High-quality human and rat spermatozoal RNA isolation for functional genomic studies.

Sperm RNA is a sensitive monitoring endpoint for male reproductive toxicants, and a potential biomarker to assess male infertility and sperm quality. However, isolation of sperm RNA is a challenging procedure due to the heterogeneous population of cells present in the ejaculate, the low yield of RNA per spermatozoon, and the absence of 18S and 28S ribosomal RNA subunits. The unique biology of spermatozoa has created some uncertainty in the field about RNA isolation methods, indicating the need for rigorous quality control checks to ensure reproducibility of data generated from sperm RNA. Therefore, we developed a reliable and effective protocol for RNA isolation from rat and human spermatozoa that delivers highly purified and intact RNA, verified using RNA-specific electrophoretic chips and molecular biology approaches such as RT-PCR and Western blot analysis. The sperm RNA isolation technique was optimized using rat spermatozoa and then adapted to human spermatozoa. Three steps in the sperm isolation procedure, epididymal fluid collection, sperm purification, and spermatozoon RNA extraction, were evaluated and assessed. The sperm RNA extraction methodology consists of collection of rat epididymal fluid with repeated needle punctures of the epididymis, somatic cell elimination using detergent-based somatic cell lysis buffer (SCLB) and the use of RNA isolation Kit. Rat sperm heads are more resistant to disruption than human spermatozoa, necessitating the addition of mechanical lysis with microbeads and heat in the rat protocol, whereas the human sperm protocol only required lysis buffer. In conclusion, this methodology results in reliable and consistent isolation of high-quality sperm RNA. Using this technique will aid in translation of data collected from animal models, and reproducibility of clinical assessment of male factor fertility using RNA molecular biomarkers.

Sexual dimorphism of AMBRA1-related autistic features in human and mouse.

Ambra1 is linked to autophagy and neurodevelopment. Heterozygous Ambra1 deficiency induces autism-like behavior in a sexually dimorphic manner. Extraordinarily, autistic features are seen in female mice only, combined with stronger Ambra1 protein reduction in brain compared to males. However, significance of AMBRA1 for autistic phenotypes in humans and, apart from behavior, for other autism-typical features, namely early brain enlargement or increased seizure propensity, has remained unexplored. Here we show in two independent human samples that a single normal AMBRA1 genotype, the intronic SNP rs3802890-AA, is associated with autistic features in women, who also display lower AMBRA1 mRNA expression in peripheral blood mononuclear cells relative to female GG carriers. Located within a non-coding RNA, likely relevant for mRNA and protein interaction, rs3802890 (A versus G allele) may affect its stability through modification of folding, as predicted by in silico analysis. Searching for further autism-relevant characteristics in Ambra1+/- mice, we observe reduced interest of female but not male mutants regarding pheromone signals of the respective other gender in the social intellicage set-up. Moreover, altered pentylentetrazol-induced seizure propensity, an in vivo readout of neuronal excitation-inhibition dysbalance, becomes obvious exclusively in female mutants. Magnetic resonance imaging reveals mild prepubertal brain enlargement in both genders, uncoupling enhanced brain dimensions from the primarily female expression of all other autistic phenotypes investigated here. These data support a role of AMBRA1/Ambra1 partial loss-of-function genotypes for female autistic traits. Moreover, they suggest Ambra1 heterozygous mice as a novel multifaceted and construct-valid genetic mouse model for female autism.

Intra- and inter-individual differences in human sperm DNA methylation.

There is growing evidence that sperm DNA methylation is important in maintaining proper sperm health and function. Previous studies have associated sperm DNA methylation levels with sperm quality and function, however, little is known regarding the intra- and inter-individual variability in sperm methylation levels. This study characterizes this variation. Sperm epigenetic differences between successive semen samples from 12 patients were examined to identify the intra- and inter-individual differences globally across the genome, and in specifically defined genomic regions using the Illumina Infinium HumanMethylation450 BeadChips. Methylation analysis identified a bimodal distribution in the methylation levels that were non-uniformly distributed across the different genomic regions. The methylation levels were highly correlated in both the intra- and inter-individual comparisons. The intra-individual methylation levels were more highly correlated than the inter-individual comparison both globally and across the defined genomic regions, demonstrating that sperm DNA methylation levels are relatively stable between semen sample collections.

