Kimberly L. Stark

Serotonin1A receptor acts during development to establish normal anxiety-like behaviour in the adult.

Serotonin is implicated in mood regulation, and drugs acting via the serotonergic system are effective in treating anxiety and depression. Specifically, agonists of the serotonin1A receptor have anxiolytic properties, and knockout mice lacking this receptor show increased anxiety-like behaviour. Here we use a tissue-specific, conditional rescue strategy to show that expression of the serotonin1A receptor primarily in the hippocampus and cortex, but not in the raphe nuclei, is sufficient to rescue the behavioural phenotype of the knockout mice. Furthermore, using the conditional nature of these transgenic mice, we suggest that receptor expression during the early postnatal period, but not in the adult, is necessary for this behavioural rescue. These findings show that postnatal developmental processes help to establish adult anxiety-like behaviour. In addition, the normal role of the serotonin1A receptor during development may be different from its function when this receptor is activated by therapeutic intervention in adulthood.

Impaired hippocampal–prefrontal synchrony in a genetic mouse model of schizophrenia

Abnormalities in functional connectivity between brain areas have been postulated as an important pathophysiological mechanism underlying schizophrenia. In particular, macroscopic measurements of brain activity in patients suggest that functional connectivity between the frontal and temporal lobes may be altered. However, it remains unclear whether such dysconnectivity relates to the aetiology of the illness, and how it is manifested in the activity of neural circuits. Because schizophrenia has a strong genetic component, animal models of genetic risk factors are likely to aid our understanding of the pathogenesis and pathophysiology of the disease. Here we study Df(16)A+/– mice, which model a microdeletion on human chromosome 22 (22q11.2) that constitutes one of the largest known genetic risk factors for schizophrenia. To examine functional connectivity in these mice, we measured the synchronization of neural activity between the hippocampus and the prefrontal cortex during the performance of a task requiring working memory, which is one of the cognitive functions disrupted in the disease. In wild-type mice, hippocampal–prefrontal synchrony increased during working memory performance, consistent with previous reports in rats. Df(16)A+/– mice, which are impaired in the acquisition of the task, showed drastically reduced synchrony, measured both by phase-locking of prefrontal cells to hippocampal theta oscillations and by coherence of prefrontal and hippocampal local field potentials. Furthermore, the magnitude of hippocampal–prefrontal coherence at the onset of training could be used to predict the time it took the Df(16)A+/– mice to learn the task and increased more slowly during task acquisition. These data suggest how the deficits in functional connectivity observed in patients with schizophrenia may be realized at the single-neuron level. Our findings further suggest that impaired long-range synchrony of neural activity is one consequence of the 22q11.2 deletion and may be a fundamental component of the pathophysiology underlying schizophrenia.

Palmitoylation-dependent neurodevelopmental deficits in a mouse model of 22q11 microdeletion

Individuals with 22q11.2 microdeletions have cognitive deficits and a high risk of developing schizophrenia. Here we provide evidence that primary hippocampal neurons from a mouse model of 22q11.2 deletion (Df(16)A+/− mice) have decreased density of dendritic spines and glutamatergic synapses, as well as impaired dendritic growth. These deficits were prevented by introduction of the enzymatically active ZDHHC8 palmitoyltransferase encoded by a gene in the 22q11.2 locus, and they were also observed in primary cultures from Zdhhc8-deficient mice. Many of these deficits were also present in the hippocampi of adult Df(16)A+/− and Zdhhc8-deficient mice. Finally, we provide evidence that PSD95 is one of the substrates of ZDHHC8. Our analysis reveals that 22q11.2 microdeletion results in deficits in neuronal development and suggests that impaired neuronal protein palmitoylation contributes to many of these deficits.

The 22q11.2 microdeletion: Fifteen years of insights into the genetic and neural complexity of psychiatric disorders

Over the last fifteen years it has become established that 22q11.2 deletion syndrome (22q11DS) is a true genetic risk factor for schizophrenia. Carriers of deletions in chromosome 22q11.2 develop schizophrenia at rate of 25–30% and such deletions account for as many as 1–2% of cases of sporadic schizophrenia in the general population. Access to a relatively homogeneous population of individuals that suffer from schizophrenia as the result of a shared etiological factor and the potential to generate etiologically valid mouse models provides an immense opportunity to better understand the pathobiology of this disease. In this review we survey the clinical literature associated with the 22q11.2 microdeletions with a focus on neuroanatomical changes. Then, we highlight results from work modeling this structural mutation in animals. The key biological pathways disrupted by the mutation are discussed and how these changes impact the structure and function of neural circuits is described.

Derepression of a Neuronal Inhibitor due to miRNA Dysregulation in a Schizophrenia-Related Microdeletion

22q11.2 microdeletions result in specific cognitive deficits and schizophrenia. Analysis of Df(16)A(+/-) mice, which model this microdeletion, revealed abnormalities in the formation of neuronal dendrites and spines, as well as altered brain microRNAs. Here, we show a drastic reduction of miR-185, which resides within the 22q11.2 locus, to levels more than expected by a hemizygous deletion, and we demonstrate that this reduction alters dendritic and spine development. miR-185 represses, through an evolutionarily conserved target site, a previously unknown inhibitor of these processes that resides in the Golgi apparatus and shows higher prenatal brain expression. Sustained derepression of this inhibitor after birth represents the most robust transcriptional disturbance in the brains of Df(16)A(+/-) mice and results in structural alterations in the hippocampus. Reduction of miR-185 also has milder age- and region-specific effects on the expression of some Golgi-related genes. Our findings illuminate the contribution of microRNAs in psychiatric disorders and cognitive dysfunction.

