus, social activity was also affected by induction of the fragment early in development, and no such effects were noted because of adult expression of DISC1-cc. Possible alterations in neuronal structure and function during development were analyzed to explain the behavioral deficits. Induction of expression of the fragment at day 7 but not in adulthood produced attenuated dendritic complexity in mice. Notably, this reduction in dendritic arborization was associated with reduced basal synaptic transmission, although presynaptic functions seemed to remain unaffected by activation of the DISC1-cc protein.
Taken together, this study was the first to convincingly demonstrate that disruption of DISC1 functions during early postnatal period but not adulthood can produce a set of behavioral, neuroanatomical, and neurophysiolo-gical alterations that are consistent with aspects of schizophrenia and mood disorders. The weak points of the study include expression of the DISC1-cc fragment that is the deleted C-terminal portion of human DISC1, a brief expression of the fragment, and manipulations with postnatal expression only.
Another successful example of using inducible genetic mouse model to explore the developmental etiology of schizophrenia-like neurobehavioral abnormalities has been reported by Kelly and associates (Kelly et al., 2009). Their study sought to evaluate a role of the G-protein subunit Gas in the pathophysiology of neurobehavioral schizophrenia-like abnormalities in mice. Recently, a polymorphism that increases mRNA levels of Gas was associated with schizophrenia (Kelly et al., 2009).
The bi-transgenic model used is based on the Tet-off system in which the Gas transgene with a hemagglutinin epitope that is placed downstream the tetracycline operator (tetO). Gas transgenic mice was found to express high levels of the transgene restricted to the cortex, hippocampus, dorsal striatum and ventral striatum, and low levels in the amygdala and in the inferior colliculus. Expectedly, expression of the transgene produced increased levels of Gas mRNA in those regions. As a result, Gas mice had elevated basal and GTPgs-stimulated adenylate cyclase activity.
The authors analyzed the relative contributions of developmental vs. postnatal expression of the transgene to schizophrenia-related behavioral alterations. Gas mice exhibited unaltered startle responses, but their PPI was significantly attenuated. Notably, both Gasdev (developmental expression) and Gasadult (adult expression only) mice had similar PPI compared to control littermates, suggesting both developmental and adulthood over-expression of Gas were critical for manifestation of the PPI deficit. Administration of a typical antipsychotic, haloperidol (1 mg/kg, i.p.) was able to ameliorate the PPI deficits in Gas mice. Compared to control animals, Gas mice exhibited increased horizontal locomotor activity and a trend toward increased rearing activity in open field. Interestingly, Gasadult mice, but not
Gasdev mice, demonstrated increased horizontal movements, suggesting that expression of the transgene during adulthood produced locomotor hyperactivity.
Developmental expression of Gas was necessary and sufficient to produce spatial learning deficits in adult mice. When hippocampus-dependent spatial learning and memory in the hidden-platform Morris water maze were assayed, both Gas mice (expression throughout development and adulthood) and Gasdev (developmental expression only) mice displayed significantly greater escape latencies during training compared to control littermates, indicating impaired spatial learning. In contrast, Gasadult mice with expression during adulthood only did not differ from Gasdox mice treated with DOX (no expression) in learning rate in the test. Poorer memory of the location of the hidden platform was also detected in Gas and Gasdev mice. Gasadult mice (adult expression only) displayed an intermediate phenotype, while Gasdox mice (no expression) had no alterations in this test, suggesting that the cognitive effects observed in Gas mice were likely related to expression of the transgene rather than non-specific gene insertion effects.
As Gasadult mice did not have any changes in spatial learning but were inferior in the impaired spatial memory phase of MWM, the authors hypothesized that adult over-expression of Gas might selectively affect memory retrieval. To address this hypothesis, a one-trial Pavlovian fear conditioning task was used. Gas and control mice showed comparable amounts of freezing during the training session, suggesting that over-expression of the transgene did not alter the pain sensitivity in mice. Cue-dependent short-term and long-term conditional fear was not changed in transgenic mice compared to control littermates. In contrast, Gas mice displayed significantly impaired short-term and long-term contextual fear that is hippocampus-dependent. When expression of the transgene was present only during development, no abnormalities were seen in long-term memory for contextual fear. However, adult expression of the transgene was associated with lower levels of contextual fear.
Using an elegant experimental design, the authors tried to evaluate if the effects of Gas on contextual fear were related to selective alterations in acquisition, consolidation or retrieval. They compared control littermates to Gas mice that expressed the transgene during training but not retrieval by administering DOX beginning 24 h after training until the day of the retrieval test (i.e., Gastraining group) or expressing the transgene during retrieval but not training (DOX was given 2.5 weeks before training until 24 h following training; Gasretrieval group). In order to have sufficient time for changing the expression status of the transgene, freezing to the training context was measured 2.5 weeks following training. Gastraining and control animals had similar levels of freezing. In contrast, compared to non-expressing control mice, freezing behavior in Gasretrieval mice was significantly attenuated.