Revisiting adult neurogenesis and the role of erythropoietin for neuronal and oligodendroglial differentiation in the hippocampus.

Recombinant human erythropoietin (EPO) improves cognitive performance in neuropsychiatric diseases ranging from schizophrenia and multiple sclerosis to major depression and bipolar disease. This consistent EPO effect on cognition is independent of its role in hematopoiesis. The cellular mechanisms of action in brain, however, have remained unclear. Here we studied healthy young mice and observed that 3-week EPO administration was associated with an increased number of pyramidal neurons and oligodendrocytes in the hippocampus of ~20%. Under constant cognitive challenge, neuron numbers remained elevated until >6 months of age. Surprisingly, this increase occurred in absence of altered cell proliferation or apoptosis. After feeding a 15N-leucine diet, we used nanoscopic secondary ion mass spectrometry, and found that in EPO-treated mice, an equivalent number of neurons was defined by elevated 15N-leucine incorporation. In EPO-treated NG2-Cre-ERT2 mice, we confirmed enhanced differentiation of preexisting oligodendrocyte precursors in the absence of elevated DNA synthesis. A corresponding analysis of the neuronal lineage awaits the identification of suitable neuronal markers. In cultured neurospheres, EPO reduced Sox9 and stimulated miR124, associated with advanced neuronal differentiation. We are discussing a resulting working model in which EPO drives the differentiation of non-dividing precursors in both (NG2+) oligodendroglial and neuronal lineages. As endogenous EPO expression is induced by brain injury, such a mechanism of adult neurogenesis may be relevant for central nervous system regeneration.

Behavioral alterations and changes in Ca/calmodulin kinase II levels in the striatum of connexin36 deficient mice.

Gap junctions (GJ) are intercellular channels which directly connect the cytoplasm of adjacent cells. GJ allow direct cell-to-cell communication via the diffusion of ions, metabolites and second messengers such as IP(3). The connexin36 (Cx36) protein has been detected in GJ between interneurons of the hippocampus, cerebral cortex, striatum, amygdala, the inferior olive, cerebellum and other brain structures, such as the olfactory bulb. Cx36 knockout (Cx36 KO) mice display changes in synchronous network oscillations in the hippocampus, neocortex and inferior olive and exhibit impaired spatial alternation and one-trial object recognition in a Y-maze. Here, we further characterized the behavioral changes induced by Cx36 deficiency in the mouse by using different behavioral measures and experimental procedures. Additionally, we examined the effects of Cx36 deficiency on acetylcholine esterase (AChE) activity and calcium calmodulin kinase II alpha (CaMKII) protein levels in the striatum. The homozygous Cx36 KO mice displayed increased locomotion and running speed in the open-field, reduced object exploration and impaired one-trial object-place recognition. Furthermore, they exhibited more anxiety-like behavior as compared to the heterozygous controls in the light-dark box. Homozygous Cx36 KO mice exhibited reduced CaMKII levels in the striatum as compared to the heterozygous mice. AChE activity in the striatum was not significantly different between groups. The present results suggest that Cx36 deficiency in the mouse leads to reduced CaMKII levels in the striatum and behavioral changes in open-field activity, anxiety-related behavior in the light-dark box and one-trial object-place recognition.

Deletion of connexin45 in mouse neurons disrupts one-trial object recognition and alters kainate-induced gamma-oscillations in the hippocampus.

