Using Rotenone to Model Parkinson's Disease in Mice: A Review of the Role of Pharmacokinetics.

Rotenone is a naturally occurring toxin that inhibits complex I of the mitochondrial electron transport chain. Several epidemiological studies have shown an increased risk of Parkinson's disease (PD) in individuals exposed chronically to rotenone, and it has received great attention for its ability to reproduce many critical features of PD in animal models. Laboratory studies of rotenone have repeatedly shown that it induces in vivo substantia nigra dopaminergic cell loss, a hallmark of PD neuropathology. Additionally, rotenone induces in vivo aggregation of α-synuclein, the major component of Lewy bodies and Lewy neurites found in the brain of PD patients and another hallmark of PD neuropathology. Some in vivo rotenone models also reproduce peripheral signs of PD, such as reduced intestinal motility and peripheral α-synuclein aggregation, both of which are thought to precede classical signs of PD in humans, such as cogwheel rigidity, bradykinesia, and resting tremor. Nevertheless, variability has been noted in cohorts of animals exposed to the same rotenone exposure regimen and also between cohorts exposed to similar doses of rotenone. Low doses, administered chronically, may reproduce PD symptoms and neuropathology more faithfully than excessively high doses, but overlap between toxicity and parkinsonian motor phenotypes makes it difficult to separate if behavior is examined in isolation. Rotenone degrades when exposed to light or water, and choice of vehicle may affect outcome. Rotenone is metabolized extensively in vivo, and choice of route of exposure influences greatly the dose used. However, male rodents may be capable of greater metabolism of rotenone, which could therefore reduce their total body exposure when compared with female rodents. The pharmacokinetics of rotenone has been studied extensively, over many decades. Here, we review these pharmacokinetics and models of PD using this important piscicide.

Profiling of Acylcarnitines in First Episode Psychosis before and after Antipsychotic Treatment.

Acylcarnitines (ACs) have been shown to have a potential to activate pro-inflammatory signaling pathways and to foster the development of insulin resistance. The first task of the current study was to study the full list of ACs (from C2 to C18) in first episode psychosis (FEP) patients before and after antipsychotic treatment. The second task was to relate ACs to inflammatory and metabolic biomarkers established in the same patient cohort as in our previous studies. Serum levels of ACs were determined with the AbsoluteIDQ p180 kit (BIOCRATES Life Sciences AG, Innsbruck, Austria) using the flow injection analysis tandem mass spectrometry ([FIA]-MS/MS) as well as liquid chromatography ([LC]-MS/MS) technique. Identification and quantification of the metabolites was achieved using multiple reactions monitoring along with internal standards. The comparison of ACs in antipsychotic-naïve first-episode psychosis (FEP) patients (N = 38) and control subjects (CSs, N = 37) revealed significantly increased levels of long-chain ACs (LCACs) C14:1 (p = 0.0001), C16 (p = 0.00002), and C18:1 (p = 0.000001) in the patient group. These changes of LCACs were associated with augmented levels of CARN palmitoyltransferase 1 (CPT-1) (p = 0.006). By contrast, the level of short-chain AC (SCAC) C3 was significantly reduced (p = 0.00003) in FEP patients. Seven months of antipsychotic drug treatment ameliorated clinical symptoms in patients (N = 36) but increased significantly their body mass index (BMI, p = 0.001). These changes were accompanied by significantly reduced levels of C18:1 (p = 0.00003) and C18:2 (p = 0.0008) as well as increased level of C3 (p = 0.01). General linear model revealed the relation of LCACs (C16, C16:1, and C18:1) to the inflammatory markers (epidermal growth factor, IL-2, IL-4, IL-6), whereas SCAC C3 was linked to the metabolic markers (leptin, C-peptide) and BMI. FEP was associated with an imbalance of ACs in patients because the levels of several LCACs were significantly higher and the levels of several SCACs were significantly reduced compared with CSs. This imbalance was modified by 7 months of antipsychotic drug treatment, reversing the levels of both LCACs and SCACs to that established for CSs. This study supports the view that ACs have an impact on both inflammatory and metabolic alterations inherent for FEP.

The IgLON family of cell adhesion molecules expressed in developing neural circuits ensure the proper functioning of the sensory system in mice.

