Hippocampal and Cortical Primary Cilia Are Required for Aversive Memory in Mice

et al. (2014) Hippocampal and Cortical Primary Cilia Are Required for Aversive Memory in Mice. PLoS ONE 9(9): e106576. doi:10.1371/journal.pone.0106576 Hippocampal and Cortical Primary Cilia Are Required for Aversive Memory in Mice Nicolas F. Berbari 0 Erik B. Malarkey 0 S. M. Zaki R. Yazdi 0 Andrew D. McNair 0 Jordyn M. Kippe 0 Mandy J. Croyle 0 Timothy W. Kraft 0 Bradley K. Yoder 0 Alexandra Kavushansky, Technion - Israel Institute of Technology, Israel 0 1 Department of Cell, Developmental, and Integrative Biology, The University of Alabama at Birmingham , Birmingham , Alabama, United States of America, 2 Department of Vision Sciences, The University of Alabama at Birmingham , Birmingham, Alabama , United States of America It has been known for decades that neurons throughout the brain possess solitary, immotile, microtubule based appendages called primary cilia. Only recently have studies tried to address the functions of these cilia and our current understanding remains poor. To determine if neuronal cilia have a role in behavior we specifically disrupted ciliogenesis in the cortex and hippocampus of mice through conditional deletion of the Intraflagellar Transport 88 (Ift88) gene. The effects on learning and memory were analyzed using both Morris Water Maze and fear conditioning paradigms. In comparison to wild type controls, cilia mutants displayed deficits in aversive learning and memory and novel object recognition. Furthermore, hippocampal neurons from mutants displayed an altered paired-pulse response, suggesting that loss of IFT88 can alter synaptic properties. A variety of other behavioral tests showed no significant differences between conditional cilia mutants and controls. This type of conditional allele approach could be used to distinguish which behavioral features of ciliopathies arise due to defects in neural development and which result from altered cell physiology. Ultimately, this could lead to an improved understanding of the basis for the cognitive deficits associated with human cilia disorders such as Bardet-Biedl syndrome, and possibly more common ailments including depression and schizophrenia. - Funding: Funding provided by National Institutes of Health RO1 (RO1 DK075996) to BKY and UAB Neuroscience Behavioral Assessment Core (P30 NS47466). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. . These authors contributed equally to this work. Nearly every cell in the body possesses a primary cilium, a small microtubule based appendage now known to serve as a signaling hub for a diverse set of pathways [1]. The use of model systems such as Chlamydomonas reinhardtii and Caenorhabditis elegans has revealed much about how cilia are assembled through a highly conserved process known as intraflagellar transport (IFT) and disrupting ciliary proteins in neurons has been shown to impact behavior in C. elegans (for a review see [2]). However, whether primary cilia have any role in the neurophysiology of mammalian neurons has not been well described. Collectively, the research done in these model systems and more recently in mouse mutants and human patients with ciliary defects have revealed that the primary cilium is a highly complex sensory and signaling center throughout both embryonic development and for adult tissue homeostasis [3]. Clinically, a growing list of human genetic syndromes, termed ciliopathies, has been identified as having cilia dysfunction as their root cause [4]. Multiple ciliopathies (e.g. Bardet-Biedl syndrome, Alstro m syndrome, Joubert syndrome) present with both neurodevelopmental and cognitive deficits. Furthermore, cilia perturbation in mouse models can lead to neurodevelopmental phenotypes including cerebellar malformations, altered neuronal migration, and defects in adult neurogenesis [58]. The phenotypes observed in these mouse models closely mimic the clinical features observed in human ciliopathy patients. As such mouse models are very important tools for understanding the cellular and physiological underpinnings responsible for the cognitive and behavioral deficits in ciliopathy patients Another possible connection between cilia and regulation of behavior was made by Einstein et al. who demonstrated that disruption of the ciliary localized G-Protein Coupled Receptor somatostatin receptor 3 (Sstr3) caused deficits in novel object recognition [9]. However, a direct role for cilia in this process was not evaluated. Here, we extend this analysis by analyzing the behavioral and cognitive consequences of disrupting IFT/cilia in the hippocampus and cortex of the mouse brain. Ift88D/D;Emx1-Cre mutants have cilia loss specific to the hippocampus and cortex To determine the effects of hippocampal and cortical cilia loss on behavior we utilized a previously described conditional allele of a gene required for cilia formation and maintenance, Intraflagellar transport 88 (hereafter referred to as Ift88flox/flox) [10]. The Ift88flox/flox mice were crossed with mice carrying the transgene Empty spiracles homeobox 1 promoter driven Cre recombinase (hereafter referred to as Emx1-Cre) where Cre is expressed specifically in the telencephalon, [11]. To confirm the specificity of the Cre line and the efficacy of Ift88 loss, we used PCR, immunoblotting, Cre reporter analysis, and immunofluorescence microscopy. Region specific PCR and Western blotting revealed that the mutant allele (hereafter Ift88D/D) and IFT88 protein loss occurred only in the olfactory bulb, hippocampus, and cortex but not in control regions such as the hypothalamus and cerebellum (Figure 1A and B). Emx1-Cre specificity was further confirmed using a Rosa26 Cre reporter that expresses b-galactosidase only in cells in which Cre was active (Figure 1C and File S1) [12]. Direct analysis of cilia using antibodies to the cilia marker adenylate cyclase III (ACIII) showed a marked reduction in cilia number occurred only in the regions of Cre expression (Figure 2A, B and File S2, S3, S4) [13]. We further confirmed that the Emx1-Cre was not promiscuously expressed or associated with other phenotypes associated with early neurodevelopmental roles of cilia. For example, in the eye photoreceptor degeneration has been observed in Bbs mutants [14]. To rule out the presence of vision deficits in Ift88D/D;Emx1-Cre mutants electrorentinogram analysis was performed and the dark adapted threshold b-wave, and maximal b-wave were not different between the wild type and mutant animals; likewise the light adapted a- and b-waves were also not different (File S5). We subsequently performed genotyping by PCR on whole eye DNA to test directly for the presence of the mutant allele (File S5). These results indicate that adult Ift88D/D; Emx1-Cre mutants were not visually impaired. Upon establishing the spatial specificity and efficacy of (...truncated)