Hendrik Luuk

Distribution of Wfs1 protein in the central nervous system of the mouse and its relation to clinical symptoms of the Wolfram syndrome

Mutations in the coding region of the WFS1 gene cause Wolfram syndrome, a rare multisystem neurodegenerative disorder of autosomal recessive inheritance. Patients with Wolfram syndrome display considerable clinical pleiomorphism, and symptoms such as neurological complications and psychiatric disorders are common. In the present study we have characterized Wfs1 expression pattern in the mouse central nervous system by using a combination of immunohistochemistry on wild‐type mice and X‐Gal staining of Wfs1 knockout mice with targeted insertion of the lacZ reporter. We identified a robust enrichment of Wfs1 protein in the central extended amygdala and ventral striatum. Prominent Wfs1 expression was seen in the hippocampal CA1 region, parasubiculum, superficial part of the second and third layers of the prefrontal cortex and proisocortical areas, hypothalamic magnocellular neurosecretory system, and central auditory pathway. Wfs1 expression was also detected in numerous brainstem nuclei and in laminae VIII and IX of the spinal cord. Wfs1‐positive nerve fibers were found in the medial forebrain bundle, reticular part of the substantia nigra, globus pallidus, posterior caudate putamen, lateral lemniscus, alveus, fimbria, dorsal hippocampal commissure, subiculum, and to a lesser extent in the central sublenticular extended amygdala, compact part of substantia nigra, and ventral tegmental area. The neuroanatomical findings suggest that the lack of Wfs1 protein function can be related to several neurological and psychiatric symptoms found in Wolfram syndrome. Enrichment of Wfs1 protein in the central extended amygdala suggests a role in the modulation of anxiety and fear. J. Comp. Neurol. 509:642–660, 2008.

Wfs1-deficient mice display impaired behavioural adaptation in stressful environment

Wfs1-deficient mice were generated by disrupting the 8th exon of Wfs1 gene. Reproduction rates of homozygous Wfs1-deficient mice were slightly below the expected values, they displayed intolerance to glucose and overall lower body weight. The present behavioural study was performed in female Wfs1-deficient mice due to their milder metabolic disturbances. Non-fasting blood glucose levels did not differ between homozygous Wfs1-deficient mice and wild-type littermates. While there was no difference in baseline plasma corticosterone, exposure to stress induced a nearly three-fold elevation of corticosterone in Wfs1-deficient mice in relation to wild-type littermates. Wfs1-deficient mice did not display obvious shortcomings in sensory and motor functioning as exemplified by intact responses in conditioned learning paradigms and rota-rod test. Locomotor activity of Wfs1-deficient mice was significantly lower only in brightly lit environment. Short-term isolation had a significant anxiogenic-like effect on the behaviour of Wfs1-deficient mice in dark/light exploration test. Lower exploratory activity of Wfs1-deficient mice in the plus-maze was antagonised by pre-treatment with diazepam (1 mg/kg), a GABA(A) receptor agonist. Wfs1-deficient mice displayed increased anxiety-like behaviour in hyponeophagia test. The locomotor stimulatory effects of amphetamine (2.5-7.5 mg/kg) and apomorphine (3 mg/kg) were significantly attenuated and facilitated, respectively, in Wfs1-deficient mice. There were no differences between Wfs1-deficient mice and wild-types in forced swimming behaviour and conditioned fear responses. Subtle impairments in reversal learning were apparent in Wfs1-deficient mice in the Morris water maze. Altogether, the present study demonstrates impaired behavioural adaptation of Wfs1-deficient mice in stress-inducing situations. It is likely that Wfs1 protein plays a major role in the behavioural adaptation mechanisms to novel and stressful environments.

A screen for genes induced in the amygdaloid area during cat odor exposure

The aim of a present study was to identify the genes activated or inactivated in the amygdaloid area after the exposure to cat odor. Cat odor exposure was used to induce the ethologically relevant anxiety reaction in male rats. Differential expression of genes was analyzed using the cDNA Representational Difference Analysis (cDNA RDA). Differentially expressed mRNAs were identified by sequencing combined with database search and subsequently verified by dot blot analysis. Exposure of rats to cat odor induced avoidance of odor stimulus and suppressed the exploratory activity of animals. We found that during the cat odor exposure several genes with various functions were activated in the amygdaloid area of rat. Moreover, reverse subtraction resulted in a different set of genes that are inactivated during anxiety response. These genes can be classified according to their function as the neurotransmission related, enzymes, cell cycle regulating proteins and transcription factors. We found that during anxiety response the genes participating directly or indirectly in the synthesis of neurotransmitters (carboxypeptidase E, tyrosine 3‐monooxygenase/tryptophan 5‐mono‐oxygenase activation protein, wolframin) were up regulated. Moreover, a number of genes involved in the signal transduction (Rho GTPase, neurochondrin, Ca/calmodulin‐dependent protein kinase) were also activated. Additionally, reverse subtraction in control animals identified several up regulated genes having the antagonistic action to these genes (nischarin, Rab geranylgeranyl transferase). In conclusion, we were able to define the possible pathways linked to the regulation of anxiety response.