Deletions and malfunctions of the IgLON family of cell adhesion molecules are associated with anatomical, behavioral, and metabolic manifestations of neuropsychiatric disorders. We have previously shown that IgLON genes are expressed in sensory nuclei/pathways and that IgLON proteins modulate sensory processing. Here, we examined the expression of IgLON alternative promoter-specific isoforms during embryonic development and studied the sensory consequences of the anatomical changes when one of the IgLON genes, Negr1, is knocked out. At the embryonal age of E12.5 and E13.5, various IgLONs were distributed differentially and dynamically in the developing sensory areas within the central and peripheral nervous system, as well as in limbs and mammary glands. Sensory tests showed that Negr1 deficiency causes differences in vestibular function and temperature sensitivity in the knockout mice. Sex-specific differences were noted across olfaction, vestibular functioning, temperature regulation, and mechanical sensitivity. Our findings highlight the involvement of IgLON molecules during sensory circuit formation and suggest Negr1's critical role in somatosensory processing.

Lsamp⁻/⁻ mice display lower sensitivity to amphetamine and have elevated 5-HT turnover.

In mice, the limbic system-associated membrane protein (Lsamp) gene has been implicated in locomotion, anxiety, fear reaction, learning, social behaviour and adaptation. Human data links the LSAMP gene to several psychiatric disorders and completed suicide. Here, we investigated changes in major monoamine systems in mice lacking the Lsamp gene. First, the locomotor and rewarding effects of amphetamine were studied in Lsamp(-/-) mice and Lsamp(+/+) mice. Second, monoamine levels in major brain regions in response to saline and amphetamine injections were measured and, third, the expression levels of dopamine system-related genes in the brain were studied in these mice. Lsamp(-/-) mice displayed lower sensitivity to amphetamine in the motility box. Likewise, in the place preference test, the rewarding effect of amphetamine was absent in Lsamp(-/-) mice. In all brain regions studied, Lsamp(-/-) mice displayed lower serotonin (5-HT) baseline levels, but a greater 5-HT turnover rate, and amphetamine increased the level of 5-HT and lowered 5-HT turnover to a greater extent in Lsamp(-/-) mice. Finally, Lsamp(-/-) mice had lower level of dopamine transporter (DAT) mRNA in the mesencephalon. In conclusion, Lsamp-deficiency leads to increased endogenous 5-HT-ergic tone and enhanced 5-HT release in response to amphetamine. Elevated 5-HT function and reduced activity of DAT are the probable reasons for the blunted effects of amphetamine in these mice. Lsamp(-/-) mice are a promising model to study the neurobiological mechanisms of deviant social behaviour and adaptation impairment observed in many psychiatric disorders.

Dopamine System, NMDA Receptor and EGF Family Expressions in Brain Structures of Bl6 and 129Sv Strains Displaying Different Behavioral Adaptation.

C57BL/6NTac (Bl6) and 129S6/SvEvTac (129Sv) mice display different coping strategies in stressful conditions. Our aim was to evaluate biomarkers related to different adaptation strategies in the brain of male 129Sv and Bl6 mice. We focused on signaling pathways related to the dopamine (DA) system, N-methyl-D-aspartate (NMDA) receptor and epidermal growth factor (EGF) family, shown as the key players in behavioral adaptation. Mice from Bl6 and 129Sv lines were divided into either home cage controls (HCC group) or exposed to repeated motility testing and treated with saline for 11 days (RMT group). Distinct stress responses were reflected in severe body weight loss in 129Sv and the increased exploratory behavior in Bl6 mice. Besides that, amphetamine caused significantly stronger motor stimulation in Bl6. Together with the results from gene expression (particularly Maob), this study supports higher baseline activity of DA system in Bl6. Interestingly, the adaptation is reflected with opposite changes of DA markers in dorsal and ventral striatum. In forebrain, stress increased the gene expressions of Egf-Erbb1 and Nrg1/Nrg2-Erbb4 pathways more clearly in 129Sv, whereas the corresponding proteins were significantly elevated in Bl6. We suggest that not only inhibited activity of the DA system, but also reduced activity of EGF family and NMDA receptor signaling underlies higher susceptibility to stress in 129Sv. Altogether, this study underlines the better suitability of 129Sv for modelling neuropsychiatric disorders than Bl6.

Expression and impact of Lsamp neural adhesion molecule in the serotonergic neurotransmission system.

