Altered neocortical rhythmic activity states in Fmr1 KO mice are due to enhanced mGluR5 signaling and involve changes in excitatory circuitry

Despite the pronounced neurological deficits associated with mental retardation and autism, the degree to which neocortical circuit function is altered remains unknown. Here, we study changes in neocortical network function in the form of persistent activity states in the mouse model of fragile X syndrome--the Fmr1 knock-out (KO). Persistent activity states, or UP states, in the neocortex underlie the slow oscillation which occurs predominantly during slow-wave sleep, but may also play a role during awake states. We show that spontaneously occurring UP states in the primary somatosensory cortex are 38-67% longer in Fmr1 KO slices. In vivo, UP states reoccur with a clear rhythmic component consistent with that of the slow oscillation and are similarly longer in the Fmr1 KO. Changes in neocortical excitatory circuitry likely play the major role in this alteration as supported by three findings: (1) longer UP states occur in slices of isolated neocortex, (2) pharmacologically isolated excitatory circuits in Fmr1 KO neocortical slices display prolonged bursting states, and (3) selective deletion of Fmr1 in cortical excitatory neurons is sufficient to cause prolonged UP states whereas deletion in inhibitory neurons has no effect. Excess signaling mediated by the group 1 glutamate metabotropic receptor, mGluR5, contributes to the longer UP states. Genetic reduction or pharmacological blockade of mGluR5 rescues the prolonged UP state phenotype. Our results reveal an alteration in network function in a mouse model of intellectual disability and autism which may impact both slow-wave sleep and information processing during waking states.     

Ascorbate treatment attenuates the Huntington behavioral phenotype in mice

The R6/2 mouse line expresses exon 1 of the human gene for Huntington disease (HD) and shows behavioral symptoms as early as 6 weeks of age. In the striatum, a forebrain target of HD, these animals show a behavior-related deficit in extracellular ascorbate, the deprotonated form of vitamin C. We report here that this deficit may contribute to the HD behavioral phenotype. Regular injections of ascorbate (300 mg/kg/day, 4 days/week) beginning at symptom onset restored the behavior-related release of ascorbate in striatum and also improved behavioral responding. Compared to vehicle, ascorbate treatment significantly attenuated the neurological motor signs of HD without altering overall motor activity. Ascorbate regulation of striatal function appears key for understanding HD.     

Spatiotemporal expression of PSD‐95 in Fmr1 knockout mice brain

To investigate and compare the spatial and temporal expression of post‐synaptic density‐95 (PSD‐95) in Fmr1 knockout mice (the animal model of fragile X syndrome, FXS) and wild‐type mice brain, on postnatal day 7 (P7), P14, P21, P28 and P90, mice from each group were decapitated, and three principal brain regions (cerebral cortex, hippocampus and cerebellum) were obtained and stored for later experiments. PSD‐95 mRNA in the three brain areas was analyzed with quantitative RT‐PCR. PSD‐95 protein was measured by immunohistochemical staining and Western blot. In the three principal brain areas of Fmr1 knockout mice and wild‐type mice, the expression of PSD‐95 mRNA and protein were detected at the lowest levels on P7, and then significantly increased on P14, reaching the peak levels in adolescents or adults. Moreover, it was found that PSD‐95 mRNA and protein in the hippocampus were significantly decreased in Fmr1 knockout mice during the developmental period (P7, P14, P21 and P28) as well as at adulthood (P90) (P < 0.05, and P < 0.01, respectively). However, there was no significant difference of expression of PSD‐95 in the cortex and cerebellum between Fmr1 knockout and wild mice. The expression of PSD‐95 in the hippocampus might be regulated by fragile X mental retardation protein (FMRP) during mice early developmental and adult periods. It is suggested that impairment of PSD‐95 is possibly involved in hippocampal‐dependent learning defects, which are common in people with FXS.     

An altered neonatal behavioral phenotype in Mecp2 mutant mice

We examined somatic growth, somatosensory reflexes, and ultrasonic calls from postnatal day 3 to day 18 in Mecp2 mutant mice, a mouse model of Rett syndrome. Both Mecp2 null male and Mecp2 heterozygous female mice exhibited normal somatic growth, but transient delays in the development of some reflexes relative to sex-matched wild-type mice. Both Mecp2 null male and heterozygous female mice exhibited dramatic increases in ultrasonic vocalizations in response to social isolation; these differences were evident as early as postnatal day 5. These results suggest very early abnormalities in sensory reflex development and behavioral responsiveness in the Mecp2 mutants that may provide a target for early therapeutic intervention.     