In addition to the behavioral tests, the study sought to analyze the effects of over-expression of the transgene on the brain morphology. MRI experiments found that Gas expression was associated with a dramatic enlargement of the lateral ventricles, which was found to be independent of changed total brain volumes that were moderately reduced. Curiously, suppression of expression of the transgene during adulthood for as long as 6 weeks was not able to reverse the ventricular enlargement. By regulating timing of expression of the transgene, the authors found that developmental expression led to a striking enlargement of the lateral ventricles and an associated significant reduction in sizes of the dorsal and ventral striatum, and, to a lesser extent, frontal cortex and dorsal hippocampus. Similar to developmental expression, adult expression of Gas also produced significantly enlarged lateral ventricles accompanied by a smaller dorsal and ventral striatum. Interestingly, the authors noted that the extent of the change in the lateral ventricles and ventral striatum was less prominent in Gasadult than Gasdev mice, suggesting the major contribution to the brain pathology of developmental expression of the transgene.
The authors also performed pharmacological and biochemical studies that suggested that the Gas-induced behavioral deficits may be associated with compensatory decline in levels of hippocam-pal and cortical cyclic AMP (cAMP). These decreases in cAMP may be responsible for reduced activation of the guanine exchange factor Epac (also known as RapGEF 3/4) since the select Epac agonist, 8-pCPT-20-O-Me-cAMP, increased PPI and improved memory in mice. Thus, the authors hypothesize that the developmental impact of increased Gas expression could lead to a specific phenotypic manifestation and that Epac could be a novel target for the treatment of both developmentally regulated and non-devel-opmentally regulated symptoms associated with schizophrenia
Increased activity of D2 receptors (D2Rs) in the striatum has been linked to the pathophysiology of schizophrenia (Meisenzahl et al., 2007). To determine directly the behavioral and physiological consequences of increased D2R function in the striatum, Kellendonk et al. (2006) have generated transgenic mice with inducible expression of D2R restricted to the striatum (Kellendonk et al., 2006; Drew et al., 2007; Bach et al., 2008). I will only briefly describe this study and model. For more details, the reader is referred to Chapter 7.
Transgenic D2 receptors were functional and transgenic mice had 15% higher receptor-binding capacity than their littermates reminiscent of the 12% increase observed in schizophrenic patients (Laruelle et al., 1996). D2R transgenic mice did not exhibit reliable alterations in locomotor activity in open field, sensorimotor gating as tested by PPI, or anxiety assayed elevated plus maze. In contrast, in a series of very elegant and sophisticated cognitive tests, the authors observed moderate but significant abnormalities, consistent with cognitive impairments found in patients with schizophrenia, particularly deficits on tests of executive function, traditionally considered related to frontal lobe dysfunctions (Ross et al., 2006).
Another type of executive function abnormal in subjects with schizophrenia is attentional set-shifting and cognitive and behavioral flexibility (Kellendonk et al., 2009). An attentional set-shifting paradigm based on olfactory discrimination was used (Birrell and Brown, 2000; Colacicco et al., 2002). Although transgenic mice demonstrated normal performance in the original odor-reward association task, they were found to show significantly increased latencies to choose between the two odors during the reversal trials, indicating a subtle deficit in flexibility to reverse the original rule. D2 receptor over-expression was found to produce increased dopamine levels, decreased dopamine turnover, and increased D1 receptor activation in the medial PFC, factors which could contribute to the behavioral abnormalities.
The major advantageous feature of inducible systems is the ability to evaluate timing of the effects of expression of a transgene in question. To determine if the observed behavioral abnormalities resulted from to on-going hyperactivity of DA neurotransmission in the striatum or were rather related to developmental effects of over-expression of D2 receptors, mice were given DOX food to shut off expression in the transgene. This 2-week DOX treatment did not change the pre-existing cognitive deficit despite normalization of the binding capacity of D2 receptor in the striatum. The authors conclude that "the concurrent expression of trans-genic D2 receptors in the adult animal is not responsible for the cognitive deficit; chronic or developmental expression of the receptors is sufficient to cause it" (Kellendonk et al., 2006). In order to evaluate the contribution of developmental over-expression of D2 receptors to the behavioral deficit, expression of the transgene was turned off at birth. D2 transgenic mice were found to continue to exhibit the impairment in the working memory task. Thus, the persistence of this deficit strongly suggests the critical contribution of over-expression during development.
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