Limbic system associated membrane protein (Lsamp) is a neural adhesion protein which has been recently found to be differentially expressed between serotonergic neuron subtypes. We have previously shown elevated serotonin (5-HT) turnover rate in Lsamp-deficient mice. The purpose of the current study was to elucidate the role of Lsamp in serotonergic neurotransmission. Chronic (18 days) administration of serotonin reuptake inhibitor (SSRI) escitalopram (10 mg/kg) significantly increased general activity in wild-type mice in the open field and protected exploration in Lsamp-/- mice in the elevated-plus maze. An important psychopathology-related endophenotype, elevated 5-HT turnover in the brain of Lsamp-deficient mice, was reproduced in the saline group. Escitalopram restored the elevated 5-HT turnover of Lsamp-deficient mice to a level comparable with their wild-type littermates, suggesting that high 5-HT turnover in mutants is mediated by the increased activity of serotonin transporter (SERT protein encoded by Slc6a4 gene). The baseline level of Slc6a4 transcript was not changed in Lsamp-deficient mice, however, our immunohistochemical analysis showed partial co-expression of Lsamp with both SERT and Tph2 proteins in raphe. Overactivity of SERT in Lsamp-/- mice is further supported by significant elevation of Maoa transcript and increase of DOPAC, another Mao A product, specifically in the raphe. Again, elevation of DOPAC was reduced to the level of wild-type by chronic SSRI treatment. The activity of Lsamp gene promoters varied in 5-HT producing nuclei: both Lsamp 1a and 1b promoters were active in the dorsal raphe; most of the expression in the median raphe was from 1b promoter, whereas Lsamp 1a promoter was almost exclusively active in the caudal subgroup of raphe nuclei. We suggest that Lsamp may have an impact on the integrity of serotonergic synapses, which is possibly the neurochemical basis of the anxiety- and sociability-related phenotype in Lsamp-deficient mice.

Increased sensitivity to psychostimulants and GABAergic drugs in Lsamp-deficient mice.

Lsamp, in combinations with other members of the IgLON family of cell adhesion molecules, promotes and inhibits neurite outgrowth and synapse formation during development. Mice lacking Lsamp gene display decreased social behaviour, hyperactivity; decreased anxiety level, alongside with altered balance in GABAA receptor α1 and α2 subunits; and decreased sensitivity to amphetamine, alongside with elevated serotonin function. In human studies, Lsamp has been associated with several psychiatric diseases, including schizophrenia, and suicide. Here, we provide a more thorough characterization of the pharmacological phenotype of Lsamp-deficient mice, including testing for sensitivity to morphine, cocaine, MK-801 and ketamine. More thorougly, sensitivity to GABA modulators (diazepam, alprazolam, ethanol, pentobarbital, TP003, and SL651498) was assessed. In brief, Lsamp-deficient mice were more sensitive to the locomotor activating effects of cocaine and morphine, and hypersensitive to the sedative and muscle relaxant effects of GABA modulators, most likely reflecting enhanced function of α1 and α5 subunits of the GABAA receptor. No gross differences in sensitivity to NMDA receptor modulators were observed. Thus, as the lack of Lsamp gene leads to widespread imbalances in major neurotransmitter systems in the brain accompanied by remarkable changes in behavioural phenotype as well, Lsamp-deficient mice are a promising model for mimicking psychiatric disorders.

Neural cell adhesion molecule Negr1 deficiency in mouse results in structural brain endophenotypes and behavioral deviations related to psychiatric disorders.

Neuronal growth regulator 1 (NEGR1) belongs to the immunoglobulin (IgLON) superfamily of cell adhesion molecules involved in cortical layering. Recent functional and genomic studies implicate the role of NEGR1 in a wide spectrum of psychiatric disorders, such as major depression, schizophrenia and autism. Here, we investigated the impact of Negr1 deficiency on brain morphology, neuronal properties and social behavior of mice. In situ hybridization shows Negr1 expression in the brain nuclei which are central modulators of cortical-subcortical connectivity such as the island of Calleja and the reticular nucleus of thalamus. Brain morphological analysis revealed neuroanatomical abnormalities in Negr1-/- mice, including enlargement of ventricles and decrease in the volume of the whole brain, corpus callosum, globus pallidus and hippocampus. Furthermore, decreased number of parvalbumin-positive inhibitory interneurons was evident in Negr1-/- hippocampi. Behaviorally, Negr1-/- mice displayed hyperactivity in social interactions and impairments in social hierarchy. Finally, Negr1 deficiency resulted in disrupted neurite sprouting during neuritogenesis. Our results provide evidence that NEGR1 is required for balancing the ratio of excitatory/inhibitory neurons and proper formation of brain structures, which is prerequisite for adaptive behavioral profiles. Therefore, Negr1-/- mice have a high potential to provide new insights into the neural mechanisms of neuropsychiatric disorders.