Neuronal gap junctions, allowing fast intercellular electrotonic signal transfer, have been implicated in mechanisms governing learning and memory processes. We have examined conditional neuron-directed (Cx45fl/fl:Nestin-Cre) connexin45 deficient mice in terms of behavioral and electrophysiological correlates of learning and memory. Behavioral habituation to a novel environment and motor learning were not changed in these mice. Novel object recognition after delays of up to 60min was impaired in neuronal Cx45 deficient mice. However, object-place recognition was not significantly different from controls. Analysis of enhanced green fluorescent reporter protein expression controlled by the endogenous mouse Cx45 promoter in the brain of neuronal Cx45 deficient mice suggested that Cx45 is expressed in the perirhinal cortex and the CA3 subregion of the hippocampus. The neuronal Cx45 deficient mice were also examined for aberrations in the generation and synchronization of network oscillations in the hippocampus. General excitability, synaptic short time plasticity, and spontaneous high-frequency oscillations (sharp-wave ripples) in the hippocampus were not different from controls. However, bath stimulation of hippocampal slices with kainate induced significantly lower gamma-oscillation amplitudes in the CA3, but not in the CA1 subfield of the neuronal Cx45 deficient mice. Additionally, they exhibited a significantly larger full width half maximum of the frequency distribution in the CA1 subfield as compared to the controls. In conclusion, the neuron-directed deletion of Cx45 impaired one-trial novel object recognition and altered kainate-induced gamma-oscillations possibly via the disruption of inter-neuronal gap junctional communication in the hippocampus or perirhinal cortex.

Neuronal histamine and the interplay of memory, reinforcement and emotions.

The biogenic amine histamine is an important neurotransmitter-neuromodulator in the central nervous system that has been implicated in a variety of biological functions including thermo- and immunoregulation, food intake, seizures, arousal, anxiety, reward and memory. The review of the pertinent literature indicates that the majority of findings are compatible with the appraisal that the inhibition of histaminergic neurotransmission impairs learning and memory formation, decreases cortical activation and arousal, has a suppressive effect on behavioral measures of fear and anxiety, exponentiates the rewarding effects of drugs of abuse and intracranial brain stimulation. In contrast, the stimulation of histaminergic neurotransmission can ameliorate learning and memory impairments that are associated with various experimental deficit models and pathological conditions. Clinical investigations with patients suffering from neurodegenerative diseases such as Alzheimer's and Parkinson's disease demonstrate pathological alterations in the brain's histaminergic system, which, in some cases are correlated with the severity of cognitive deficits. The role of the brain's histamine system in episodic memory formation and the potential of histamine-related drugs to ameliorate cognitive deficits in early stages of neurodegenerative diseases are discussed.

Neurokinin-2 receptor antagonism in medial septum influences temporal-order memory for objects and forebrain cholinergic activity.

UNLABELLED: In the mammalian brain the neurokinin NK(2) receptors are predominantly located in the hippocampus, thalamus, septum and frontal cortex. It has been shown that administration of the NK(2) receptor agonist, neurokinin A (NKA), into the medial septum of rats increases extracellular levels of acetylcholine (ACh) in the hippocampus and that NK(2) receptor antagonism blocks this increase. Therefore, given the prominent role of hippocampal ACh in information processing, we hypothesized that NK(2) receptor antagonism in the medial septum would negatively affect learning and memory via its influence on the cholinergic neurons of the basal forebrain. We investigated the action of local application of the peptidic NK(2) receptor antagonist, Bz-Ala-Ala-D-Trp-Phe-D-Pro-Pro-Nle-NH (1, 10 and 100pmol), into the medial septum on object memory for temporal order and spatial location using an object novelty paradigm. By means of in vivo microdialysis and HPLC analyses, we also examined the influence of NK(2) receptor antagonism in the medial septum on ACh in major cholinergic projection areas of the basal forebrain, namely, hippocampus, frontal cortex and amygdala. RESULTS: Injection of vehicle alone into the medial septum impaired memory for temporal order and spatial location of objects. Application of 1pmol of the NK(2) receptor antagonist partially reversed this deficit by reinstating memory for temporal order. Injection of 10pmol of the NK(2) receptor antagonist into the medial septum decreased levels of ACh in the hippocampus (at 30min post-injection), and frontal cortex (at 30 and 80min post-injection) in comparison to vehicle. However, this apparent decrease was the result of the blockade of a saline-induced increase in ACh levels.

Histamine H1 receptor knockout mice exhibit impaired spatial memory in the eight-arm radial maze.

