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Expressions of voltage-gated sodium channels Nav1.1 and Nav1.2, encoded by SCN1A and SCN2A genes, respectively, have been reported to be mutually exclusive in most brain regions. In juvenile and adult neocortex, Nav1.1 is predominantly expressed in inhibitory neurons while Nav1.2 is in excitatory neurons. Although a distinct subpopulation of layer V (L5) neocortical excitatory neurons were also reported to express Nav1.1, their nature has been uncharacterized. In hippocampus, Nav1.1 has been proposed to be expressed only in inhibitory neurons. By using newly generated transgenic mouse lines expressing Scn1a promoter-driven green fluorescent protein (GFP), here we confirm the mutually exclusive expressions of Nav1.1 and Nav1.2 and the absence of Nav1.1 in hippocampal excitatory neurons. We also show that Nav1.1 is expressed in inhibitory and a subpopulation of excitatory neurons not only in L5 but all layers of neocortex. By using neocortical excitatory projection neuron markers including FEZF2 for L5 pyramidal tract (PT) and TBR1 for layer VI (L6) cortico-thalamic (CT) projection neurons, we further show that most L5 PT neurons and a minor subpopulation of layer II/III (L2/3) cortico-cortical (CC) neurons express Nav1.1 while the majority of L6 CT, L5/6 cortico-striatal (CS), and L2/3 CC neurons express Nav1.2. These observations now contribute to the elucidation of pathological neural circuits for diseases such as epilepsies and neurodevelopmental disorders caused by SCN1A and SCN2A mutations.

The mechanisms coordinating the sensing and seeking of social contacts are unclear. Here, the authors show that amylin-calcitonin receptor signalling in the media preoptic area mediates affiliative social behaviors in female mice.

Abstract Reduced expression of glutamate decarboxylase 67 (GAD67), encoded by the Gad1 gene, is a consistent finding in postmortem brains of patients with several psychiatric disorders, including schizophrenia, bipolar disorder and major depressive disorder. The dysfunction of GAD67 in the brain is implicated in the pathophysiology of these psychiatric disorders; however, the neurobiological consequences of GAD67 dysfunction in mature brains are not fully understood because the homozygous Gad1 knockout is lethal in newborn mice. We hypothesized that the tetracycline-controlled gene expression/suppression system could be applied to develop global GAD67 knockdown mice that would survive into adulthood. In addition, GAD67 knockdown mice would provide new insights into the neurobiological impact of GAD67 dysfunction. Here, we developed Gad1 tTA/STOP−tetO biallelic knock-in mice using Gad1 STOP−tetO and Gad1 tTA knock-in mice, and compared them with Gad1 +/+ mice. The expression level of GAD67 protein in brains of Gad1 tTA/STOP−tetO mice treated with doxycycline (Dox) was decreased by approximately 90%. The GABA content was also decreased in the brains of Dox-treated Gad1 tTA/STOP−tetO mice. In the open-field test, Dox-treated Gad1 tTA/STOP−tetO mice exhibited hyper-locomotor activity and decreased duration spent in the center region. In addition, acoustic startle responses were impaired in Dox-treated Gad1 tTA/STOP−tetO mice. These results suggest that global reduction in GAD67 elicits emotional abnormalities in mice. These GAD67 knockdown mice will be useful for elucidating the neurobiological mechanisms of emotional abnormalities, such as anxiety symptoms associated with psychiatric disorders.