Different housing conditions alter the behavioural phenotype of CCK(2) receptor-deficient mice.

The behavioural phenotype of mice, lacking CCK(2) receptors, has varied across studies conducted not only in different laboratories, but also across studies published by the same laboratory. The present study was designed to elucidate the phenotype of CCK(2) receptor-deficient mice housed in two different environmental conditions within the same laboratory. Environmental enrichment was used as an alternative environment to standard laboratory conditions. Significant genotype by environment interaction was observed in the plus-maze, hot-plate, restraint-induced analgesia and water maze test. While mice, lacking CCK(2) receptors, housed in standard conditions were more anxious, displayed stronger restraint-induced analgesia and performed worse in the water maze when compared to corresponding wild-type littermates, none of these phenotypes were observed in mice, housed in enriched conditions. By contrast, in the hot-plate test, rota-rod and locomotor activity test a genotype-dependent phenotype was observed in mice housed in enriched, but not in standard conditions. Moreover, the phenotype of CCK(2) receptor-deficient mice established in the hot-plate test and rota-rod was sex-specific. These results suggest that thorough and labour-consuming study of mutation-induced behavioural phenotype is necessary not only in different genetic backgrounds but also the substantial variation of phenotype due to sex- and environment-related factors have to be explored.

Metabolic profile associated with distinct behavioral coping strategies of 129Sv and Bl6 mice in repeated motility test.

We investigated the metabolic outcome of different coping strategies in 129S6/SvEvTac (129Sv) and C57BL/6Ntac (Bl6) strains. Two different batches of male 129Sv and Bl6 mice were used. One batch was not subjected to any behavioral manipulations (home cage control; HCC), whereas the other batch was treated with saline for 11 days and exposed after every treatment to the motor activity measurement (repeated motility tested; RMT). Bl6 RMT mice displayed a robust increase in number of rearings during repeated testing. 129Sv RMT mice experienced significant loss of body weight, but showed enhanced weight gain in HCC batch compared to Bl6. Serum metabolites (acylcarnitines, amino acids, biogenic amines, hexoses, glycerophospholipids and sphingolipids) were determined with AbsoluteIDQ p180 kit. Results of the metabolomic study revealed prominent peculiarities between strains in two different conditions. Comparison of both batches of mice demonstrated that in Bl6 biogenic amines (acetyl-ornithine, alpha-amionadipic acid, carnosine) and lysophosphatidylcholine PC(16:1/0:0) dominated. However in 129Sv acylcarnitine C5 clearly dominated, indicating shift towards short-chain acylcarnitines. Stable strain-specific ratios also emerged for both lines, ratio of glycine/PC ae C38:2 for Bl6 and ratios of C5/C0 as well as PC(16:0/0:0)/PC(16:1/0:0) for 129Sv. The described metabolic changes probably reflect different behavioral coping strategies of 129Sv and Bl6 mice.

Repeated Administration of D-Amphetamine Induces Distinct Alterations in Behavior and Metabolite Levels in 129Sv and Bl6 Mouse Strains.

