Disrupted circadian expression of ß-arrestin 2 affects reward-related µ-opioid receptor function in alcohol dependence.

There is increasing evidence for a daily rhythm of µ-opioid receptor (MOR) efficacy and the development of alcohol dependence. Previous studies show that ß-arrestin 2 (bArr2) has an impact on alcohol intake, at least partially mediated via modulation of MOR signaling, which in turn mediates the alcohol rewarding effects. Considering the interplay of circadian rhythms on MOR and alcohol dependence, we aimed to investigate bArr2 in alcohol dependence at different time points of the day/light cycle on the level of bArr2 mRNA (in situ hybridization), MOR availability (receptor autoradiography), and MOR signaling (Damgo-stimulated G-protein coupling) in the nucleus accumbens of alcohol-dependent and non-dependent Wistar rats. Using a microarray data set we found that bArr2, but not bArr1, shows a diurnal transcription pattern in the accumbens of naïve rats with higher expression levels during the active cycle. In 3-week abstinent rats, bArr2 is up-regulated in the accumbens at the beginning of the active cycle (ZT15), whereas no differences were found at the beginning of the inactive cycle (ZT3) compared with controls. This effect was accompanied by a specific down-regulation of MOR binding in the active cycle. Additionally, we detect a higher receptor coupling during the inactive cycle compared with the active cycle in alcohol-dependent animals. Together, we report daily rhythmicity for bArr2 expression linked to an inverse pattern of MOR, suggesting an involvement for bArr2 on circadian regulation of G-protein coupled receptors in alcohol dependence. The presented data may have implications for the development of novel bArr2-related treatment targets for alcoholism.

Pianp deficiency links GABAB receptor signaling and hippocampal and cerebellar neuronal cell composition to autism-like behavior.

Pianp (also known as Leda-1) is a type I transmembrane protein with preferential expression in the mammalian CNS. Its processing is characterized by proteolytic cleavage by a range of proteases including Adam10, Adam17, MMPs, and the γ-secretase complex. Pianp can interact with Pilrα and the GB1a subunit of the GABAB receptor (GBR) complex. A recent case description of a boy with global developmental delay and homozygous nonsense variant in PIANP supports the hypothesis that PIANP is involved in the control of behavioral traits in mammals. To investigate the physiological functions of Pianp, constitutive, global knockout mice were generated and comprehensively analyzed. Broad assessment did not indicate malformation or malfunction of internal organs. In the brain, however, decreased sizes and altered cellular compositions of the dentate gyrus as well as the cerebellum, including a lower number of cerebellar Purkinje cells, were identified. Functionally, loss of Pianp led to impaired presynaptic GBR-mediated inhibition of glutamate release and altered gene expression in the cortex, hippocampus, amygdala, and hypothalamus including downregulation of Erdr1, a gene linked to autism-like behavior. Behavioral phenotyping revealed that Pianp deficiency leads to context-dependent enhanced anxiety and spatial learning deficits, an altered stress response, severely impaired social interaction, and enhanced repetitive behavior, which all represent characteristic features of an autism spectrum disorder-like phenotype. Altogether, Pianp represents a novel candidate gene involved in autism-like behavior, cerebellar and hippocampal pathology, and GBR signaling.

CACNA1C gene regulates behavioral strategies in operant rule learning.

Behavioral experiments are usually designed to tap into a specific cognitive function, but animals may solve a given task through a variety of different and individual behavioral strategies, some of them not foreseen by the experimenter. Animal learning may therefore be seen more as the process of selecting among, and adapting, potential behavioral policies, rather than mere strengthening of associative links. Calcium influx through high-voltage-gated Ca2+ channels is central to synaptic plasticity, and altered expression of Cav1.2 channels and the CACNA1C gene have been associated with severe learning deficits and psychiatric disorders. Given this, we were interested in how specifically a selective functional ablation of the Cacna1c gene would modulate the learning process. Using a detailed, individual-level analysis of learning on an operant cue discrimination task in terms of behavioral strategies, combined with Bayesian selection among computational models estimated from the empirical data, we show that a Cacna1c knockout does not impair learning in general but has a much more specific effect: the majority of Cacna1c knockout mice still managed to increase reward feedback across trials but did so by adapting an outcome-based strategy, while the majority of matched controls adopted the experimentally intended cue-association rule. Our results thus point to a quite specific role of a single gene in learning and highlight that much more mechanistic insight could be gained by examining response patterns in terms of a larger repertoire of potential behavioral strategies. The results may also have clinical implications for treating psychiatric disorders.