Spatial learning, contextual fear conditioning and conditioned emotional response in Fmr1 knockout mice

Fmr1 knockout mice are an animal model for fragile X syndrome, the most common form of heritable mental retardation in humans. Fmr1 knockout mice exhibit macro-orchidism and cognitive and behavioural deficits reminiscent of the human phenotype. In the present study additional behavioural and cognitive testing was performed. Knockouts and control littermates were subjected to a spatial learning test using a plus-shaped water maze. Animals had to learn the position of a hidden escape platform during training trials. The position of this platform was changed during subsequent reversal trials. Previously reported deficits in reversal learning were replicated, but we also observed significant differences during the acquisition trials. A plus-shaped water maze experiment with daily changing platform positions failed to provide clear evidence for a working memory impairment, putatively underlying the spatial learning deficits. Two different test settings were used to examine the reported deficit of Fmr1 knockout mice in fear conditioning. Conditioned fear responses were observed in a contextual fear test, and the ability to acquire an emotional response was tested by means of response suppression in a conditioned emotional response procedure. Neither protocol revealed significant differences between controls and knockouts.     

Altered mossy fiber distributions in adult Fmr1 (FVB) knockout mice

The fragile-X mental retardation protein (FMRP) is greatly reduced or absent in individuals with fragile-X mental retardation syndrome, a common, heritable form of mental retardation. Morphological studies suggest that this protein functions in normal synapse maturation and neuronal plasticity. Examination of human brain autopsy tissue has shown that fragile-X patients exhibit long, thin spines more frequently, and stubby mushroom-shaped spines less frequently, than these two types of spines are seen in normal autopsy tissue. Fragile-X tissue also has a greater density of these spines along dendrites, which suggests a possible failure of synapse elimination. Fmr1 knockout mice and wild-type littermates brains were processed for Timm staining, which reveals the zinc-rich terminals of the dentate gyrus, the mossy fibers. The Fmr1 knockout mice exhibited a pattern of Timm granule-staining within the stratum oriens of subfield CA3 and the inner molecular layer that was significantly different than staining seen in wild-type animals. The sources and consequences of the altered terminal staining are unclear, but are discussed in relation to immature synapse morphology, a failure of normal regression of synapses, and a potential biological penalty of such a failure to regress.     

Correlations between genotype, ultrastructural morphology and clinical phenotype in the neuronal ceroid lipofuscinoses

The neuronal ceroid lipofuscinoses (NCLs) are a group of severe neurodegenerative diseases with onset usually in childhood and characterised by the intracellular accumulation of autofluorescent storage material. Within the last decade, mutations that cause NCL have been found in six human genes (CLN1, CLN2, CLN3, CLN5, CLN6 and CLN8). Mutations in two additional genes cause disease in animal models that share features with NCL-CTSD in sheep and mice and PPT2 in mice. Approximately 160 NCL disease-causing mutations have now been described (listed and fully cited in the NCL Mutation Database, ). Most mutations result in a classic morphology and disease phenotype, but some mutations are associated with disease that is of later onset, less severe or protracted in its course, or with atypical morphology. Seven common mutations exist, some having a worldwide distribution and others associated with families originating from specific geographical regions. This review attempts to correlate the gene, disease-causing mutation, morphology and clinical phenotype for each type of NCL.     

微管相关蛋白1B在Fmr1基因敲除小鼠脑组织内的表达变化

目的 探讨脆性X智能低下蛋白（fragile X mental retardation protein,FMRP）对微管相关蛋白1B（microtubuleassociated protein 1B,MAP1B）是否具有调控作用。方法应用免疫组化、免疫印记和原位杂交的方法,对1周龄和6周龄的Fmr1基因敲除型（KO）和同龄野生型（WT）小鼠脑组织MAP1B及MAP1B mRNA进行分析。结果免疫组化的结果显示 ：6周龄KO小鼠各个脑区MAP1B的平均光密度值（MOD）值均显著低于同龄WT小鼠（P 〈 0.05）,1周龄KO小鼠仅在小脑和海马显著降低（P 〈 0.01） ;各脑区MAP1B的MOD值在6周龄小鼠均比同基因型的1周龄小鼠显著降低（P 〈 0.05）。免疫印记和原位杂交结果分别显示MAP1B及MAP1B mRNA在KO小鼠的海马组织均显著降低（P 〈0.05）。结论MAP1B和MAP1B mRNA在Fmr1基因敲除小鼠脑组织的表达均显著减少,提示FMRP可能正性调节MAP1B的表达。     

微管相关蛋白1B在Fmr1基因敲除小鼠脑组织内的表达变化(英文)