The main goal of the study was to characterize the behavioral and metabolomic profiles of repeated administration (for 11 days) of d-amphetamine (AMPH, 3 mg/kg i. p.), indirect agonist of dopamine (DA), in widely used 129S6/SvEvTac (129Sv) and C57BL/6NTac (Bl6) mouse strains. Acute administration of AMPH (acute AMPH) induced significantly stronger motor stimulation in Bl6. However, repeated administration of AMPH (repeated AMPH) caused stronger motor sensitization in 129Sv compared acute AMPH. Body weight of 129Sv was reduced after repeated saline and AMPH, whereas no change occurred in Bl6. In the metabolomic study, acute AMPH induced an elevation of isoleucine and leucine, branched chain amino acids (BCAA), whereas the level of hexoses was reduced in Bl6. Both BCAAs and hexoses remained on level of acute AMPH after repeated AMPH in Bl6. Three biogenic amines [asymmetric dimethylarginine (ADMA), alpha-aminoadipic acid (alpha-AAA), kynurenine] were significantly reduced after repeated AMPH. Acute AMPH caused in 129Sv a significant reduction of valine, lysophosphatidylcholines (lysoPC a C16:0, lysoPC a C18:2, lysoPC a C20:4), phosphatidylcholine (PC) diacyls (PC aa C34:2, PC aa C36:2, PC aa C36:3, PC aa C36:4) and alkyl-acyls (PC ae C38:4, PC ae C40:4). However, repeated AMPH increased the levels of valine and isoleucine, long-chain acylcarnitines (C14, C14:1-OH, C16, C18:1), PC diacyls (PC aa C38:4, PC aa C38:6, PC aa C42:6), PC acyl-alkyls (PC ae C38:4, PC ae C40:4, PC ae C40:5, PC ae C40:6, PC ae C42:1, PC ae C42:3) and sphingolipids [SM(OH)C22:1, SM C24:0] compared to acute AMPH in 129Sv. Hexoses and kynurenine were reduced after repeated AMPH compared to saline in 129Sv. The established changes probably reflect a shift in energy metabolism toward lipid molecules in 129Sv because of reduced level of hexoses. Pooled data from both strains showed that the elevation of isoleucine and leucine was a prominent biomarker of AMPH-induced behavioral sensitization. Simultaneously a significant decline of hexoses, citrulline, ADMA, and kynurenine occurred. The reduced levels of kynurenine, ADMA, and citrulline likely reflect altered function of N-methyl-D-aspartate (NMDA) and NO systems caused by repeated AMPH. Altogether, 129Sv strain displays stronger sensitization toward AMPH and larger variance in metabolite levels than Bl6.

The combined impact of IgLON family proteins Lsamp and Neurotrimin on developing neurons and behavioral profiles in mouse.

Cell surface neural adhesion proteins are critical components in the complex orchestration of cell proliferation, apoptosis, and neuritogenesis essential for proper brain construction and behavior. We focused on the impact of two plasticity-associated IgLON family neural adhesion molecules, Neurotrimin (Ntm) and Limbic system associated membrane protein (Lsamp), on mouse behavior and its underlying neural development. Phenotyping neurons derived from the hippocampi of Lsamp-/-, Ntm-/- and Lsamp-/-Ntm-/- mice was performed in parallel with behavioral testing. While the anatomy of mutant brains revealed no gross changes, the Ntm-/- hippocampal neurons exhibited premature sprouting of neurites and manifested accelerated neurite elongation and branching. We propose that Ntm exerts an inhibitory impact on neurite outgrowth, whereas Lsamp appears to be an enhancer of the said process as premature neuritogenesis in Ntm-/- neurons is apparent only in the presence of Lsamp. We also show interplay between Lsamp and Ntm in regulating tissue homeostasis: the impact of Ntm on cellular proliferation was dependent on Lsamp, and Lsamp appeared to be a positive regulator of apoptosis in the presence of Ntm. Behavioral phenotyping indicated test-specific interactions between Lsamp and Ntm. The phenotypes of single mutant lines, such as reduced swimming speed in Morris water maze and increased activity in the elevated plus maze, were magnified in Lsamp-/-Ntm-/- mice. Altogether, evidence both from behavioral experiments and cultured hippocampal cells show combined and differential interactions between Ntm and Lsamp in the formation of hippocampal circuits and behavioral profiles. We demonstrate that mutual interactions between IgLON molecules regulate the initiation of neurite sprouting at very early ages, and even cell-autonomously, independent of their regulation of cell-cell adhesion.

Altered Expression Profile of IgLON Family of Neural Cell Adhesion Molecules in the Dorsolateral Prefrontal Cortex of Schizophrenic Patients.

