Hippocampal phenotypes in kalirin-deficient mice

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Abstract

Regulation of forebrain cellular structure and function by small GTPase pathways is crucial for normal and pathological brain development and function. Kalirin is a brain-specific activator of Rho-like small GTPases implicated in neuropsychiatric disorders. We have recently demonstrated key roles for kalirin in cortical synaptic transmission, dendrite branching, spine density, and working memory. However, little is known about the impact of the complete absence of kalirin on the hippocampus in mice. We thus investigated hippocampal function, structure, and associated behavioral phenotypes in KALRN knockout (KO) mice we have recently generated. Here we show that KALRN KO mice had modest impairments in hippocampal LTP, but normal hippocampal synaptic transmission. In these mice, both context and cue-dependent fear conditioning were impaired. Spine density and dendrite morphology in hippocampal pyramidal neurons were not significantly affected in the KALRN KO mice, but small alterations in the gross morphology of the hippocampus were detected. These data suggest that hippocampal structure and function are more resilient to the complete loss of kalirin, and reveal impairments in fear learning. These studies allow the comparison of the phenotypes of different kalirin mutant mice and shed light on the brain region-specific functions of small GTPase signaling.

Introduction

Kalirin is a brain-specific guanine-nucleotide exchange factor (GEF) for the Rho family of small GTPases (Johnson et al., 2000, Penzes et al., 2000, Penzes and Jones, 2008). Members of the Rho subfamily of Ras-like small GTPases are central regulators of actin cytoskeletal dynamics in neurons, regulating the development and morphology of dendrites and spines (Nakayama et al., 2000, Nakayama and Luo, 2000, Tashiro et al., 2000, Threadgill et al., 1997, Wong et al., 2000). Their essential role in regulating spine morphology and human cognition, including learning and memory, is supported by the fact that many types of mental retardation (MR) are associated with altered spine morphogenesis. Mutations in genes encoding proteins in the Rho GTPases signaling pathways have also been associated with MR (Dierssen and Ramakers, 2006).

In adult mice, kalirin expression is mainly restricted to the cerebral cortex and hippocampus (Ma et al., 2001) and is not detectable outside of the brain. Several alternatively spliced forms are generated from a single KALRN gene. Kalirin-7 is the most abundant isoform in the adult brain, and is enriched in postsynaptic densities (PSD) of dendritic spines where it controls their morphology (Penzes et al., 2000, Penzes et al., 2001b). The less abundant kalirin isoforms, kalirin-9 and kalirin-12, are localized in the soma and in the processes of young neurons (Penzes et al., 2001a). The Rac1 activating GEF1 domain of kalirin is present in all isoforms, while the RhoA-activating GEF2 domain is present only in kalirin-9 and kalirin-12 (Penzes et al., 2001a). While kalirin-9 and -12 are expressed early in postnatal development, kalirin-7 expression is not detectable at birth, and increases after P7-10 (Ma et al., 2003, Xie et al., 2007).

We have shown that in cultured cortical neurons kalirin plays an important role in activity-dependent spine plasticity, synaptic expression and maintenance of AMPA receptors (AMPAR), and AMPAR-mediated synaptic transmission (Xie et al., 2007). Kalirin-7 is also required in cortical pyramidal neurons for spine morphogenesis downstream of the trans-synaptic adhesion molecules ephrinB–EphB (Penzes et al., 2003) and N-cadherin (Xie et al., 2008) and 5-HT2A serotonin receptors (Jones et al., 2009). We have recently reported the generation of a KALRN knockout (KALRN KO) mouse, which exhibited impairment in both spine and dendrite morphogenesis in vivo, associated with a working memory deficit (Cahill et al., 2009).