Abstract The insular cortex (IC) is the primary gustatory cortex, and it is a critical structure for encoding and retrieving the conditioned taste aversion (CTA) memory. In the CTA, consumption of an appetitive tastant is associated with aversive experience such as visceral malaise, which results in avoidance of consuming a learned tastant. Previously, we showed that levels of the cyclic-AMP-response-element-binding protein (CREB) determine the insular cortical neurons that proceed to encode a conditioned taste memory. In the amygdala and hippocampus, it is shown that CREB and neuronal activity regulate memory allocation and the neuronal mechanism that determines the specific neurons in a neural network that will store a given memory. However, cellular mechanism of memory allocation in the insular cortex is not fully understood. In the current study, we manipulated the neuronal activity in a subset of insular cortical and/or basolateral amygdala (BLA) neurons in mice, at the time of learning; for this purpose, we used an hM3Dq designer receptor exclusively activated by a designer drug system (DREADD). Subsequently, we examined whether the neuronal population whose activity is increased during learning, is reactivated by memory retrieval, using the expression of immediate early gene c-fos. When an hM3Dq receptor was activated only in a subset of IC neurons, c-fos expression following memory retrieval was not significantly observed in hM3Dq-positive neurons. Interestingly, the probability of c-fos expression in hM3Dq-positive IC neurons after retrieval was significantly increased when the IC and BLA were co-activated during conditioning. Our findings suggest that functional interactions between the IC and BLA regulates CTA memory allocation in the insular cortex, which shed light on understanding the mechanism of memory allocation regulated by interaction between relevant brain areas.

Dopamine neurons play crucial roles in pleasure, reward, memory, learning, and fine motor skills and their dysfunction is associated with various neuropsychiatric diseases. Dopamine receptors are the main target of treatment for neurologic and psychiatric disorders. Antipsychotics that antagonize the dopamine D2 receptor (DRD2) are used to alleviate the symptoms of these disorders but may also sometimes cause disabling side effects such as parkinsonism (catalepsy in rodents). Here we show that GPR143, a G-protein-coupled receptor for L-3,4-dihydroxyphenylalanine (L-DOPA), expressed in striatal cholinergic interneurons enhances the DRD2-mediated side effects of haloperidol, an antipsychotic agent. Haloperidol-induced catalepsy was attenuated in male Gpr143 gene-deficient (Gpr143-/y) mice compared with wild-type (Wt) mice. Reducing the endogenous release of L-DOPA and preventing interactions between GPR143 and DRD2 suppressed the haloperidol-induced catalepsy in Wt mice but not Gpr143-/y mice. The phenotypic defect in Gpr143-/y mice was mimicked in cholinergic interneuron-specific Gpr143-/y (Chat-cre;Gpr143flox/y) mice. Administration of haloperidol increased the phosphorylation of ribosomal protein S6 at Ser240/244 in the dorsolateral striatum of Wt mice but not Chat-cre;Gpr143flox/y mice. In Chinese hamster ovary cells stably expressing DRD2, co-expression of GPR143 increased cell surface expression level of DRD2, and L-DOPA application further enhanced the DRD2 surface expression. Shorter pauses in cholinergic interneuron firing activity were observed after intrastriatal stimulation in striatal slice preparations from Chat-cre; Gpr143flox/y mice compared with those from Wt mice. Together, these findings provide evidence that GPR143 regulates DRD2 function in cholinergic interneurons and may be involved in parkinsonism induced by antipsychotic drugs. [ABSTRACT FROM AUTHOR]

Spike and wave discharge (SWD) activity is seen during absence seizures and is thought to be thalamocortical in origin. Here, the authors show that SWDs are initiated through the impaired corticostriatal excitatory transmissions onto striatal fast spiking interneurons.

Abstract Background Mutations of the SCN2A gene encoding a voltage-gated sodium channel alpha-II subunit Nav1.2 are associated with neurological disorders such as epilepsy, autism spectrum disorders, intellectual disability, and schizophrenia. However, causal relationships and pathogenic mechanisms underlying these neurological defects, especially social and psychiatric features, remain to be elucidated. Methods We investigated the behavior of mice with a conventional or conditional deletion of Scn2a in a comprehensive test battery including open field, elevated plus maze, light-dark box, three chambers, social dominance tube, resident-intruder, ultrasonic vocalization, and fear conditioning tests. We further monitored the effects of the positive allosteric modulator of AMPA receptors CX516 on these model mice. Results Conventional heterozygous Scn2a knockout mice (Scn2a KO/+) displayed novelty-induced exploratory hyperactivity and increased rearing. The increased vertical activity was reproduced by heterozygous inactivation of Scn2a in dorsal-telencephalic excitatory neurons but not in inhibitory neurons. Moreover, these phenotypes were rescued by treating Scn2a KO/+ mice with CX516. Additionally, Scn2a KO/+ mice displayed mild social behavior impairment, enhanced fear conditioning, and deficient fear extinction. Neuronal activity was intensified in the medial prefrontal cortex of Scn2a KO/+ mice, with an increase in the gamma band. Conclusions Scn2a KO/+ mice exhibit a spectrum of phenotypes commonly observed in models of schizophrenia and autism spectrum disorder. Treatment with the CX516 ampakine, which ameliorates hyperactivity in these mice, could be a potential therapeutic strategy to rescue some of the disease phenotypes.