BACKGROUND AND PURPOSE: In the mammalian brain, histaminergic neurotransmission is mediated by the postsynaptic histamine H1 and H2 receptors and the presynaptic H3 autoreceptor, which also acts as a heteroreceptor. The H1 receptor has been implicated in spatial learning and memory formation. However, pharmacological and lesion studies have revealed conflicting results. To examine the involvement of histamine H1 receptor in spatial reference and working memory formation, H1 receptor knockout mice (KO) were tested in the eight-arm radial maze. Previously, we found that the H1 receptor-KO mice showed reduced emotionality when confronted with spatial novelty. As it is known that emotions can have an impact on spatial learning and memory performance, we also evaluated H1 receptor-KO mice in terms of emotional behaviour in the light-dark box. EXPERIMENTAL APPROACH: Mice lacking the H1 receptor and wild-type mice (WT) were tested for spatial reference and working memory in an eight-arm radial maze with three arms baited and one trial per day. Emotional behaviour was measured using the light-dark test. KEY RESULTS: The H1 receptor-KO mice showed impaired spatial reference and working memory in the radial maze task. No significant differences between H1 receptor-KO and WT mice were observed in the light-dark test. CONCLUSIONS AND IMPLICATIONS: The spatial memory deficits of the H1 receptor-KO mice might be due to the reported changes in cholinergic neurochemical parameters in the frontal cortex and the CA1 subregion of the hippocampus, to impaired synaptic plasticity in the hippocampus, and/or to a dysfunctional brain reward/reinforcement system.

Episodic-like and procedural memory impairments in histamine H1 Receptor knockout mice coincide with changes in acetylcholine esterase activity in the hippocampus and dopamine turnover in the cerebellum.

We investigated episodic-like (ELM) and procedural memory (PM) in histamine H1 receptor knockout (H1R-KO) mice. In order to relate possible behavioral deficits to neurobiological changes, we examined H1R-KO and wild-type (WT) mice in terms of acetylcholine esterase (AChE) activity in subregions of the hippocampus and AChE and tyrosine hydroxylase (TH) expression in the striatum. Furthermore, we analyzed acetylcholine (ACh), 5-HT and dopamine (DA) levels, including metabolites, in the cerebellum of H1R-KO and WT mice. The homozygous H1R-KO mice showed impaired ELM as compared with the heterozygous H1R-KO and WT mice. The performance of homozygous H1R-KO mice in the ELM task was primarily driven by familiarity-based memory processes. While the homozygous H1R-KO mice performed similar to the heterozygous H1R-KO and WT mice during the acquisition of a PM, as measured with an accelerating rotarod, after a retention interval of 7 days their performance was impaired relative to the heterozygous H1R-KO and WT mice. These findings suggest that, both, ELM and long-term PM are impaired in the homozygous H1R-KO mice. Neurochemical assays revealed that the H1R-KO mice had significantly lower levels of AChE activity in the dentate gyrus (DG) and CA1 subregions of the hippocampus as compared with the WT mice. The homozygous H1R-KO mice also displayed significantly reduced dihydroxyphenylacetic acid (DOPAC) levels and a reduced DOPAC/DA ratio in the cerebellum, suggesting that the DA turnover in the cerebellum is decelerated in homozygous H1R-KO mice. In conclusion, homozygous H1R-KO mice display severe long-term memory deficits in, both, ELM and PM, which coincide with changes in AChE activity in the hippocampus as well as DA turnover in the cerebellum. The importance of these findings for Alzheimer's (AD) and Parkinson's disease (PD) is discussed.

Connexin31.1 deficiency in the mouse impairs object memory and modulates open-field exploration, acetylcholine esterase levels in the striatum, and cAMP response element-binding protein levels in the striatum and piriform cortex.

Neuronal gap junctions in the brain, providing intercellular electrotonic signal transfer, have been implicated in physiological and behavioral correlates of learning and memory. In connexin31.1 (Cx31.1) knockout (KO) mice the coding region of the Cx31.1 gene was replaced by a LacZ reporter gene. We investigated the impact of Cx31.1 deficiency on open-field exploration, the behavioral response to an odor, non-selective attention, learning and memory performance, and the levels of memory-related proteins in the hippocampus, striatum and the piriform cortex. In terms of behavior, the deletion of the Cx31.1 coding DNA in the mouse led to increased exploratory behaviors in a novel environment, and impaired one-trial object recognition at all delays tested. Despite strong Cx31.1 expression in the peripheral and central olfactory system, Cx31.1 KO mice exhibited normal behavioral responses to an odor. We found increased levels of acetylcholine esterase (AChE) and cAMP response element-binding protein (CREB) in the striatum of Cx31.1 KO mice. In the piriform cortex the Cx31.1 KO mice had an increased heterogeneity of CREB expression among neurons. In conclusion, gap-junctions featuring the Cx31.1 protein might be involved in open-field exploration as well as object memory and modulate levels of AChE and CREB in the striatum and piriform cortex.