Type-1 astrocyte-like stem cells harboring Cacna1d gene deletion exhibit reduced proliferation and decreased neuronal fate choice.

In the central nervous system, CaV 1.2 and CaV 1. 3 constitute the main L-type voltage-gated calcium channels (LTCCs) coupling membrane depolarization to gene transcription. We have previously demonstrated that inducible disruption of Cav1.2 in type-1 astrocyte-like stem cells of the adult dentate gyrus (DG) impairs hippocampal neurogenesis in a cell-autonomous fashion. To address the role of Cav1.3 channels (encoded by the Cacna1d gene), we here generated TgGLAST-CreERT2 /Cacna1dfl/fl /RCE:loxP mice which facilitate inducible deletion of Cacna1d in tandem with induction of EGFP expression in type-1 cells, allowing tracking of recombined cells and their descendants. Neurosphere cultures derived from fluorescence-activated cell sorting sorted Cacna1d-deficient (Cacna1d-/- /EGFP) hippocampal neural precursor cells (NPCs) exhibited a significant decrease in proliferative activity. Further, under differentiation conditions, Cacna1d deficiency conferred an increase in astrogenesis at the expense of neurogenesis. In like manner, type-1 cells lacking Cacna1d showed reduced proliferation in the dentate gyrus (DG) in vivo. Moreover, Cacna1d deficiency resulted in a significant decrease in the number of newly born cells adopting a neuronal fate. Finally, massive excitation induced by repeated electroconvulsive seizures rescued the proliferation defect of Cacna1d-/- /EGFP type-1 cells. Together, the effects of Cacna1d gene deletion closely recapitulate our earlier findings on the role of Cav1.2 channels expressed by type-1 cells. Similar to Cav1.2 channels, Cav1.3 channels on type-1 cells boost type-1 cell proliferation and promote subsequent neuronal fate choice.

Postnatal subventricular zone progenitors switch their fate to generate neurons with distinct synaptic input patterns.

New granule cell neurons (GCs) generated in the neonatal and adult subventricular zone (SVZ) have distinct patterns of input synapses in their dendritic domains. These synaptic input patterns determine the computations that the neurons eventually perform in the olfactory bulb. We observed that GCs generated earlier in postnatal life had acquired an 'adult' synaptic development only in one dendritic domain, and only later-born GCs showed an 'adult' synaptic development in both dendritic domains. It is unknown to what extent the distinct synaptic input patterns are already determined in SVZ progenitors and/or by the brain circuit into which neurons integrate. To distinguish these possibilities, we heterochronically transplanted retrovirally labeled SVZ progenitor cells. Once these transplanted progenitors, which mainly expressed Mash1, had differentiated into GCs, their glutamatergic input synapses were visualized by genetic tags. We observed that GCs derived from neonatal progenitors differentiating in the adult maintained their characteristic neonatal synapse densities. Grafting of adult SVZ progenitors to the neonate had a different outcome. These GCs formed synaptic densities that corresponded to neither adult nor neonatal patterns in two dendritic domains. In summary, progenitors in the neonatal and adult brain generate distinct GC populations and switch their fate to generate neurons with specific synaptic input patterns. Once they switch, adult progenitors require specific properties of the circuit to maintain their characteristic synaptic input patterns. Such determination of synaptic input patterns already at the progenitor-cell level may be exploited for brain repair to engineer neurons with defined wiring patterns.

Deletion of psychiatric risk gene Cacna1c impairs hippocampal neurogenesis in cell-autonomous fashion.