Relation between increased anxiety and reduced expression of alpha1 and alpha2 subunits of GABAA receptors in Wfs1-deficient mice

Mutations in the coding region of the WFS1 gene cause Wolfram syndrome, a rare multisystem neurodegenerative disorder of autosomal recessive inheritance. In clinical studies a relation between mutations in the Wfs1 gene and increased susceptibility for mood disorders has been established. According to our previous studies, mice lacking Wfs1 gene displayed increased anxiety in stressful environment. As the GABA-ergic system plays a significant role in the regulation of anxiety, we analyzed the expression of GABA-related genes in the forebrain structures of wild-type and Wfs1-deficient mice. Experimentally naïve Wfs1-deficient animals displayed a significant down-regulation of alpha1 (Gabra1) and alpha2 (Gabra2) subunits of GABA(A) receptors in the temporal lobe and frontal cortex. Exposure of wild-type mice to the elevated plus-maze decreased levels of Gabra1 and Gabra2 genes in the temporal lobe. A similar tendency was also established in the frontal cortex of wild-type animals exposed to behavioral test. In Wfs1-deficient mice the elevated plus-maze exposure did not induce further changes in the expression of Gabra1 and Gabra2 genes. By contrast, the expression of Gad1 and Gad2 genes, enzymes responsible for the synthesis of GABA, was not significantly affected by the exposure of mice to the elevated plus-maze or by the invalidation of Wfs1 gene. Altogether, the present study demonstrates that increased anxiety of Wfs1-deficient mice is probably linked to reduced expression of Gabra1 and Gabra2 genes in the frontal cortex and temporal lobe.

Cat odour exposure increases the expression of wolframin gene in the amygdaloid area of rat.

The aim of the present study was to find the genes expressed in the amygdaloid area after exposure to cat odour. Cat odour exposure was used to induce the ethologically relevant fear response in male rats. The differential expression of genes was analyzed using the cDNA Representational Difference Analysis (RDA). The differentially expressed clones were identified by sequencing and database search. Reverse transcription-polymerase chain reaction (RT-PCR) was applied to confirm the differences found by the RDA. Exposure of rats to cat odour induced avoidance of odour stimulus and suppressed the exploratory activity of animals. We found that during the cat odour exposure, several genes with various functions were activated in the amygdaloid area. Among the identified genes, we found the activation of the wolframin gene. RT-PCR confirmed quantitative elevation in the levels of wolframin transcripts in the amygdaloid area. This study supports the role of wolframin in the regulation of emotional behaviour.

Distinct changes in the behavioural effects of morphine and naloxone in CCK2 receptor-deficient mice

The effects of morphine, mu-opioid receptor agonist, and naloxone, a non-selective opioid receptor antagonist, in the locomotor activity and place conditioning tests were studied in the CCK(2) receptor-deficient male mice. The exposure of mice to the motility boxes for 3 consecutive days induced a significant inhibition of locomotor activity in the wild-type (+/+) mice compared to homozygous (-/-) animals. The administration of naloxone (10 mg/kg i.p.) to animals, adapted to the motility boxes, induced a significant reduction of locomotor activity in the homozygous (-/-), but not in the wild-type (+/+) mice. Treatment of habituated mice with morphine (10 mg/kg i.p.) caused a stronger increase of locomotor activity in the wild-type (+/+) mice compared to the homozygous (-/-) littermates. In the place preference test the pairing of the preferred side with naloxone (1 and 10 mg/kg i.p.) induced a dose-dependent place aversion in the wild-type (+/+) mice. The treatment with naloxone was less effective in the homozygous (-/-) mice, because the high dose of naloxone (10 mg/kg) tended to shift the preference. The pairing of morphine (3 mg/kg i.p.) injections with the non-preferred side induced a significant place preference both in the wild-type (+/+) and homozygous (-/-) mice. The increased density of opioid receptors was established in the striatum of homozygous (-/-) mice, but not in the other forebrain structures. In conclusion, the targeted invalidation of CCK(2) receptors induces a dissociation of behavioural effects of morphine and naloxone. Morphine-induced place preference remained unchanged, whereas hyper-locomotion was less pronounced in the mutant mice compared to the wild-type (+/+) littermates. By contrast, naloxone-induced place aversion was weaker, but naloxone caused a stronger inhibition of locomotor activity in the homozygous (-/-) mice than in the wild-type (+/+) animals. These behavioural alterations can be explained in the light of data that the targeted mutation of CCK(2) receptors induces distinct changes in the properties of opioid receptors in various brain structures.

Restricted expression of Neuroglobin in the mouse retina and co-localization with Melanopsin and Tyrosine Hydroxylase.