The histamine H1-receptor mediates the motivational effects of novelty.

Novelty-induced arousal has motivational effects and can reinforce behavior. The mechanisms by which novelty acts as a reinforcer are unknown. Novelty-induced arousal can be either rewarding or aversive dependent on its intensity and the preceding state of arousal. The brain's histamine system has been implicated in both arousal and reinforcement. Histamine and histamine-1-receptor (H1R) agonists induced arousal and wakefulness in humans and rodents, e.g. by stimulating cortical acetylcholine (ACh) release. The H1R has also been implicated in processes of brain reward via interactions with the nigrostriatal- and mesolimbic dopamine (DA) systems. We asked whether the motivational effects of novelty-induced arousal are compromised in H1R knockout (KO) mice. The H1R-KO mice failed to develop a conditioned place-preference induced by novel objects. Even though they still explore novel objects, their reinforcing value is diminished. Furthermore, they showed impaired novelty-induced alternation in the Y-maze. Rearing activity and emotional behavior in a novel environment was also altered in H1R-KO mice, whereas object-place recognition was unaffected. The H1R-KO mice had higher ACh concentrations in the frontal cortex and amygdala (AMY). In the latter, the H1R-KO mice had also increased levels of DA, but a lower dihydrophenylacetic acid/DA ratio. Furthermore, the H1R-KO mice had also increased tyrosine hydroxylase immunoreactivity in the basolateral anterior, basolateral ventral and cortical AMY nuclei. We conclude that the motivational effects of novelty are diminished in H1R-KO mice, possibly due to reduced novelty-induced arousal and/or a dysfunctional brain reward system.

NR2C by NR2B subunit exchange in juvenile mice affects emotionality and 5-HT in the frontal cortex.

The N-methyl-D-aspartate receptor (NMDA-R) has been inter alia implicated in synaptic plasticity, brain development and emotional processes. The NMDA-R is a multiprotein complex composed of NR1, NR2 and/or NR3 subunits. We generated NR2C-2B mutant mice in which an insertion of NR2B cDNA into the gene locus of the NR2C gene replaced NR2C by NR2B expression throughout the brain. This NR2C-2B mutant was used to examine whether an NMDA-R subunit exchange in juvenile mice would affect emotional behaviors and acetylcholine (ACh), dopamine (DA) and serotonin (5-HT) content in the frontal cortex (FC) and brain structures, which are part of the brain defense system, such as the periaqueductal grey matter (PAG). Juvenile, 1-month-old NR2C-2B mice showed increased open arm avoidance in the elevated plus-maze and increased fear-induced immobility. In terms of brain neurochemistry, NR2C-2B mice showed an increase in 5-HT levels in the FC at the age of 2 months. A correlational analysis revealed that mice with low open arms avoidance had high levels of ACh in the PAG but reduced 5-HT levels in the FC. Animals which showed high levels of fear-induced immobility also had high levels of 5-HT in the FC. These results suggest that the replacement of subunit NR2C by NR2B in juvenile mice increases anxiety- and fear-related behaviors possibly due to changes in FC-5-HT and PAG-ACh levels.

The case for episodic memory in animals.

The conscious recollection of unique personal experiences in terms of their details (what), their locale (where) and temporal occurrence (when) is known as episodic memory and is thought to require a 'self-concept', autonoetic awareness/conciousness, and the ability to subjectively sense time. It has long been held that episodic memory is unique to humans, because it was accepted that animals lack a 'self-concept', 'autonoetic awareness', and the ability to 'subjectively sense time'. These assumptions are now being questioned by behavioral evidence showing that various animal species indeed show behavioral manifestations of different features of episodic memory such as, e.g. 'metacognition', 'conscious recollection' of past events, 'temporal order memory', 'mental time travel' and have the capacity to remember personal experiences in terms of what happened, where and when. The aim of this review is to provide a comprehensive overview on the current progress in attempts to model different prerequisites and features of human episodic memory in animals and to identify possible neural substrates of animal episodic memory. The literature covered includes behavioral and physiological studies performed with different animal species, such as non-human primates, rodents, dolphins and birds. The search for episodic memory in animals has forced researchers to define objective behavioral criteria by which different features of episodic memory can be operationalized experimentally and assessed in both animals and humans. This is especially important because the current definition of episodic memory in terms of mentalistic constructs such as 'self', 'autonoetic awareness/consciousness', and 'subjectively sensed time', not only hinders animal research on the neurobiology of episodic memory but also research with healthy human subjects as well as neuropsychiatric patients with impaired language or in children with less-developed verbal abilities.