Ca2+ is a universal signal transducer which fulfills essential functions in cell development and differentiation. CACNA1C, the gene encoding the alpha-1C subunit (i.e., Cav 1.2) of the voltage-dependent l-type calcium channel (LTCC), has been implicated as a risk gene in a variety of neuropsychiatric disorders. To parse the role of Cav 1.2 channels located on astrocyte-like stem cells and their descendants in the development of new granule neurons, we created TgGLAST-CreERT2 /Cacna1cfl/fl /RCE:loxP mice, a transgenic tool that allows cell-type-specific inducible deletion of Cacna1c. The EGFP reporter was used to trace the progeny of recombined type-1 cells. FACS-sorted Cacna1c-deficient neural precursor cells from the dentate gyrus showed reduced proliferative activity in neurosphere cultures. Moreover, under differentiation conditions, Cacna1c-deficient NPCs gave rise to fewer neurons and more astroglia. Similarly, under basal conditions in vivo, Cacna1c gene deletion in type-1 cells decreased type-1 cell proliferation and reduced the neuronal fate-choice decision of newly born cells, resulting in reduced net hippocampal neurogenesis. Unexpectedly, electroconvulsive seizures completely compensated for the proliferation deficit of Cacna1c deficient type-1 cells, indicating that there must be Cav 1.2-independent mechanisms of controlling proliferation related to excitation. In the aggregate, this is the first report demonstrating the presence of functional L-type 1.2 channels on type-1 cells. Cav 1.2 channels promote type-1 cell proliferation and push the glia-to-neuron ratio in the direction of a neuronal fate choice and subsequent neuronal differentiation. Cav 1.2 channels expressed on NPCs and their progeny possess the ability to shape neurogenesis in a cell-autonomous fashion.

Effects of p75NTR deficiency on cholinergic innervation of the amygdala and anxiety-like behavior.

The p75 neurotrophin receptor (p75NTR) is a low-affinity receptor that is capable of binding neurotrophins. Two different p75NTR knockout mouse lines are available either with a deletion in Exon III (p75NTRExIII-/- ) or in Exon IV (p75NTRExIV-/- ). In p75NTRExIII knockout mice, only the full-length p75NTR is deleted, whereas in p75NTRExIV knockout mice, the full-length as well as the truncated isoform of the receptor is deleted. Deletion of p75NTR has been shown to affect, among others, the septohippocampal cholinergic innervation pattern and neuronal plasticity within the hippocampus. We hypothesize that deletion of p75NTR also alters the morphology and physiology of a further key structure of the limbic system, the amygdala. Our results indicate that deletion of p75NTR also increases cholinergic innervation in the basolateral amygdala in adult as well as aged p75NTRExIII-/- and p75NTRExIV-/- mice. The p75NTRExIV-/- mice did not display altered long-term potentiation (LTP) in the basolateral amygdala as compared to age-matched control littermates. However, p75NTRExIII-/- mice display stronger LTP in the basolateral amygdala compared to age-matched controls. Bath-application of K252a (a trk antagonist) did not inhibit the induction of LTP in the basolateral amygdala, but reduced the level of LTP in p75NTRExIII-/- mice to levels seen in respective controls. Moreover, p75NTRExIII-/- mice display altered behavior in the dark/light box. Thus, deletion of p75NTR in mice leads to physiological and morphological changes in the amygdala and altered behavior that is linked to the limbic system.

Losing Control: Excessive Alcohol Seeking after Selective Inactivation of Cue-Responsive Neurons in the Infralimbic Cortex.

Loss of control over drinking is a key deficit in alcoholism causally associated with malfunction of the medial prefrontal cortex (mPFC), but underlying molecular and cellular mechanisms remain unclear. Cue-induced reinstatement of alcohol seeking activates a subset of mPFC neurons in rats, identified by their common expression of the activity marker cFos and comprised of both principal and interneurons. Here, we used cFos-lacZ and pCAG-lacZ transgenic rats for activity-dependent or nonselective inactivation of neurons, respectively, which by their lacZ encoded ß-galactosidase activity convert the inactive prodrug Daun02 into the neurotoxin daunorubicin. We report that activity-dependent ablation of a neuronal ensemble in the infralimbic but not the prelimbic subregion induced excessive alcohol seeking. The targeted neuronal ensemble was specific for the cue-induced response because stress-induced reinstatement was not affected in these animals. Importantly, nonselective inactivation of infralimbic neurons, using pCAG-lacZ rats, was without functional consequence on the cue-induced reinstatement task. Thus, inhibitory control over alcohol seeking is exerted by distinct functional ensembles within the infralimbic cortex rather than by a general inhibitory tone of this region on the behavioral output. This indicates a high level of functional compartmentation within the rat mPFC whereat many functional ensembles could coexist and interact within the same subregion. SIGNIFICANCE STATEMENT: Hebb's (1949) idea of memories as being represented in local neuronal networks is supported by identification of transiently stable activity patterns within subgroups of neurons. However, it is difficult to link individual networks to specific memory tasks, for example a learned behavior. By a novel approach of activity-dependent ablation, here we identify a specific neuronal ensemble located in the infralimbic subregion of the medial prefrontal cortex that controls a seeking response for alcohol in rats. Our data demonstrate that functional output depends on specific neuronal ensembles within a given brain region rather than on the global activity of that region, which raises important questions about the interpretation of numerous earlier experiments using site-directed silencing or stimulation for elucidating brain function.