Another recent study examined mice with a deletion of the exon encoding the short C-terminal domain targeting kalirin-7 to spines (Δkalirin-7 mice) (Ma et al., 2008). Interestingly, KALRN KO mice and Δkalirin-7 mutant mice show several phenotypic differences. Notably, Δkalirin-7 mice exhibit a slight reduction in hippocampal spine density, which contrasts with our previous finding that full KALRN knockout affected neither hippocampal Rac1-GTP levels nor hippocampal spine density. KALRN KO mice show a reduction in active Rac1 levels in the cortex, while active Rac1 levels were unaltered in the Δkalirin-7 cortex. Similar to KALRN KO mice, cortical cultures from Δkalirin-7 mice showed a reduced spine density. However, while cortical spine density was reduced in vivo in KALRN KO mice, cortical spine density in vivo in Δkalirin-7 has not been reported. Δkalirin-7 and KALRN KO mice also showed differing behavioral phenotypes. KALRN KO mice showed locomotor hyperactivity and deficits in spatial working memory, both of which were unimpaired in Δkalirin-7 mutants. Contextual fear conditioning was impaired in Δkalirin-7 mice, but has not been analyzed in KALRN KO mice. These findings indicate that the complete absence of kalirin versus the targeted deletion of kalirin-7 produce some non-overlapping deficits. While our previous studies on the KALRN KO mice focused on the cerebral cortex and cortex-associated behavior, hippocampal structure and function has not been analyzed in detail in these mice. On the other hand, the studies on Δkalirin-7 mice focused mainly on the hippocampus. It is thus necessary to further investigate KALRN KO mice to understand these differences.

We therefore set out to investigate in more detail hippocampal function, structure, and associated behavioral phenotypes in KALRN KO mice. These studies will allow a more complete comparison of the phenotypes of KALRN KO and Δ-kalirin-7, and would shed light on the brain region-specific functions of Rho GTPase signaling. Surprisingly, we found that contrary to the Δkalirin-7 mice, KALRN KO mice had only modest impairments in hippocampal LTP, along with normal hippocampal synaptic transmission. However, in these mice, both context and cue-dependent fear conditioning were impaired. Spine density and dendrite morphology in hippocampal pyramidal neurons were not significantly affected in the KALRN KO mice, but small alterations in the gross morphology of the hippocampus were detected. These data reveal important and unexpected differences between different kalirin mutant mice, and suggest that basal hippocampal structure and function are more resilient to the full loss of kalirin.

Section snippets

Normal basal synaptic transmission along with modest impairment of hippocampal plasticity in KALRN KO mice

We have previously shown that loss of kalirin results in reduced glutamatergic synaptic transmission in the cortex (Cahill et al., 2009). However, hippocampal function has not been investigated in KALRN KO mice. In addition, Ma et al. have reported impaired hippocampal long-term potentiation (LTP) in Δ-kalirin-7 mutant mice (Ma et al., 2008). To determine whether homozygous deletion of KALRN had a functional effect on hippocampal synaptic transmission or plasticity thereof, a series of

Discussion

In this study we report that complete absence of kalirin resulted in modestly impaired hippocampal plasticity without an impairment in synaptic transmission, an impairment in both context and cue-dependent fear conditioning, but no significant effects on spine density or dendrite morphology. In addition, we detected a reduction in overall hippocampal size, associated with reduced cell numbers. Taken together, these data suggest that kalirin is important for hippocampal plasticity, but that in

Generation of the KALRN KO mice

Design and generation of the KALRN null mice has been described in detail previously (Cahill et al., 2009). Briefly, a targeting construct was designed in which exons 27-28 was replaced by the neo cassette under an independent PGK promoter. The PGK-neo cassette was inserted in reverse orientation and contained a loxP sites at each end to allow for excision. KALRN null mice were generated from ES cells by inGenious Targeting Laboratory (Stony Brook, NY) using standard methods. PCR analysis using

Acknowledgments

We thank Kelly Jones and Igor Rafalovich for editing. This work was supported by grants from NIH-NIMH (R01MH071316), National Alliance for Autism Research (NAAR), National Alliance for Research on Schizophrenia and Depression (NARSAD), and Alzheimer's Association (to P.P.), NIH-NIMH (MH078064) and Dunbar Funds (to J.R.), NIH-NIMH (MH57014) and Evelyn F. McKnight (to J.D.S. and C.A.M.), NIH-NIMH (P01 MH074866) and NIH-NINDS (R37 NS034696) (to D.J.S.) and training grants (NINDS 5T32NS041234) to

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    These authors contributed equally to this work.

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