Cholinergic cells in the nucleus basalis of mice express the N-methyl-D-aspartate-receptor subunit NR2C and its replacement by the NR2B subunit enhances frontal and amygdaloid acetylcholine levels.

It is known that glutamatergic and cholinergic systems interact functionally at the level of the cholinergic basal forebrain. The N-methyl-d-aspartate receptor (NMDA-R) is a multiprotein complex composed of NR1, NR2 and/or NR3 subunits. The subunit composition of NMDA-R of cholinergic cells in the nucleus basalis has not yet been investigated. Here, by means of choline acetyl transferase and NR2B or NR2C double staining, we demonstrate that mice express both the NR2C and NR2B subunits in nucleus basalis cholinergic cells. We generated NR2C-2B mutant mice in which an insertion of NR2B cDNA into the gene locus of the NR2C gene replaced NR2C by NR2B expression throughout the brain. This NR2C-2B mutant was used to examine whether a subunit exchange in cholinergic neurons would affect acetylcholine (ACh) content in several brain structures. We found increased ACh levels in the frontal cortex and amygdala in the brains of NR2C-2B mutant mice. Brain ACh has been implicated in neuroplasticity, novelty-induced arousal and encoding of novel stimuli. We therefore assessed behavioral habituation to novel environments and objects as well as object recognition in NR2C-2B subunit exchange mice. The behavioral analysis did not indicate any gross behavioral alteration in the mutant mice compared with the wildtype mice. Our results show that the NR2C by NR2B subunit exchange in mice affects ACh content in two target areas of the nucleus basalis.

Transient overexpression of the 5-HT1A receptor impairs water-maze but not hole-board performance.

Previously, we showed that mice that overexpress the 5-HT(1A) receptor transiently from embryonic to perinatal stages show reduced anxiety and changes in brain serotonin turnover as adults. Here, we investigated the long-term effects of the temporary overexpression of the 5-HT(1A) receptor during early embryonic and perinatal development on the performance in two memory tasks. In the hole-board test mice that were homozygous for the transgene showed similar behavioral habituation but increased locomotion compared to heterozygous mice. In contrast water-maze performance of homozygous mice was impaired compared to heterozygous mice. These results suggest that a transient overexpression of 5-HT(1A) receptor during embryonic and perinatal development has detrimental effects on water-maze performance at adult stages.

Stimulus complexity dependent memory impairment and changes in motor performance after deletion of the neuronal gap junction protein connexin36 in mice.

Gap junction channels, composed of connexin (Cx) proteins, are conduits for intercellular communication and metabolic exchange in the central nervous system. Connexin36 (Cx36) is expressed in distinct subpopulations of neurons throughout the mammalian brain. Deletion of the Cx36 gene in the mouse affected power and frequency of gamma and sharp wave-ripple oscillations, putative correlates of memory engram inscription. Here, we present a behavioral analysis of Cx36-deficient mice. Activity patterns, exploratory- and anxiety-related responses were largely unaffected by elimination of Cx36, while sensorimotor capacities and learning and memory processes were impaired. Repeated testing on the rotarod suggested that the Cx36-deficient mice showed slower motor-coordination learning. After a retention interval of 24 h the Cx36-deficient mice showed habituation to an open-field, but failed to habituate to a more complex spatial environment (Y-maze). A more pronounced memory impairment was found when Cx36 knockout mice had to remember recently explored objects. Cx36-deficient mice were unable to recognize objects after short delays of 15 and 45 min. These data suggest that lack of Cx36 induces memory impairments that vary in dependence of the complexity of the stimuli presented. Our results suggest that neuronal gap junctions incorporating Cx36 play a role in learning and memory.