γ-Aminobutyric A receptor (GABA(A)R) regulates aquaporin 4 expression in the subependymal zone: relevance to neural precursors and water exchange.

Activation of γ-aminobutyric A receptors (GABA(A)Rs) in the subependymal zone (SEZ) induces hyperpolarization and osmotic swelling in precursors, thereby promoting surface expression of the epidermal growth factor receptor (EGFR) and cell cycle entry. However, the mechanisms underlying the GABAergic modulation of cell swelling are unclear. Here, we show that GABA(A)Rs colocalize with the water channel aquaporin (AQP) 4 in prominin-1 immunopositive (P(+)) precursors in the postnatal SEZ, which include neural stem cells. GABA(A)R signaling promotes AQP4 expression by decreasing serine phosphorylation associated with the water channel. The modulation of AQP4 expression by GABA(A)R signaling is key to its effect on cell swelling and EGFR expression. In addition, GABA(A)R function also affects the ability of neural precursors to swell in response to an osmotic challenge in vitro and in vivo. Thus, the regulation of AQP4 by GABA(A)Rs is involved in controlling activation of neural stem cells and water exchange dynamics in the SEZ.

Morphological and behavioral characterization of adult mice deficient for SrGAP3.

SrGAP3 belongs to the family of Rho GTPase proteins. These proteins are thought to play essential roles in development and in the plasticity of the nervous system. SrGAP3-deficient mice have recently been created and approximately 10 % of these mice developed a hydrocephalus and died shortly after birth. The others survived into adulthood, but displayed neuroanatomical alteration, including increased ventricular size. We now show that SrGAP3-deficient mice display increased brain weight together with increased hippocampal volume. This increase was accompanied by an increase of the thickness of the stratum oriens of area CA1 as well as of the thickness of the molecular layer of the dentate gyrus (DG). Concerning hippocampal adult neurogenesis, we observed no significant change in the number of proliferating cells. The density of doublecortin-positive cells also did not vary between SrGAP3-deficient mice and controls. By analyzing Golgi-impregnated material, we found that, in SrGAP3-deficient mice, the morphology and number of dendritic spines was not altered in the DG. Likewise, a Sholl-analysis revealed no significant changes concerning dendritic complexity as compared to controls. Despite the distinct morphological alterations in the hippocampus, SrGAP3-deficient mice were relatively inconspicuous in their behavior, not only in the open-field, nest building but also in the Morris water-maze. However, the SrGAP3-deficient mice showed little to no interest in burying marbles; a behavior that is seen in some animal models related to autism, supporting the view that SrGAP3 plays a role in neurodevelopmental disorders.

Synthetic microRNA-mediated downregulation of Nogo-A in transgenic rats reveals its role as regulator of synaptic plasticity and cognitive function.

We have generated a transgenic rat model using RNAi and used it to study the role of the membrane protein Nogo-A in synaptic plasticity and cognition. The membrane protein Nogo-A is expressed in CNS oligodendrocytes and subpopulations of neurons, and it is known to suppress neurite growth and regeneration. The constitutively expressed polymerase II-driven transgene was composed of a microRNA-targeting Nogo-A placed into an intron preceding the coding sequence for EGFP, thus quantitatively labeling cells according to intracellular microRNA expression. The transgenic microRNA in vivo efficiently reduced the concentration of Nogo-A mRNA and protein preferentially in neurons. The resulting significant increase in long-term potentiation in both hippocampus and motor cortex indicates a repressor function of Nogo-A in synaptic plasticity. The transgenic rats exhibited prominent schizophrenia-like behavioral phenotypes, such as perseveration, disrupted prepulse inhibition, and strong withdrawal from social interactions. This fast and efficient microRNA-mediated knockdown provides a way to silence gene expression in vivo in transgenic rats and shows a role of Nogo-A in regulating higher cognitive brain functions.