Astrocytes elicit transient Ca2+ elevations induced by G protein-coupled receptors (GPCRs), yet their role in vivo remains unknown. To address this, transgenic mice with astrocytic expression of the optogenetic Gq-type GPCR, Optoα1AR, were established, in which transient Ca2+ elevations similar to those in wild type mice were induced by brief blue light illumination. Activation of cortical astrocytes resulted in an adenosine A1 receptor-dependent inhibition of neuronal activity. Moreover, sensory stimulation with astrocytic activation induced long-term depression of sensory evoked response. At the behavioral level, repeated astrocytic activation in the anterior cortex gradually affected novel open field exploratory behavior, and remote memory was enhanced in a novel object recognition task. These effects were blocked by A1 receptor antagonism. Together, we demonstrate that GPCR-triggered Ca2+ elevation in cortical astrocytes has causal impacts on neuronal activity and behavior.

Dravet syndrome is a severe infantile-onset epileptic encephalopathy which begins with febrile seizures and is caused by heterozygous loss-of-function mutations of the voltage-gated sodium channel gene SCN1A. We designed a CRISPR-based gene therapy for Scn1a-haplodeficient mice using multiple guide RNAs (gRNAs) in the promoter regions together with the nuclease-deficient Cas9 fused to transcription activators (dCas9-VPR) to trigger the transcription of SCN1A or Scn1a in vitro. We tested the effect of this strategy in vivo using an adeno-associated virus (AAV) mediated system targeting inhibitory neurons and investigating febrile seizures and behavioral parameters. In both the human and mouse genes multiple guide RNAs (gRNAs) in the upstream, rather than downstream, promoter region showed high and synergistic activities to increase the transcription of SCN1A or Scn1a in cultured cells. Intravenous injections of AAV particles containing the optimal combination of 4 gRNAs into transgenic mice with Scn1a-haplodeficiency and inhibitory neuron-specific expression of dCas9-VPR at four weeks of age increased Nav1.1 expression in parvalbumin-positive GABAergic neurons, ameliorated their febrile seizures and improved their behavioral impairments. Although the usage of transgenic mice and rather modest improvements in seizures and abnormal behaviors hamper direct clinical application, our results indicate that the upregulation of Scn1a expression in the inhibitory neurons can significantly improve the phenotypes, even when applied after the juvenile stages. Our findings also suggest that the decrease in Nav1.1 is directly involved in the symptoms seen in adults with Dravet syndrome and open a way to improve this condition.

Summary: Microglia, the resident immune cells of the central nervous system, accumulate along subcerebral projection axons and support neuronal survival during the early postnatal period. It remains unknown how microglia follow an axon-specific distribution pattern to maintain neural circuits. Here, we investigated the mechanisms of microglial accumulation along subcerebral projection axons that were necessary for microglial accumulation in the internal capsule. Screening of molecules involved in this accumulation of microglia to axons of layer V cortical neurons identified netrin-G1, a member of the netrin family of axon guidance molecules with a glycosyl-phosphatidylinositol anchor. Deletion or knockdown of the netrin-G1 gene Ntng1 reduced microglial accumulation and caused loss of cortical neurons. Netrin-G1 ligand-Ngl1 knockout-mice-derived microglia showed reduced accumulation along the axons compared with wild-type microglia. Thus, microglia accumulate around the subcerebral projection axons via NGL1-netrin-G1 signaling and support neuronal survival. Our observations unveil bidirectional neurotrophic interactions between neurons and microglia. : The mechanism underlying microglial accumulation along subcerebral projection axons to maintain neural circuits during the early postnatal period remains largely unknown. Fujita et al. demonstrate that microglial NGL1 and neuronal netrin-G1 signaling mediates microglial accumulation around the subcerebral projection axons and supports neuronal survival. Keywords: brain, microglia, neuron, axon, netrin-G1