Neural adhesion proteins are crucial in the development and maintenance of functional neural connectivity. Growing evidence suggests that the IgLON family of neural adhesion molecules LSAMP, NTM, NEGR1, and OPCML are important candidates in forming the susceptibility to schizophrenia (SCZ). IgLON proteins have been shown to be involved in neurite outgrowth, synaptic plasticity and neuronal connectivity, all of which have been shown to be altered in the brains of patients with the diagnosis of schizophrenia. Here we optimized custom 5'-isoform-specific TaqMan gene-expression analysis for the transcripts of human IgLON genes to study the expression of IgLONs in the dorsolateral prefrontal cortex (DLPFC) of schizophrenic patients (n = 36) and control subjects (n = 36). Uniform 5'-region and a single promoter was confirmed for the human NEGR1 gene by in silico analysis. IgLON5, a recently described family member, was also included in the study. We detected significantly elevated levels of the NEGR1 transcript (1.33-fold increase) and the NTM 1b isoform transcript (1.47-fold increase) in the DLPFC of schizophrenia patients compared to healthy controls. Consequent protein analysis performed in male subjects confirmed the increase in NEGR1 protein content both in patients with the paranoid subtype and in patients with other subtypes. In-group analysis of patients revealed that lower expression of certain IgLON transcripts, mostly LSAMP 1a and 1b, could be related with concurrent depressive endophenotype in schizophrenic patients. Additionally, our study cohort provides further evidence that cannabis use may be a relevant risk factor associated with suicidal behaviors in psychotic patients. In conclusion, we provide clinical evidence of increased expression levels of particular IgLON family members in the DLPFC of schizophrenic patients. We propose that alterations in the expression profile of IgLON neural adhesion molecules are associated with brain circuit disorganization in neuropsychiatric disorders, such as schizophrenia. In the light of previously published data, we suggest that increased level of NEGR1 in the frontal cortex may serve as molecular marker for a wider spectrum of psychiatric conditions.

Neuronal Growth and Behavioral Alterations in Mice Deficient for the Psychiatric Disease-Associated Negr1 Gene.

Neuronal growth regulator 1 (NEGR1), a member of the immunoglobulin superfamily cell adhesion molecule subgroup IgLON, has been implicated in neuronal growth and connectivity. In addition, genetic variants in or near the NEGR1 locus have been associated with obesity and more recently with learning difficulties, intellectual disability and psychiatric disorders. However, experimental evidence is lacking to support a possible link between NEGR1, neuronal growth and behavioral abnormalities. Initial expression analysis of NEGR1 mRNA in C57Bl/6 wildtype (WT) mice by in situ hybridization demonstrated marked expression in the entorhinal cortex (EC) and dentate granule cells. In co-cultures of cortical neurons and NSC-34 cells overexpressing NEGR1, neurite growth of cortical neurons was enhanced and distal axons occupied an increased area of cells overexpressing NEGR1. Conversely, in organotypic slice co-cultures, Negr1-knockout (KO) hippocampus was less permissive for axons grown from EC of ß-actin-enhanced green fluorescent protein (EGFP) mice compared to WT hippocampus. Neuroanatomical analysis revealed abnormalities of EC axons in the hippocampal dentate gyrus (DG) of Negr1-KO mice including increased numbers of axonal projections to the hilus. Neurotransmitter receptor ligand binding densities, a proxy of functional neurotransmitter receptor abundance, did not show differences in the DG of Negr1-KO mice but altered ligand binding densities to NMDA receptor and muscarinic acetylcholine receptors M1 and M2 were found in CA1 and CA3. Activity behavior, anxiety-like behavior and sensorimotor gating were not different between genotypes. However, Negr1-KO mice exhibited impaired social behavior compared to WT littermates. Moreover, Negr1-KO mice showed reversal learning deficits in the Morris water maze and increased susceptibility to pentylenetetrazol (PTZ)-induced seizures. Thus, our results from neuronal growth assays, neuroanatomical analyses and behavioral assessments provide first evidence that deficiency of the psychiatric disease-associated Negr1 gene may affect neuronal growth and behavior. These findings might be relevant to further evaluate the role of NEGR1 in cognitive and psychiatric disorders.

Cat odor exposure induces distinct changes in the exploratory behavior and Wfs1 gene expression in C57Bl/6 and 129Sv mice.

129Sv and C57Bl/6 (Bl6) strains are two most widely used inbred mice strains for generation of transgenic animals. The present study confirms the existence of substantial differences in the behavior of these two mice strains. The exploratory behavior of Bl6 mice in a novel environment was significantly higher compared to 129Sv mice. The exposure of mice to cat odor-induced an anxiety-like state in Bl6, but not in 129Sv mice. The levels of Wfs1 gene expression did not differ in the prefrontal cortex, mesolimbic area and temporal lobe of experimentally naive Bl6 and 129Sv mice. However, after cat odor exposure the expression of Wfs1 gene was significantly lower in the mesolimbic area and temporal lobe of Bl6 mice compared to 129Sv strain. Dynamics of Wfs1 gene expression and exploratory behavior suggest that the down-regulation of Wfs1 gene in Bl6 mice might be related to the increased anxiety. Further studies are needed to test the robustness and possible causal relationship of this finding.