Activation of the sympathetic nervous system mediates hypophagic and anxiety-like effects of CB1 receptor blockade.

Complex interactions between periphery and the brain regulate food intake in mammals. Cannabinoid type-1 (CB1) receptor antagonists are potent hypophagic agents, but the sites where this acute action is exerted and the underlying mechanisms are not fully elucidated. To dissect the mechanisms underlying the hypophagic effect of CB1 receptor blockade, we combined the acute injection of the CB1 receptor antagonist rimonabant with the use of conditional CB1-knockout mice, as well as with pharmacological modulation of different central and peripheral circuits. Fasting/refeeding experiments revealed that CB1 receptor signaling in many specific brain neurons is dispensable for the acute hypophagic effects of rimonabant. CB1 receptor antagonist-induced hypophagia was fully abolished by peripheral blockade of ß-adrenergic transmission, suggesting that this effect is mediated by increased activity of the sympathetic nervous system. Consistently, we found that rimonabant increases gastrointestinal metabolism via increased peripheral ß-adrenergic receptor signaling in peripheral organs, including the gastrointestinal tract. Blockade of both visceral afferents and glutamatergic transmission in the nucleus tractus solitarii abolished rimonabant-induced hypophagia. Importantly, these mechanisms were specifically triggered by lipid-deprivation, revealing a nutrient-specific component acutely regulated by CB1 receptor blockade. Finally, peripheral blockade of sympathetic neurotransmission also blunted central effects of CB1 receptor blockade, such as fear responses and anxiety-like behaviors. These data demonstrate that, independently of their site of origin, important effects of CB1 receptor blockade are expressed via activation of peripheral sympathetic activity. Thus, CB1 receptors modulate bidirectional circuits between the periphery and the brain to regulate feeding and other behaviors.

SrGAP3 knockout mice display enlarged lateral ventricles and specific cilia disturbances of ependymal cells in the third ventricle.

In several mouse models of mental retardation, ventricular enlargements have been observed. Mutation in the SrGAP3 gene residing on chromosome 3p25 has previously been associated with intellectual disability in humans. In addition, SrGAP3 is related to Rho-GAPs signaling pathways, which play essential roles in the development and plasticity of the nervous system. About 10 % of postnatal homozygous SrGAP3-deficient mice die due to hydrocephalus, whereas the remaining mice survive into adulthood but display enlarged ventricles. We analyze the ventricular enlargement of these mice by performing a post-mortem MRI approach. We found a more than 15-fold enlargement of the lateral ventricles of homozygous SrGAP3-deficient mice. Moreover, we demonstrate that this phenotype was not accompanied by a stenosis of the aqueduct. Instead, SrGAP3 knockout mice displayed reduced densities of cilia of ependymal cells in These third ventricle compared to age-matched controls. This results indicate that the ventricular enlargement may be due to ciliopathy.

Retrocochlear function of the peripheral deafness gene Cacna1d.

Hearing impairment represents the most common sensory deficit in humans. Genetic mutations contribute significantly to this disorder. Mostly, only malfunction of the ear is considered. Here, we assessed the role of the peripheral deafness gene Cacna1d, encoding the L-type channel Ca(v)1.3, in downstream processing of acoustic information. To this end, we generated a mouse conditional Cacna1d-eGFP(flex) allele. Upon pairing with Egr2::Cre mice, Ca(v)1.3 was ablated in the auditory brainstem, leaving the inner ear intact. Structural assessment of the superior olivary complex (SOC), an essential auditory brainstem center, revealed a dramatic volume reduction (43-47%) of major nuclei in young adult Egr2::Cre;Cacna1d-eGFP(flex) mice. This volume decline was mainly caused by a reduced cell number (decline by 46-56%). Abnormal formation of the lateral superior olive was already present at P4, demonstrating an essential perinatal role of Ca(v)1.3 in the SOC. Measurements of auditory brainstem responses demonstrated a decreased amplitude in the auditory nerve between 50 and 75 dB stimulation in Egr2::Cre;Cacna1d-eGFP(flex) knockout mice and increased amplitudes in central auditory processing centers. Immunohistochemical studies linked the amplitude changes in the central auditory system to reduced expression of K(v)1.2. No changes were observed for K(v)1.1, KCC2, a determinant of inhibitory neurotransmission, and choline acetyltransferase, a marker of efferent olivocochlear neurons. Together, these analyses identify a crucial retrocochlear role of Ca(v)1.3 and demonstrate that mutations in deafness genes can affect sensory cells and neurons alike. As a corollary, hearing aids have to address central auditory processing deficits as well.