The hippocampus, a region critical for memory and spatial navigation, has been implicated in delay discounting, the decline in subjective reward value when a delay is imposed. However, how delay information is encoded in the hippocampus is poorly understood. Here, we recorded from CA1 of mice performing a delay-discounting decision-making task, where delay lengths, delay positions, and reward amounts were changed across sessions, and identified subpopulations of CA1 neurons that increased or decreased their firing rate during long delays. The activity of both delay-active and -suppressed cells reflected delay length, delay position, and reward amount; but manipulating reward amount differentially impacted the two populations, suggesting distinct roles in the valuation process. Further, genetic deletion of the N-methyl-D-aspartate (NMDA) receptor in hippocampal pyramidal cells impaired delay-discount behavior and diminished delay-dependent activity in CA1. Our results suggest that distinct subclasses of hippocampal neurons concertedly support delay-discounting decisions in a manner that is dependent on NMDA receptor function.

Summary: Multisensory integration (MSI) is a fundamental emergent property of the mammalian brain. During MSI, perceptual information encoded in patterned activity is processed in multimodal association cortex. The systems-level neuronal dynamics that coordinate MSI, however, are unknown. Here, we demonstrate intrinsic hub-like network activity in the association cortex that regulates MSI. We engineered calcium reporter mouse lines based on the fluorescence resonance energy transfer sensor yellow cameleon (YC2.60) expressed in excitatory or inhibitory neurons. In medial and parietal association cortex, we observed spontaneous slow waves that self-organized into hubs defined by long-range excitatory and local inhibitory circuits. Unlike directional source/sink-like flows in sensory areas, medial/parietal excitatory and inhibitory hubs had net-zero balanced inputs. Remarkably, multisensory stimulation triggered rapid phase-locking mainly of excitatory hub activity persisting for seconds after the stimulus offset. Therefore, association cortex tends to form balanced excitatory networks that configure slow-wave phase-locking for MSI. Video Abstract: : Kuroki et al. performed cell-type-specific, wide-field FRET-based calcium imaging to visualize cortical network activity induced by multisensory inputs. They observed phase-locking of cortical slow waves in excitatory neuronal hubs in association cortical areas that may underlie multisensory integration. Keywords: wide-field calcium imaging, multisensory integration, cortical slow waves, association cortex, phase locking, fluorescence resonance energy transfer, spontaneous activity, excitatory neuron, inhibitory neuron, mouse

Cytoplasmic aggregation of fused in sarcoma (FUS) is detected in brain regions affected by amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), which compose the disease spectrum, FUS proteinopathy. To understand the pathomechanism of ALS-FTD-associated FUS, we examined the behavior and cellular properties of an ALS mouse model overexpressing FUS with nuclear localization signal deletion. Mutant FUS transgenic mice showed hyperactivity, social interactional deficits, and impaired fear memory retrieval, all of which are compatible with FTD phenotypes. Histological analyses showed decreased dendritic spine and synaptic density in the frontal cortex before neuronal loss. Examination of cultured cells confirmed that mutant but not wild-type FUS was associated with decreased dendritic growth, mRNA levels, and protein synthesis in dendrites. These data suggest that cytoplasmic FUS aggregates impair dendritic mRNA trafficking and translation, in turn leading to dendritic homeostasis disruption and the development of FTD phenotypes.

While transcranical direct current stimulation (tDCS) is used in clinical setting, its cellular mechanism of action is unclear. Here, Hajime Hirase and colleagues visualize cellular response in mouse brain to tDCS and show robust astrocyte activation that coincide with plasticity changes.

