Behavioral, neurochemical and morphological changes induced by the overexpression of munc18-1a in brain of mice: relevance to schizophrenia

Abstract

Overexpression of the mammalian homolog of the unc-18 gene (munc18-1) has been described in the brain of subjects with schizophrenia. Munc18-1 protein is involved in membrane fusion processes, exocytosis and neurotransmitter release. A transgenic mouse strain that overexpresses the protein isoform munc18-1a in the brain was characterized. This animal displays several schizophrenia-related behaviors, supersensitivity to hallucinogenic drugs and deficits in prepulse inhibition that reverse after antipsychotic treatment. Relevant brain areas (that is, cortex and striatum) exhibit reduced expression of dopamine D(1) receptors and dopamine transporters together with enhanced amphetamine-induced in vivo dopamine release. Magnetic resonance imaging demonstrates decreased gray matter volume in the transgenic animal. In conclusion, the mouse overexpressing brain munc18-1a represents a new valid animal model that resembles functional and structural abnormalities in patients with schizophrenia. The animal could provide valuable insights into phenotypic aspects of this psychiatric disorder.

Figure 1

Characterization of Munc18-OE transgenic mice. (a) Quantitative real-time PCR study showed significant increased mRNA levels of munc18-1a in the brain cortex of Munc18-OE mice (unpaired t-test *P<0.05 versus wild-type (WT) controls). (b) Representative autoradiograms showing the distribution of munc18-1a mRNA in coronal sections of WT controls (A, B, C) and transgenic Munc18 mice (Á, , Ć). The specificity labeling was demonstrated by the displacement with unlabeled oligonucleotide (Á, , Ć). Note the increase of mRNA signal in cerebellum and piriform cortex. Pir, piriform cortex; CA1, CA1 field of hippocampus; CA3, CA3 field of hippocampus; DG, dentate gyrus; CB Cx, Cerebellum cortex. Bar: 2 mm. (c) Expression of mRNA encoding for munc18-1a in Munc18-OE and WT control mice evaluated by in situ hybridization. Significant increases of munc18-1a mRNA levels were obtained in different brain regions of Munc18-OE (unpaired t-test *P<0.05, **P<0.01 and ***P<0.005 versus WT control group). (d) Increased immunodensity of munc18-1a protein in the brain cortex of Munc18-OE mice (unpaired t-test **P<0.005 versus WT controls). Representative immunoblots of munc18-1a and β-actin in WT controls and Munc18-OE mice. (e) As a negative control, no differences were found in munc18-1b protein expression between Munc18-OE and WT controls. (f) Representative images of munc18-1a distribution in WT (left panels) and Munc18-OE (right panels) at 10 × , green (munc18-1a) and blue (nuclei) in cortex (cx), hippocampus (hp) and cerebellum (cb). (g) Increased immunofluorescence in different brain areas of Munc18-OE mice (unpaired t-test **P<0.005 and ***P<0.0001 versus WT controls); cx, cortex; hp, hippocampus; cb, cerebellum; str, striatum. Data are mean±s.e.m.; the number of animals is indicated in parentheses or inside plot bars.

Figure 2

Overexpression of munc18-1a leads to schizophrenia-related behaviors. (a, b) Decreased central locomotion following exposure to a novel environment and increased exploration of the peripheral area in Munc18-OE transgenic mice (repeated-measures analysis of variance (ANOVA) followed by Bonferroni post hoc test, **P<0.01 and ***P<0.001 versus wild-type (WT) controls). (c) Increased locomotor activity of the transgenic mice during the first 5 min in novelty-induced hyperactivity test (unpaired t-test *P<0.05 versus WT controls). No differences between Munc18-OE and WT controls were found in total distance traveled in open field. (d) Munc18-OE mice displayed anxiety-like behavior in the elevated plus maze, as shown by the decreased time in open-arms (unpaired t-test *P<0.05 versus WT controls) and the decreased number of entries into open-arms (e) (unpaired t-test **P<0.01 versus WT) (f) The social interaction test revealed decreased time of interaction elicited by transgenic Munc18-OE mice (unpaired t-test **P<0.01 versus WT controls). (g) Increased sensitivity to hallucinogenic-like action in Munc18-OE mice as shown by the increased number of head-twitch responses to DOI (1 mg kg⁻¹, i.p.) administration (unpaired t-test *P<0.05 versus WT controls). (h) Transgenic Munc18-OE mice exhibited altered episodic memory as shown in the novel-object recognition test. Decreased score in memory for objects (what), their locations (where), and the order in which they were experienced (when) (unpaired t-test *P<0.05 and **P<0.005 versus WT controls). (i) Munc18-OE mice showed impaired prepulse inhibition (PPI) of the acoustic startle reflex across prepulse intensities (two-way analysis of variance (ANOVA) ***P<0.001 for genotype factor; unpaired t-test *P<0.05 and **P<0.005 versus WT controls). Impairment of the PPI was completely reversed in the transgenic mice after clozapine (1.5 mg kg⁻¹ i.p.) administration (unpaired t-test ^#P<0.05 versus transgenic animals with saline). No differences were found in the startle amplitude between Munc18-OE and WT controls. Data are mean±s.e.m.; the number of animals is indicated in parentheses or inside the plot bars.