The role of L-type voltage-gated calcium channels Cav1.2 and Cav1.3 in normal and pathological brain function.

The use of specific activators and inhibitors that penetrate the central nervous system has suggested an essential functional role of L-type calcium channels (LTCC) in several important physiological processes of the brain, including the modulation of the mesoaccumbal dopamine signalling pathway, synaptic transmission of auditory stimuli and synaptic plasticity of neutral and aversive learning and memory processes. However, the lack of selectivity of available pharmacological agents towards the most prominent LTCC isoforms in the brain, namely Cav1.2 and Cav1.3, has hampered the elucidation of the precise contribution made by each specific channel isoform within these specific physiological processes. Modern genetic approaches, both in rodents and in human, have recently enhanced our understanding of the selective functional roles of Cav1.2 and Cav1.3 channels. In rodents, the characterisation of global and conditional isoform-specific knockouts suggests a contribution of Cav1.2 channels in spatial memory formation, whereas Cav1.3 channels seem to be involved in the consolidation of fear memories and in neurodegenerative mechanisms associated with the development of Parkinson's disease. With regard to the molecular mechanisms underlying drug addiction, Cav1.3 channels are necessary for the development and Cav1.2 channels for the expression of cocaine and amphetamine behavioural sensitisation. In humans, both the identification of naturally occurring LTCC variants ("channelopathies") and unbiased genome-wide association studies have linked LTCCs to working memory performance in healthy individuals and schizophrenic patients. Individually, CACNA1C polymorphisms and CACNA1D variants have been linked to a variety of psychiatric diseases and to congenital deafness, respectively. However, the contribution of individual LTCCs and their polymorphisms to human brain function and diseases remains unclear, necessitating the use of isoform-specific pharmacological agents.

Nogo-A downregulation impairs place avoidance in the Carousel maze but not spatial memory in the Morris water maze.

Nogo-A protein is an important inhibitor of axonal growth, which also regulates neuronal plasticity in the CNS. Mutations in the gene encoding Nogo-A or abnormalities in Nogo-A expression are linked to neuropsychiatric disorders such as schizophrenia. The present study assesses the impact of constitutively reduced expression of Nogo-A on place navigation in a novel transgenic rat model. Two spatial paradigms were used: (1) A battery of tests in the Carousel maze requiring continuous processing of spatial information with increasing demands for the segregation of reference frames and behavioral flexibility and (2) a delayed-matching-to-place version of the Morris water maze (MWM), which requires place navigation and is sensitive to deficits in one-trial-encoded place representation. The Carousel maze testing revealed a subtle but significant impairment in management of reference frames. Matching-to-place learning in the Morris water maze was unaffected, suggesting an intact representation of an unmarked goal. Our results show that Nogo-A deficiency leads to cognitive deficit in processing of the reference frames. Such a deficit may be the result of neuro-developmental alterations resulting from Nogo-A deficiency.

Genetic fate mapping of type-1 stem cell-dependent increase in newborn hippocampal neurons after electroconvulsive seizures.