A major concern in neuroscience is how cognitive ability in adulthood is affected and regulated by developmental mechanisms. The molecular bases of cognitive development are not well understood. We provide evidence for the involvement of the α2 isoform of Rac-specific guanosine triphosphatase (GTPase)-activating protein (RacGAP) α-chimaerin (chimerin) in this process. We generated and analyzed mice with global and conditional knockouts of α-chimaerin and its isoforms (α1-chimaerin and α2-chimaerin) and found that α-chimaerin plays a wide variety of roles in brain function and that the roles of α1-chimaerin and α2-chimaerin are distinct. Deletion of α2-chimaerin, but not α1-chimaerin, beginning during early development results in an increase in contextual fear learning in adult mice, whereas learning is not altered when α2-chimaerin is deleted only in adulthood. Our findings suggest that α2-chimaerin acts during development to establish normal cognitive ability in adulthood.

Opalin, a central nervous system-specific myelin protein phylogenetically unique to mammals, has been suggested to play a role in mammalian-specific myelin. To elucidate the role of Opalin in mammalian myelin, we disrupted the Opalin gene in mice and analyzed the impacts on myelination and behavior. Opalin-knockout (Opalin-/-) mice were born at a Mendelian ratio and had a normal body shape and weight. Interestingly, Opalin-/- mice had no obvious abnormalities in major myelin protein compositions, expression of oligodendrocyte lineage markers, or domain organization of myelinated axons compared with WT mice (Opalin+/+) mice. Electron microscopic observation of the optic nerves did not reveal obvious differences between Opalin+/+ and Opalin-/- mice in terms of fine structures of paranodal loops, transverse bands, and multi-lamellae of myelinated axons. Moreover, sensory reflex, circadian rhythm, and locomotor activity in the home cage, as well as depression-like behavior, in the Opalin-/- mice were indistinguishable from the Opalin+/+ mice. Nevertheless, a subtle but significant impact on exploratory activity became apparent in Opalin-/- mice exposed to a novel environment. These results suggest that Opalin is not critical for central nervous system myelination or basic sensory and motor activities under conventional breeding conditions, although it might be required for fine-tuning of exploratory behavior.

Summary The membrane-anchored metalloproteinase-regulator RECK has been characterized as a tumor suppressor. Here we report that mice with reduced Reck-expression show limb abnormalities including right-dominant, forelimb-specific defects in postaxial skeletal elements. The forelimb buds of low-Reck mutants have an altered dorsal ectoderm with reduced Wnt7a and Igf2 expression, and hypotrophy in two signaling centers (i.e., ZPA and AER) that are essential for limb outgrowth and patterning. Reck is abundantly expressed in the anterior mesenchyme in normal limb buds; mesenchyme-specific Reck inactivation recapitulates the low-Reck phenotype; and some teratogens downregulate Reck in mesenchymal cells. Our findings illustrate a role for Reck in the mesenchymal-epithelial interactions essential for mammalian development.

S100B is a Ca2+ binding protein and is typically associated with brain and CNS disorders. However, the role of S100B in an inflammatory situation is not clear. The aim of the study was to determine whether S100B is likely to influence inflammation through its effect on macrophages. A murine macrophage cell line (RAW 264.7) and primary bone marrow derived macrophages were used for in vitro studies and a model of retinal inflammatory disease in which pathogenesis is highly dependent on macrophage infiltration, Experimental Autoimmune Uveoretinitis, for in vitro study. Experimental Autoimmune Uveoretinitis is a model for the human disease posterior endogenous uveoretinitis, a potentially blinding condition, with an autoimmune aetiology, that mainly affects the working age group. To date the involvement of S100B in autoimmune uveoretinitis has not been investigated. Real-time PCR array analysis on RAW 246.7 cells indicated up-regulation of gene expression for various cytokines/chemokines in response to S100B, IL-1β and CCL22 in particular and this was confirmed by real-time PCR. In addition flow cytometry and ELISA confirmed up-regulation of protein production in response to S100B for pro-IL-1β and CCL22 respectively. This was the case for both RAW 264.7 cells and bone marrow derived macrophages. Induction of EAU with retinal antigen in mice in which S100B had been deleted resulted in a significantly reduced level of disease compared to wild-type mice, as determined by topical endoscopic fundus imaging and histology grading. Macrophage infiltration was also significantly reduced in S100B deleted mice. Real-time PCR analysis indicated that this was associated with reduction in CCL22 and IL-1β in retinas from S100B knock-out mice. In conclusion S100B augments the inflammatory response in uveoretinitis and this is likely to be, at least in part, via a direct effect on macrophages.