Knockout Mice Reveal Opposite Roles for Serotonin 1A and 1B Receptors in Prepulse Inhibition

The serotonergic system is involved in the modulation of prepulse inhibition (PPI) and habituation of startle, which are deficient in schizophrenia patients. PPI is the reduction in startle amplitude that occurs when a weak “prepulse” precedes a startling stimulus by 30–500 msec. The roles of 5-HT1A and 5-HT1B receptors in modulating PPI and habituation were examined using wild-type (WT), 5-HT1A knockout (1AKO), and 5-HT1B knockout (1BKO) mice. The 5-HT1A/1B agonist RU24969 reduced PPI and habituation in WT and 1AKO, but not 1BKO mice, whereas the 5-HT1A agonist 8-OH-DPAT increased PPI in WT and 1BKO, but not in 1AKO mice. Similarly, the selective 5-HT1B agonist anpirtoline reduced PPI in WT, but not in 1BKO mice. In experiments using intact 129Sv mice, the 5-HT1A agonist flesinoxan increased PPI while anpirtoline decreased PPI and habituation. Findings suggest that 5-HT1B receptor activation decreases PPI and habituation, and 5-HT1A receptor activation increases PPI in mice.

The pattern of cortical dysfunction in a mouse model of a schizophrenia-related microdeletion.

We used a mouse model of the schizophrenia-predisposing 22q11.2 microdeletion to evaluate how this genetic lesion affects cortical neural circuits at the synaptic, cellular, and molecular levels. Guided by cognitive deficits, we demonstrated that mutant mice display robust deficits in high-frequency synaptic transmission and short-term plasticity (synaptic depression and potentiation), as well as alterations in long-term plasticity and dendritic spine stability. Apart from previously reported reduction in dendritic complexity of layer 5 pyramidal neurons, altered synaptic plasticity occurs in the context of relatively circumscribed and often subtle cytoarchitectural changes in neuronal density and inhibitory neuron numbers. We confirmed the pronounced DiGeorge critical region 8 (Dgcr8)-dependent deficits in primary micro-RNA processing and identified additional changes in gene expression and RNA splicing that may underlie the effects of this mutation. Reduction in Dgcr8 levels appears to be a major driver of altered short-term synaptic plasticity in prefrontal cortex and working memory but not of long-term plasticity and cytoarchitecture. Our findings inform the cortical synaptic and neuronal mechanisms of working memory impairment in the context of psychiatric disorders. They also provide insight into the link between micro-RNA dysregulation and genetic liability to schizophrenia and cognitive dysfunction.

Evidence for altered hippocampal function in a mouse model of the human 22q11.2 microdeletion.

22q11.2 chromosomal deletions are recurrent copy number mutations that increase the risk of schizophrenia around thirty-fold. Deletion of the orthologous chromosomal region in mice offers an opportunity to characterize changes to neuronal structure and function that may account for the development of this disease. The hippocampus has been implicated in schizophrenia pathogenesis, is reduced in volume in 22q11.2 deletion carriers and displays altered neuronal structure in a mouse model of the mutation (Df(16)A(+/-) mice). Here we investigate hippocampal CA1 physiology, hippocampal-dependent spatial memory and novelty-induced hippocampal activation in Df(16)A(+/-) mice. We found normal spatial reference memory (as assayed by the Morris water maze test) as well as modest but potentially important deficits in physiology. In particular, a reduction in the level of inhibition of CA1 pyramidal neurons was observed, implying a decrease in interneuron activity. Additionally, deficits in LTP were observed using certain induction protocols. Induction of c-Fos expression by exploration of a novel environment suggested a relative sparing of CA1 and dentate gyrus function but showed a robust decrease in the number of activated CA3 pyramidal neurons in Df(16)A(+/-) mice. Overall, experiments performed in this 22q11.2 deletion model demonstrated deficits of various degrees across different regions of the hippocampus, which together may contribute to the increased risk of developing schizophrenia.

Novel strategies to probe the functions of serotonin receptors

Gene targeting has proven to be extremely powerful in various fields of biological research. Through this technique, knockout mice lacking a particular gene, and thus a particular protein, can be generated. One limitation to this technique is the fact that mice develop without the protein of interest and therefore, developmental compensations may have taken place, contributing to an observed phenotype. Inducible strategies, those which allow the timing of expression of a gene to be regulated, are currently being developed and should prove useful when applied to gene targeting technology. To begin to apply such new technologies to the field of gene targeting, we first created and tested several reporter constructions using the tetracycline inducible system. Here we describe the creation of several beta-galactosidase reporter constructions and the results of in vitro testing in Cos-7 cells. We then discuss future knockout strategies based upon our observations.

Analysis of prepulse inhibition in mouse lines overexpressing 22q11.2 orthologues.

Animal models have been useful in elucidating the genetic basis of the cognitive and behavioural phenotypes associated with the 22q11.2 microdeletions. Loss-of-function models have implicated a number of genes as playing a role in prepulse inhibition (PPI) of the startle response. Here, we report the generation and initial analysis of bacterial artificial chromosome (BAC) transgenic (Tg) mice, overexpressing genes from within the 22q11.2 locus. We used engineered BAC constructs to generate Tg lines and quantitative RT-PCR to assess levels of gene expression in each line. We assessed PPI and open-field activity in mice from two low copy number lines. In Tg-1, a line overexpressing Prodh and Vpreb2, PPI was significantly increased at prepulse levels of 78 dB and 82 dB while no differences were found in activity measures. By contrast, no significant differences were found in PPI testing of the Tg-2 line overexpressing Zdhhc8, Ranbp1, Htf9c, T10, Arvcf and Comt. Taken together with previous loss-of-function reports, these findings suggest that Prodh has a key role in modulating the degree of sensorimotor gating in mice and possibly in humans and provide additional support for an important role of this pathway in modulating behavioural deficits associated with genomic gains or losses at 22q11.2.