Deficit in emotional learning in neurotrimin knockout mice.

Neurotrimin (Ntm) belongs to the IgLON family of cell adhesion molecules with Lsamp, Obcam and kilon that regulate the outgrowth of neurites mostly by forming heterodimers. IgLONs have been associated with psychiatric disorders, intelligence, body weight, heart disease and tumours. This study provides an initial behavioural and pharmacological characterization of the phenotype of Ntm-deficient mice. We expected to see at least some overlap with the phenotype of Lsamp-deficient mice as Ntm and Lsamp are the main interaction partners in the IgLON family and are colocalized in some brain regions. However, Ntm-deficient mice displayed none of the deviations in behaviour that we have previously shown in Lsamp-deficient mice, but differently from Lsamp-deficient mice, had a deficit in emotional learning in the active avoidance task. The only overlap was decreased sensitivity to the locomotor stimulating effect of amphetamine in both knockout models. Thus, despite being interaction partners, on the behavioural level Lsamp seems to play a much more central role than Ntm and the roles of these two proteins seem to be complementary rather than overlapping.

Promoter-Specific Expression and Genomic Structure of IgLON Family Genes in Mouse.

IgLON family is composed of five genes: Lsamp, Ntm, Opcml, Negr1, and Iglon5; encoding for five highly homologous neural adhesion proteins that regulate neurite outgrowth and synapse formation. In the current study we performed in silico analysis revealing that Ntm and Opcml display similar genomic structure as previously reported for Lsamp, characterized by two alternative promotors 1a and 1b. Negr1 and Iglon5 transcripts have uniform 5' region, suggesting single promoter. Iglon5, the recently characterized family member, shares high level of conservation and structural qualities characteristic to IgLON family such as N-terminal signal peptide, three Ig domains, and GPI anchor binding site. By using custom 5'-isoform-specific TaqMan gene-expression assay, we demonstrated heterogeneous expression of IgLON transcripts in different areas of mouse brain and several-fold lower expression in selected tissues outside central nervous system. As an example, the expression of IgLON transcripts in urogenital and reproductive system is in line with repeated reports of urogenital tumors accompanied by mutations in IgLON genes. Considering the high levels of intra-family homology shared by IgLONs, we investigated potential compensatory effects at the level of IgLON isoforms in the brains of mice deficient of one or two family members. We found that the lack of IgLONs is not compensated by a systematic quantitative increase of the other family members. On the contrary, the expression of Ntm 1a transcript and NEGR1 protein was significantly reduced in the frontal cortex of Lsamp-deficient mice suggesting that the expression patterns within IgLON family are balanced coherently. The actions of individual IgLONs, however, can be antagonistic as demonstrated by differential expression of Syp in deletion mutants of IgLONs. In conclusion, we show that the genomic twin-promoter structure has impact on both anatomical distribution and intra-family interactions of IgLON family members. Remarkable variety in the activity levels of 1a and 1b promoters both in the brain and in other tissues, suggests complex functional regulation of IgLONs by alternative signal peptides driven by 1a and 1b promoters.

Gender specific effects of ethanol in mice, lacking CCK2 receptors.

Neuropeptide cholecystokinin (CCK) has been reported to suppress ethanol intake, but there is contradictory evidence about the role of CCK(2) receptors. In the present study anxiolytic, hypolocomotor and sedative effects of acute ethanol administration, but also voluntary ethanol consumption were studied in male and female mice, lacking CCK(2) receptors (-/-). Ethanol (1.0 and 2.0 g/kg) induced a significant reduction of anxiety-related behaviours in the elevated plus-maze, but this effect was statistically significant only in female homozygous mice (-/-). In male mice, lacking CCK(2) receptors (-/-), but not in their wild-type littermates (+/+), the suppression of vertical locomotor activity was caused by ethanol at a dose 0.5 g/kg. The highest dose of ethanol (2.0 g/kg) produced statistically significant reduction of horizontal locomotor activity only in female wild-type (+/+) mice, but this effect was related to increased basal activity when compared to female mutant (-/-) mice. Duration of the loss of righting reflex was not significantly affected by genotype or gender, but blood ethanol levels at regain of righting reflex were significantly lower in female homozygous mice (-/-) compared to their wild-type (+/+) littermates, indicating increased sensitivity to the sedative effect of ethanol. Ethanol intake, but not preference, at concentration 10% was significantly increased in female mice, lacking CCK(2) receptors (-/-). The present study revealed an altered response to the acute effects of ethanol in CCK(2) receptor deficient mice (-/-). These changes are gender-specific and could be attributed to the altered activity of dopaminergic system in male mice and increased activity of GABA-ergic system in female mice as established in our previous studies.