The M5 muscarinic acetylcholine receptor (M5R) has been shown to play a crucial role in mediating acetylcholine-dependent dilation of cerebral blood vessels. We show that male M5R−/− mice displayed constitutive constriction of cerebral arteries using magnetic resonance angiography in vivo. Male M5R−/− mice exhibited a significantly reduced cerebral blood flow (CBF) in the cerebral cortex, hippocampus, basal ganglia, and thalamus. Cortical and hippocampal pyramidal neurons from M5R−/− mice showed neuronal atrophy. Hippocampus-dependent spatial and nonspatial memory was also impaired in M5R−/− mice. In M5R−/− mice, CA3 pyramidal cells displayed a significantly attenuated frequency of the spontaneous postsynaptic current and long-term potentiation was significantly impaired at the mossy fiber-CA3 synapse. Our findings suggest that impaired M5R signaling may play a role in the pathophysiology of cerebrovascular deficits. The M5 receptor may represent an attractive novel therapeutic target to ameliorate memory deficits caused by impaired cerebrovascular function.

Low-threshold mechanoreceptors (LTMRs) and their cutaneous end organs convert light mechanical forces acting on the skin into electrical signals that propagate to the central nervous system. In mouse hairy skin, hair follicle-associated longitudinal lanceolate complexes, which are end organs comprising LTMR axonal endings that intimately associate with terminal Schwann cell (TSC) processes, mediate LTMR responses to hair deflection and skin indentation. Here, we characterized developmental steps leading to the formation of Aß rapidly adapting (RA)-LTMR and Ad-LTMR lanceolate complexes. During early postnatal development, Aß RA-LTMRs and Ad-LTMRs extend and prune cutaneous axonal branches in close association with nascent TSC processes. Netrin-G1 is expressed in these developing Aß RA-LTMR and Ad-LTMR lanceolate endings, and Ntng1 ablation experiments indicate that Netrin-G1 functions in sensory neurons to promote lanceolate ending elaboration around hair follicles. The Netrin-G ligand (NGL-1), encoded by Lrrc4c, is expressed in TSCs, and ablation of Lrrc4c partially phenocopied the lanceolate complex deficits observed in Ntng1 mutants. Moreover, NGL-1-Netrin-G1 signaling is a general mediator of LTMR end organ formation across diverse tissue types demonstrated by the fact that Aß RA-LTMR endings associated with Meissner corpuscles and Pacinian corpuscles are also compromised in the Ntng1 and Lrrc4c mutant mice. Thus, axon-glia interactions, mediated in part by NGL-1-Netrin-G1 signaling, promote LTMR end organ formation. [ABSTRACT FROM AUTHOR]