Figure 3

Alterations of dopaminergic system components. (a) Munc18-OE mice displayed decreased dopamine D₁ receptor density versus wild-type (WT) controls in both cortical and striatal brain regions (unpaired t-test *P<0.05 and **P<0.0001 versus WT controls). Representative immunoblots of dopamine D₁ receptor and β-actin in WT controls and Munc18-OE mice. (b) No differences were observed in dopamine D₂ receptor immunodensity between transgenic Munc18-OE and WT control mice. (c) Striatal dopamine transporter (DAT) was strongly decreased in Munc18-OE mice (unpaired t-test *P<0.001 versus WT controls). Representative immunoblots of DAT and β-actin in WT controls and Munc18-OE mice. (d) Munc18-OE mice exhibited an increase of the dopamine synthesizing enzyme tyrosine hydroxylase (TH) in both cortex and striatum (unpaired t-test *P<0.05 and ***P<0.0001 versus WT controls). Data bars of immunoblots are mean (percentage over controls)±s.e.m.; the number of animals is indicated in parentheses. (e) Increased motor activity in Munc18-OE mice (▴) versus WT controls (○) following a single injection of 5 mg kg⁻¹ i.p. of amphetamine (Amph) (repeated-measures two-way analysis of variance (ANOVA) followed by Bonferroni post hoc test, *P<0.05 and **P<0.01 versus WT). (f, g) Effect of Amph (5 mg kg⁻¹ i.p.) on extracellular DA concentrations in the prefrontal cortex (f) or striatum (g) of Munc18-OE mice (●) and their WT controls (○) expressed as percentage of DA basal concentration values (two-way ANOVA followed by Bonferroni post hoc test, *P<0.05 and ***P<0.001 versus WT controls). The mean of the six first samples were considered basal values and assumed as the 100% value. Values were expressed as percentage of the basal values. Points are mean±s.e.m.; the number of animals is indicated in parentheses.

Figure 4

Volumetric reduction in gray matter of Munc18-OE transgenic mice. (a) A reduction in volume in temporal cortex was observed, P<0.01, K=200 voxels, T=5.66 (left side) and 6.02 (right side). (b) A reduction in volume in prefrontal–olfatory–cingulate cortex was shown, P<0.01, K=200 voxels, T=8.11, as well as in (c) cerebellar lobe P<0.01, K=200 voxels, T=5.25. Images were analyzed according to voxel-based morphometry (VBM) methodology (see Materials and methods for details). Color bar indicates potency of significance level, from 0 (less potency) to 8 (more potency).

Figure 5

Apoptotic pathways and inflammatory markers. (a) No changes in Fas receptor density (glycosylated form), evaluated by western blot, were found in cortex of Munc18-OE mice. Representative immunoblots of Fas receptor and β-actin in WT controls and Munc18-OE mice (b) FADD (dimeric form) significantly increased in Munc18-OE mice (unpaired t-test *P<0.05 versus WT controls) while phosphorylated p-191 FADD (oligomeric form) remained unchanged. Representative immunoblots of FADD, p-191 FADD and β-actin in WT controls and Munc18-OE mice. (c) No changes were found in caspase-3 activity. (d) No aberrant changes were observed in the proteolytic cleavage of nuclear PARP-1. Representative immunoblots of PARP-1, 85 fragment and β-actin in WT and Munc18-OE mice. (e) An increase of inducible nitric oxide synthase (NOS2) was detected in the brain cortex of Munc18-OE (unpaired t-test *P<0.05 versus WT controls). The Munc18-OE mice did not present changes in the pro-inflammatory enzyme ciclooxygenase 2 (COX2) and in neuronal nitric oxide synthase (NOS1). Representative immunoblots of NOS2/COX2/NOS1 and β-actin in WT controls and Munc18-OE mice. (f) Lipid peroxidation was studied by measuring the levels of malondialdehyde (MDA) in the brain cortex of Munc19-OE and WT controls. No changes were found in cortical MDA of Munc18-OE mice when compared with their WT controls. Data bars are mean (percentage over controls)±s.e.m.; the number of animals is indicated in parentheses or inside the plot bars.