Cat odour exposure decreases exploratory activity and alters neuropeptide gene expression in CCK(2) receptor deficient mice, but not in their wild-type littermates.

An attempt was made to establish whether the anxiogenic effect of cat odour differs in female wild-type and CCK(2) receptor deficient mice, having different exploratory activity in the elevated plus-maze. The exposure of wild-type and homozygous CCK(2) receptor deficient mice to cat odour did not reveal substantial differences between the two genotypes. The number of contacts with the cat odour impregnated cloth was reduced and the frequency of stretch-attend postures was increased similarly in wild-type and homozygous mice. However, the following exposure of mice to the elevated plus-maze established differences as homozygous mice displayed increased exploratory activity in the plus-maze. The cat odour exposure significantly reduced exploratory activity only in homozygous mice. Together with the increased exploratory activity we established in homozygous mice significantly increased expression of the Oprm1 gene in the frontal cortex and mesencephalon. The exposure of mice to cat odour caused only minor changes in the gene expression of wild-type mice, whereas in homozygous animals a significantly increased expression of the Mc3r gene in the frontal cortex and temporal lobe, and the Pomc1 gene in the temporal lobe, mesencephalon and mesolimbic area was established. In conclusion, CCK(2) receptor deficient mice displayed reduced anxiety compared to their wild-type littermates in the plus-maze test. This behavioural effect seems to be related, at least partly, to an increased tone of opioid system in the brain. Moreover, homozygous mice respond to the exposure of cat odour with an increased anxiety. This effect seems to be related to the increased function of the melanocortin system in the brain structures of genetically modified mice.

Rats displaying distinct exploratory activity also have different expression patterns of gamma-aminobutyric acid- and cholecystokinin-related genes in brain regions.

The aim of the present study was to evaluate the expression of genes in relation to the exploratory activity of rats. Limbic system-associated membrane protein (LsAMP) gene, gamma-aminobutyric acid-(GABA)- and cholecystokinin-(CCK)-related genes were analyzed in the frontal cortex, amygdala and periaqueductal gray (PAG) after exposure of rats to exploratory challenge. Two groups of animals displaying low and high exploratory activity in the elevated plus-maze were selected for gene expression studies from the population of 43 male Wistar rats. Eight rats were taken randomly from the same cages as animals exposed to the plus-maze, but they were not subjected to the exploratory test. This home-cage control group was also used for gene expression analysis in order to explore a possible impact of the plus-maze exposure. Rats with low and high exploratory activity displayed clearly distinct profiles in gene expression. Most prominent alterations were established in the amygdala where almost all GABA-related and CCK receptor genes were two- to five-fold up-regulated in low exploratory activity rats compared to high exploratory activity and home-cage control group. The expression of several GABA-related genes was also increased in the PAG of animals displaying low exploratory activity compared to the other groups. Moreover, we found reduced expression of GABA- and CCK-related genes in all three brain regions in animals with high exploratory activity compared to the home-cage control group. In addition to these findings, we established a significantly increased expression of the LsAMP gene in the amygdala and PAG of low exploratory activity animals compared with the other groups. In conclusion, low and high exploratory activity rats differed not only by their exploratory activity but also displayed opposite gene expression patterns. Low exploratory activity of rats correlated with the up-regulation of LsAMP and GABA-related genes in the amygdala and PAG and with the up-regulation of CCK receptors in the amygdala. High exploratory activity of rats was related to a significant down-regulation of CCK receptor genes in the amygdala and PAG. These rats also displayed the reduced expression of GABA-related genes in the frontal cortex and PAG.