Avulsion of spinal nerve roots in the brachial plexus (BP) can be repaired by crossing nerve transfer via a nerve graft to connect injured nerve ends to the BP contralateral to the lesioned side. Sensory recovery in these patients suggests that the contralateral primary somatosensory cortex (S1) is activated by afferent inputs that bypassed to the contralateral BP. To confirm this hypothesis, the present study visualized cortical activity after crossing nerve transfer in mice through the use of transcranial flavoprotein fluorescence imaging. In naïve mice, vibratory stimuli applied to the forepaw elicited localized fluorescence responses in the S1 contralateral to the stimulated side, with almost no activity in the ipsilateral S1. Four weeks after crossing nerve transfer, forepaw stimulation in the injured and repaired side resulted in cortical responses only in the S1 ipsilateral to the stimulated side. At eight weeks after crossing nerve transfer, forepaw stimulation resulted in S1 cortical responses of both hemispheres. These cortical responses were abolished by cutting the nerve graft used for repair. Exposure of the ipsilateral S1 to blue laser light suppressed cortical responses in the ipsilateral S1, as well as in the contralateral S1, suggesting that ipsilateral responses propagated to the contralateral S1 via cortico-cortical pathways. Direct high-frequency stimulation of the ipsilateral S1 in combination with forepaw stimulation acutely induced S1 bilateral cortical representation of the forepaw area in naïve mice. Cortical responses in the contralateral S1 after crossing nerve transfer were reduced in cortex-restricted heterotypic GluN1 (NMDAR1) knockout mice. Functional bilateral cortical representation was not clearly observed in genetically manipulated mice with impaired cortico-cortical pathways between S1 of both hemispheres. Taken together, these findings strongly suggest that activity-dependent potentiation of cortico-cortical pathways has a critical role for sensory recovery in patients after crossing nerve transfer.

Cyclin-dependent kinase-like 2 (Cdkl2) is a cdc2-related serine/threonine protein kinase that is postnatally expressed in various brain regions, including the cerebral cortex, entorhinal cortex, hippocampus, amygdala, and dorsal thalamus. The extremely high Cdkl2 expression in these regions suggests that it has a role in cognition and emotion. Recent genetic studies indicate that mutations of Cdkl family kinases are associated with neurodevelopmental and neuropsychiatric disorders in humans. To elucidate the physiologic role of Cdkl2, we behaviorally analyzed Cdkl2LacZ/LacZ mice lacking Cdkl2. Cdkl2LacZ/LacZ mice had reduced latencies to enter the dark compartment after electric footshock in an inhibitory avoidance task and attenuated contextual fear responses when exposed to mild training conditions. Hippocampal spatial learning in the Morris water maze was slightly anomalous with mice exhibiting an abnormal swimming pattern. The aversive response in a two-way avoidance task was slightly, but not significantly, enhanced. On the other hand, Cdkl2LacZ/LacZ mice did not exhibit altered sensitivity to aversive stimuli, such as electric footshock and heat, or deficits in the elevated plus maze or rotating rod test. These findings suggest that Cdkl2 is involved in cognitive function and provide in vivo evidence for the function of Cdkl family kinases expressed in terminally differentiated neurons in mice.

Fear is one of the most potent emotional experiences and is an adaptive component of response to potentially threatening stimuli. On the other hand, too much or inappropriate fear accounts for many common psychiatric problems. Cumulative evidence suggests that the amygdala plays a central role in the acquisition, storage and expression of fear memory. Here, we developed an inducible striatal neuron ablation system in transgenic mice. The ablation of striatal neurons in the adult brain hardly affected the auditory fear learning under the standard condition in agreement with previous studies. When conditioned with a low-intensity unconditioned stimulus, however, the formation of long-term fear memory but not short-tem memory was impaired in striatal neuron-ablated mice. Consistently, the ablation of striatal neurons 24 h after conditioning with the low-intensity unconditioned stimulus, when the long-term fear memory was formed, diminished the retention of the long-term memory. Our results reveal a novel form of the auditory fear memory depending on striatal neurons at the low-intensity unconditioned stimulus.

Connexin43 (Cx43), a major component of astrocytic gap junctions, is abundantly expressed in Bergmann glial cells (BGCs) in the cerebellum, but the function of Cx43 in BGCs is largely unknown. BGCs are specialized astrocytes closely associated with Purkinje cells. Here, we review our recent studies of the role of Cx43 in gap junctional coupling between BGCs and in cerebellar function. We generated Cx43 conditional knockout mice with an S100b-Cre transgenic line (Cx43fl/fl:S100b-Cre), in which there was a significant postnatal loss of Cx43 in BGCs and cerebellar astrocytes. Gap junctional coupling between BGCs measured by dye coupling was virtually abolished in Cx43fl/fl:S100b-Cre mice. Electrophysiologic and behavioral analyses suggested that Cx43-mediated gap junctions and Cx43 hemichannels in BGCs are not necessary for the neuron-glia interactions required for cerebellum-dependent motor coordination and motor learning. These findings raise questions regarding the regional differences in the impact of the loss of Cx43 in the brain.