Electroconvulsive therapy (ECT) is a uniquely effective treatment for major depressive disorder. An increase in hippocampal neurogenesis is implicated in the recovery from depression. We used an inducible genetic mouse model in which only GFAP-expressing stem-like cells (type-1 cells) and their progeny are selectively labeled with the reporter protein ß-galactosidase to track the process of neurogenesis in the dentate gyrus over 3 months following electroconvulsive seizures (ECS), the mouse equivalent of ECT. All ECS protocols tested induced a transient increase in type-1 cell divisions. While this led to an expansion of the type-1 cell pool after high-frequency ECS sessions for 5 consecutive days (5-ECS), asymmetric divisions drove neurogenesis by giving rise to Doublecortin (DCX)-expressing neuroblasts that matured into NeuN+ neurons. Significantly, the increase in newly generated DCX+ and NeuN+ cells after 5-ECS could be traced back to proliferating type-1 cells. Low-frequency continuation ECS (c-ECS) consisting of five single ECS sessions administered every 2 weeks resulted in a similar increase in newborn neurons as the high-frequency 5-ECS protocol. Moreover, the combination of 5-ECS and c-ECS led to a further significant increase in newborn neurons, suggesting a cellular mechanism responsible for the propitious effects of high-frequency ECT followed by continuation ECT in severely depressed patients. The ability of high- and low-frequency ECS to induce normally quiescent type-1 cells to proliferate and generate new neurons sets it apart from other antidepressant treatments and may underlie the superior clinical efficacy of ECT.

SrGAP3 interacts with lamellipodin at the cell membrane and regulates Rac-dependent cellular protrusions.

SrGAP3/MEGAP is a member of the Slit-Robo GAP (srGAP) family and is implicated in repulsive axon guidance and neuronal migration through Slit-Robo-mediated signal transduction. Here we describe an inhibitory role of srGAP3 on actin dynamics, specifically on lamellipodia formation. We show that the F-BAR domain localizes srGAP3 to the leading edge of cellular protrusions whereas the SH3 domain is important for focal adhesion targeting. We report on a novel srGAP3 interaction partner, lamellipodin, which localizes with srGAP3 at the leading edge. Live-cell analyses revealed that srGAP3 influences lamellipodin-evoked lamellipodial dynamics. Furthermore, we show that mouse embryonic fibroblasts derived from homozygous srGAP3-knockout embryos display an increased cell area and lamellipodia formation that can be blocked by shRNA-mediated knockdown of lamellipodin.

Srgap3⁻/⁻ mice present a neurodevelopmental disorder with schizophrenia-related intermediate phenotypes.

Mutations in the SRGAP3 gene residing on chromosome 3p25 have previously been associated with intellectual disability. Genome-wide association studies have also revealed SRGAP3, together with genes from the same cellular network, as risk genes for schizophrenia. SRGAP3 regulates cytoskeletal dynamics through the RHO protein RAC1. RHO proteins are known to be involved in cytoskeletal reorganization during brain development to control processes such as synaptic plasticity. To elucidate the importance of SRGAP3 in brain development, we generated Srgap3-knockout mice. Ten percent of these mice developed a hydrocephalus and died before adulthood. Surviving mice showed various neuroanatomical changes, including enlarged lateral ventricles, white matter tracts, and dendritic spines together with molecular changes, including an increased basal activity of RAC1. Srgap3(-/-) mice additionally exhibited a complex behavioral phenotype. Behavioral studies revealed an impaired spontaneous alternation and social behavior, while long-term memory was unchanged. The animals also had tics. Lower locomotor activity was observed in male Srgap3(-/-) only. Srgap3(-/-) mice showed increased methylphenidate stimulation in males and an impaired prepulse inhibition in females. Together, the results show neurodevelopmental aberration in Srgap3(-/-) mice, with many of the observed phenotypes matching several schizophrenia-related intermediate phenotypes. Mutations of SRGAP3 may thus contribute to various neurodevelopmental disorders.

Conditional gene expression systems in the transgenic rat brain.

BACKGROUND: Turning gene expression on and off at will is one of the most powerful tools for the study of gene function in vivo. While several conditional systems were successful in invertebrates, in mice the Cre/loxP recombination system and the tet-controlled transcription activation system are predominant. Both expression systems allow for spatial and temporal control of gene activities, and, in the case of tet regulation, even for the reversible activation/inactivation of gene expression. Although the rat is the principal experimental model in biomedical research, in particular in studies of neuroscience, conditional rat transgenic systems are exceptionally rare in this species. RESULTS: We addressed this lack of technology, and established and thoroughly characterized CreERT2 and tTA transgenic rats with forebrain-specific transgene expression, controlled by the CaMKII alpha promoter. In addition, we developed new universal rat reporter lines for both transcription control systems and established inducible and efficient reporter gene expression in forebrain neurons. CONCLUSIONS: We demonstrate that conditional genetic manipulations in the rat brain are both feasible and practicable and outline advantages and limitations of the Tet and Cre/loxP system in the rat brain.