Neuroglobin (Ngb), a neuronal specific oxygen binding heme-globin, reported to be expressed at high levels in most layers of the murine retina. Ngbs function is presently unknown, but based on its high expression level and oxygen binding capabilities Ngb was proposed to function as an oxygen reservoir facilitating oxygen metabolism in highly active neurons or to function as a neuroprotectant. In the present study, we re-examined the expression pattern of Ngb in the retina using a highly validated antibody. Furthermore, intactness of retino-hypothalamic projections and the retinal expression level of Melanopsin and Tyrosine Hydroxylase were investigated in Ngb-null mice. Ngb-immunoreactivity was found in a few neurons of the ganglion cell and inner nuclear layers co-expressing Melanopsin and Tyrosine Hydroxylase, respectively. Ngb deficiency neither affected the level of Melanopsin and Tyrosine Hydroxylase proteins nor the intactness of PACAP-positive retinohypothalamic projections in the suprachiasmatic nucleus. Based on the present results, it seems unlikely that Ngb could have a major role in retinal oxygen homeostasis and neuronal survival under normal conditions. The present study suggests that a number of previously published reports have relied on antibodies with dubious specificity.

The redundancy of recursion and infinity for natural language

An influential line of thought claims that natural language and arithmetic processing require recursion, a putative hallmark of human cognitive processing (Chomsky in Evolution of human language: biolinguistic perspectives. Cambridge University Press, Cambridge, pp 45–61, 2010; Fitch et al. in Cognition 97(2):179–210, 2005; Hauser et al. in Science 298(5598):1569–1579, 2002). First, we question the need for recursion in human cognitive processing by arguing that a generally simpler and less resource demanding process—iteration—is sufficient to account for human natural language and arithmetic performance. We argue that the only motivation for recursion, the infinity in natural language and arithmetic competence, is equally approachable by iteration and recursion. Second, we submit that the infinity in natural language and arithmetic competence reduces to imagining infinite embedding or concatenation, which is completely independent from the ability to implement infinite processing, and thus, independent from both recursion and iteration. Furthermore, we claim that a property of natural language is physically uncountable finity and not discrete infinity.

Lsamp is implicated in the regulation of emotional and social behavior by use of alternative promoters in the brain.

Limbic system-associated membrane protein (LSAMP) is a neural cell adhesion molecule involved in neurite formation and outgrowth. The purpose of the present study was to characterize the distribution of alternatively transcribed Lsamp isoforms in the mouse brain and its implications on the regulation of behavior. Limbic system-associated membrane protein 1b transcript was visualized by using a mouse strain expressing beta-galactosidase under the control of Lsamp 1b promoter. The distribution of Lsamp 1a transcript and summarized expression of the Lsamp transcripts was investigated by non-radioactive in situ RNA hybridization analysis. Cross-validation was performed by using radioactive in situ hybridization with oligonucleotide probes. Quantitative RT-PCR was used to study correlations between the expression of Lsamp isoforms and behavioral parameters. The expression pattern of two promoters differs remarkably from the developmental initiation at embryonic day 12.5. Limbic system-associated membrane protein 1a promoter is active in “classic” limbic structures where the hippocampus and amygdaloid area display the highest expression. Promoter 1b is mostly active in the thalamic sensory nuclei and cortical sensory areas, but also in areas that regulate stress and arousal. Higher levels of Lsamp 1a transcript had significant correlations with all of the measures indicating higher trait anxiety in the elevated plus-maze test. Limbic system-associated membrane protein transcript levels in the hippocampus and ventral striatum correlated with behavioral parameters in the social interaction test. The data are in line with decreased anxiety and alterations in social behavior in Lsamp-deficient mice. We propose that Lsamp is involved in emotional and social operating systems by complex regulation of two alternative promoters.

Evidence for impaired function of dopaminergic system in Wfs1-deficient mice.

Immunohistological studies suggest abundant expression of Wfs1 protein in neurons and nerve fibers that lie in the vicinity of dopaminergic (DA-ergic) fibers and neurons. Therefore, we sought to characterize the function of DA-ergic system in Wfs1-deficient mice. In wild-type mice, amphetamine, an indirect agonist of DA, caused significant hyperlocomotion and increase in tissue DA levels in the dorsal and ventral striatum. Both effects of amphetamine were significantly blunted in homozygous Wfs1-deficient mice. Motor stimulation caused by apomorphine, a direct DA receptor agonist, was somewhat stronger in Wfs1-deficient mice compared to their wild-type littermates. However, apomorphine caused a similar reduction in levels of DA metabolites (3,4-dihydroxyphenylacetic acid and homovanillic acid) in the dorsal and ventral striatum in all genotypes. Behavioral sensitization to repeated treatment with amphetamine (2.5 mg/kg) was observed in wild-type, but not in Wfs1-deficient mice. The expression of DA transporter gene (Dat) mRNA was significantly lower in the midbrain of male and female homozygous mice compared to wild-type littermates. Altogether, the blunted effects of amphetamine and the reduced gene expression of DA transporter are probably indicative of an impaired functioning of the DA-ergic system in Wfs1-deficient mice.