Bergmann glial cells are specialized astrocytes in the cerebellum. In the mature cerebellar molecular layer, Bergmann glial processes are closely associated with Purkinje cells, enclosing Purkinje cell dendritic synapses with a glial sheath. There is intensive gap junctional coupling between Bergmann glial processes, but their significance in cerebellar functions is not known. Connexin43 (Cx43), a major component of astrocytic gap junction channels, is abundantly expressed in Bergmann glial cells. To examine the role of Cx43-mediated gap junctions between Bergmann glial cells in cerebellar functions, we generated Cx43 conditional knockout mice with the S100b-Cre transgenic line (Cx43fl/fl:S100b-Cre), which exhibited a significant loss of Cx43 in the Bergmann glial cells and astrocytes in the cerebellum with a postnatal onset. The Cx43fl/fl:S100b-Cre mice had normal cerebellar architecture. Although gap junctional coupling between the Bergmann glial cells measured by spreading of microinjected Lucifer yellow was virtually abolished in Cx43fl/fl:S100b-Cre mice, electrophysiologic analysis revealed that cerebellar long-term depression could be induced and maintained normally in thier cerebellar slices. In addition, at the behavioral level, Cx43fl/fl:S100b-Cre mice had normal motor coordination in the rotarod task and normal conditioned eyelid response. Our findings suggest that Cx43-mediated gap junctional coupling between Bergmann glial cells is not necessary for the neuron-glia interactions required for cerebellum-dependent motor coordination and motor learning.

During puberty, mouse mammary epithelial ducts invade the stromal mammary fat pad in a wave of branching morphogenesis to form a complex ductal tree. Using pharmacologic and genetic approaches, we find that mammary gland branching morphogenesis requires transient matrix metalloproteinase (MMP) activity for invasion and branch point selection. MMP-2, but not MMP-9, facilitates terminal end bud invasion by inhibiting epithelial cell apoptosis at the start of puberty. Unexpectedly, MMP-2 also represses precocious lateral branching during mid-puberty. In contrast, MMP-3 induces secondary and tertiary lateral branching of ducts during mid-puberty and early pregnancy. Nevertheless, the mammary gland is able to develop lactational competence in MMP mutant mice. Thus, specific MMPs refine the mammary branching pattern by distinct mechanisms during mammary gland branching morphogenesis.

BackgroundAmyloid-beta peptide species ending at positions 40 and 42 (Abeta40, Abeta42) are generated by the proteolytic processing of the Alzheimer's amyloid precursor protein (APP). Abeta peptides accumulate in the brain early in the course of Alzheimer's disease (AD), especially Abeta42. The cytoplasmic domain of APP regulates intracellular trafficking and metabolism of APP and its carboxyl-terminal fragments (CTFalpha, CTFbeta). The role of protein phosphorylation in general, and that of the phosphorylation state of APP at threonine-668 (Thr668) in particular, has been investigated in detail by several laboratories (including our own). Some investigators have recently proposed that the phosphorylation state of Thr668 plays a pivotal role in governing brain Abeta levels, prompting the current study.MethodologyIn order to evaluate whether the phosphorylation state of Thr668 controlled brain Abeta levels, we studied the levels and subcellular distributions of holoAPP, sAPPalpha, sAPPbeta, CTFalpha, CTFbeta, Abeta40 and Abeta42 in brains from "knock-in" mice in which a non-phosphorylatable alanyl residue had been substituted at position 668, replacing the threonyl residue present in the wild-type protein.ConclusionsThe levels and subcellular distributions of holoAPP, sAPPalpha, sAPPbeta, CTFalpha, CTFbeta, Abeta40 and Abeta42 in the brains of Thr668Ala mutant mice were identical to those observed in wild-type mice. These results indicate that, despite speculation to the contrary, the phosphorylation state of APP at Thr668 does not play an obvious role in governing the physiological levels of brain Abeta40 or Abeta42 in vivo.