Changes in motoric, exploratory and emotional behaviours and neuronal acetylcholine content and 5-HT turnover in histidine decarboxylase-KO mice.

Histamine has been implicated, inter alia, in mechanisms underlying arousal, exploratory behaviour and emotionality. Here, we investigated behavioural and neurochemical parameters related to these concepts, including open-field activity, rotarod performance and anxiety, as well as brain acetylcholine and 5-HT concentrations of mice deficient for the histidine decarboxylase (HDC) gene. These mice are unable to synthesize histamine from its precursor histidine. The HDC-knockout mice showed reduced exploratory activity in an open-field, but normal habituation to a novel environment. They behaved more anxious than the controls, as assessed by the height-fear task and the graded anxiety test, a modified elevated plus-maze. Furthermore, motor coordination on the rotarod was superior to controls. Biochemical assessments revealed that the HDC-knockout mice had higher acetylcholine concentrations and a significantly higher 5-HT turnover in the frontal cortex, but reduced acetylcholine levels in the neostriatum. These results are suggestive of important interactions between neuronal histamine and these site-specific neurotransmitters, which may be related to the behavioural changes found in the HDC-deficient animals.

Connexin30-deficient mice show increased emotionality and decreased rearing activity in the open-field along with neurochemical changes.

Gap-junction channels in the brain, formed by connexin (Cx) proteins with a distinct regional/cell-type distribution, allow intercellular electrical and metabolic communication. In astrocytes, mainly the connexins 43, 26 and 30 are expressed. In addition, connexin30 is expressed in ependymal and leptomeningeal cells, as well as in skin and cochlea. The functional implications of the astrocytic gap-junctional network are not well understood and evidence regarding their behavioural relevance is lacking. Thus, we have tested groups of Cx30-/-, Cx30+/-, and Cx30+/+ mice in the open-field, an object exploration task, in the graded anxiety test and on the rotarod. The Cx30-/- mice showed reduced exploratory activity in terms of rearings but not locomotion in the open-field and object exploration task. Furthermore, Cx30-/- mice exhibited anxiogenic behaviour as shown by higher open-field centre avoidance and corner preference. Graded anxiety test and rotarod performance was similar across groups. The Cx30-/- mice had elevated choline levels in the ventral striatum, possibly related to their aberrant behavioural phenotypes. The Cx30+/- mice had lower dopamine and metabolite levels in the amygdala and ventral striatum and lower hippocampal 5-hydroxyindole acid (5-HIAA) concentrations relative to Cx30+/+ mice. Furthermore, the Cx30+/- mice had lower acetylcholine concentrations in the ventral striatum and higher choline levels in the neostriatum, relative to Cx30+/+ mice. Our data suggest that the elimination of connexin30 can alter the reactivity to novel environments, pointing to the importance of gap-junctional signalling in behavioural processes.

NMDA-receptor antagonism via dextromethorphan and ifenprodil modulates graded anxiety test performance of C57BL/6 mice.

The majority of N-methyl-D-aspartate receptors (NMDA-R) in the adult forebrain are di- or triheteromers composed of NR1, NR2A and NR2B subunits. Subunit non-selective NMDA-R antagonists produce anxiolytic-like effects together with motor and sensory side-effects. The graded anxiety test (GAT), permits the within-task distinction of drug effects on anxiety from those on activity and perception. By testing NMDA-R subunit selective agents in the GAT it might be possible to determine whether their effects on anxiety, activity and perception are interrelated, and whether separate NMDA-R subtypes are involved. Dextromethorphan (weakly NR2A-selective) (10 and 30 mg/kg, i.p.) and ifenprodil (highly NR2B-selective) (1, 3 and 5 mg/kg, i.p.) were tested in the GAT. Both drugs failed to induce anxiolysis devoid of side-effects. However, the 10 mg/kg dose of dextromethorpan showed an anxiolytic, whereas the 30 mg/kg dose showed an anxiogenic, behavioral profile. Since the selective blockade of the NR2B subunit by ifenprodil had no clear anxiolytic effect, the anxiolytic potential of NMDA subunit non-selective agents might involve NR2A-containing receptors.