Elimination of Kalrn expression in POMC cells reduces anxiety-like behavior and contextual fear learning

Abstract

Kalirin, a Rho GDP/GTP exchange factor for Rac1 and RhoG, is known to play an essential role in the formation and maintenance of excitatory synapses and in the secretion of neuropeptides. Mice unable to express any of the isoforms of Kalrn in cells that produce POMC at any time during development (POMC cells) exhibited reduced anxiety-like behavior and reduced acquisition of passive avoidance behavior, along with sex-specific alteration in the corticosterone response to restraint stress. Strikingly, lack of Kalrn expression in POMC cells closely mimicked the effects of global Kalrn knockout on anxiety-like behavior and passive avoidance conditioning without causing the other deficits noted in Kalrn knockout mice. Our data suggest that deficits in excitatory inputs onto POMC neurons are responsible for the behavioral phenotypes observed.

Keywords: ACTH; Corticosterone; Cre-recombinase; Passive-avoidance; Pituitary.

Fig. 1. Kalirin isoforms and the Kalirin POMC-Cre mouse

A] The major isoforms of Kalirin are indicated, along with the position of the Exon 13 ablation. The black box at the COOH-terminus of Kal7 is a PDZ-binding motif. B] Real time polymerase chain reaction (qPCR) was performed on RNA from the indicated tissues as described (Mandela et al. 2012). Results are expressed with respect to the GAPDH signal in the same samples on the same 96-well plate. C] Genotyping from the tissues indicated was performed as described; the products expected from KalSR^CKO and KalSR^KO mice are indicated (Mandela et al. 2012).

Fig. 2. The POMC promoter drives Cre-recombinase expression in POMC-producing cells in the pituitary and hypothalamus

POMC-Cre female mice were crossed with homozygous Rosa26-LacZ [A, B] or Rosa26-TdTomato [C, D] male mice and tissues taken from their progeny were examined after fixing and sectioning; the X-gal reaction was used to visualize sites of LacZ expression and fluorescence was used to detect TdTomato. A and C] pituitary; B and D] arcuate nucleus of the hypothalamus (coronal sections). Ant, anterior lobe of the pituitary; I or Int, intermediate lobe of the pituitary; Arc N, arcuate nucleus of the hypothalamus. Scale bars, 200 μm.

Fig. 3. The POMC promoter drives Cre-recombinase expression in cells not producing POMC in the adult

POMC-Cre female mice were crossed with homozygous Rosa26-TdTomato male mice and tissues taken from progeny were examined for expression of TdTomato after fixation and sectioning: A] Nucleus of the solitary tract - TdTomato is detected in scattered cells in the adult; B] Hippocampus - TdTomato expressing cells are prevalent in the dentate gyrus; C] Amygdala (posteromedial and – lateral cortical amygdala, Bregma −2.0) (Paxinos and Franklin, 2008) - scattered TdTomato expressing cells are present; D] Cerebral cortex (layers numbered) and E] Cerebellum [layers as in (Tanaka et al. 2008); ML, molecular layer; PCL, Purkinje cell layer; GCL, granule cell layer; WM, white matter]. F, G, H] Higher power images of hippocampus stained for POMC product 16K fragment (F), glutamic acid decarboxylase (GAD 65/67) (G) and neuropeptide Y (NPY) (H). Expression of TdTomato indicates that the POMC promoter was active in these cells at some stage during development and that expression of all isoforms of Kalrn has been eliminated. Scale bars: A-E, 200 μm; F-H, 50 μm.

Fig. 4. Knockout of Kalirin in POMC cells increases serum corticosterone in females after restraint stress

A] Mice were restrained in a ventilated disposable 50 ml tube for 15 min (Experiment 1) or 30 min (Experiment 2). Basal corticosterone was determined from submandibular pouch sampling at least 24h before the stress, and stressed levels of corticosterone were determined using trunk blood. B] Male mice (N=11 each genotype) were timed for immobility in the tail suspension test, with no significant genotypic differences (p=0.17). C] Male and female KalSR^CKO and KalSR^POMC-KO mice (N=9 for each sex and genotype) were tested for mobility in the open field; no genotypic or sex differences were observed (RM-ANOVA p>0.6), so the data were pooled across sexes. D] Male and female KalSR^CKO and KalSR^POMC-KO mice (N=6 for each sex and genotype) were tested for time to fall from the rotarod as its speed was increased; no genotypic or sex differences were observed.

fig. 5. Knockout of Kalirin in POMC cells decreases anxiety-like behavior and acquisition of fear-based behavior in both male and female mice

A] Mice were tested for 5 min in the elevated zero maze and scored from videotapes by a blinded observer. B] Mice were tested for acquisition of fear-based behavior using a two compartment box with a footshock grid. Mice were trained once with the footshock and then tested without footshock at 2h and 24h later.

Fig. 6. Pituitary cultures and ACTH secretion

Anterior pituitary cultures were prepared separately from littermate male and female KalSR^CKO (control) and KalSR^POMC-KO mice (Mandela et al. 2012). Three different secretagogues were tested: 2 mM BaCl₂ (Expt.1), 20 nM CRH (Expt. 2), 20 nM AVP (Expt.2; a basal collection was made after CRH was removed and AVP was then applied). There were no consistent differences in stimulated secretion by sex or genotype across the experiments.