目的探讨脆性X智能低下蛋白(fragile X mental retardation protein,FMRP)对微管相关蛋白1B(microtubuleassociated protein 1B,MAP1B)是否具有调控作用。方法应用免疫组化、免疫印记和原位杂交的方法,对1周龄和6周龄的Fmr1基因敲除型(KO)和同龄野生型(WT)小鼠脑组织MAP1B及MAP1B mRNA进行分析。结果免疫组化的结果显示 :6周龄KO小鼠各个脑区MAP1B的平均光密度值(MOD)值均显著低于同龄WT小鼠(P < 0.05),1周龄KO小鼠仅在小脑和海马显著降低(P < 0.01) ;各脑区MAP1B的MOD值在6周龄小鼠均比同基因型的1周龄小鼠显著降低(P < 0.05)。免疫印记和原位杂交结果分别显示MAP1B及MAP1B mRNA在KO小鼠的海马组织均显著降低(P <0.05)。结论MAP1B和MAP1B mRNA在Fmr1基因敲除小鼠脑组织的表达均显著减少,提示FMRP 可能正性调节MAP1B的表达。     

Sensorimotor gating abnormalities in young males with fragile X syndrome and Fmr1-knockout mice

Fragile X syndrome (FXS) is the most common single gene (FMR1) disorder affecting cognitive and behavioral function in humans. This syndrome is characterized by a cluster of abnormalities including lower IQ, attention deficits, impairments in adaptive behavior and increased incidence of autism. Here, we show that young males with FXS have profound deficits in prepulse inhibition (PPI), a basic marker of sensorimotor gating that has been extensively studied in rodents. Importantly, the magnitude of the PPI impairments in the fragile X children predicted the severity of their IQ, attention, adaptive behavior and autistic phenotypes. Additionally, these measures were highly correlated with each other, suggesting that a shared mechanism underlies this complex phenotypic cluster. Studies in Fmr1-knockout mice also revealed sensorimotor gating and learning abnormalities. However, PPI and learning were enhanced rather than reduced in the mutants. Therefore, these data show that mutations of the FMR1 gene impact equivalent processes in both humans and mice. However, since these phenotypic changes are opposite in direction, they also suggest that murine compensatory mechanisms following loss of FMR1 function differ from those in humans.     

Central neuronal loss and behavioral impairment in mice lacking neurotrophin receptor p75

The neurotrophin receptor p75 is a low-affinity receptor that binds neurotrophins. To investigate the role of p75 in the survival and function of central neurons, p75 null-mutant and wild type litter mate mice were tested on behavioral tasks. Null mutants showed significant performance deficits on water maze, inhibitory avoidance, motor activity, and habituation tasks that may be attributed to cognitive dysfunction or may represent a global sensorimotor impairment. The p75 null-mutant and wild type litter mate mice were assessed for central cholinergic deficit by using quantitative stereology to estimate the total neuronal number in basal forebrain and striatum and for subpopulations expressing the high-affinity tyrosine receptor kinase A (trkA) neurotrophin receptor and choline acetyltransferase (ChAT). In the adult brain, cholinergic neurons of the basal forebrain receive target-derived trophic support, whereas cholinergic striatal neurons do not. Adult p75 null-mutant mice had significant reduction of basal forebrain volume by 25% and had a corresponding significant loss of 37% of total basal forebrain neurons. The basal forebrain population of ChAT-positive neurons in p75-deficient mice declined significantly by 27%, whereas the trkA-positive population did not change significantly. There was no significant change in striatal volume or in striatal neuronal number either in total or by cholinergic subpopulation. These results demonstrate vulnerability to the lack of p75 in adult central neurons that are neurotrophin dependent. In addition, the loss of noncholinergic central neurons in mice lacking p75 suggests a role for p75 in cell survival by an as yet undetermined mechanism. Possible direct and indirect effects of p75 loss on neuronal survival are discussed. J. Comp. Neurol. 404:1–20, 1999. © 1999 Wiley-Liss, Inc.     

LTP induction translocates cortactin at distant synapses in wild-type but not Fmr1 knock-out mice

Stabilization of long-term potentiation (LTP) depends on reorganization of the dendritic spine actin cytoskeleton. The present study tested whether this involves activity-driven effects on the actin-regulatory protein cortactin, and whether such effects are disturbed in the Fmr1 knock-out (KO) model of fragile X syndrome, in which stabilization of both actin filaments and LTP is impaired. LTP induced by theta burst stimulation (TBS) in hippocampal slices from wild-type mice was associated with rapid, broadly distributed, and NMDA receptor-dependent decreases in synapse-associated cortactin. The reduction in cortactin content was blocked by blebbistatin, while basal levels were reduced by nocodazole, indicating that cortactin's movements into and away from synapses are regulated by microtubule and actomyosin motors, respectively. These results further suggest that synapse-specific LTP influences cytoskeletal elements at distant connections. The rapid effects of TBS on synaptic cortactin content were absent in Fmr1 KOs as was evidence for activity-driven phosphorylation of the protein or its upstream kinase, ERK1/2. Phosphorylation regulates cortactin's interactions with actin, and coprecipitation of the two proteins was reduced in the KOs. We propose that, in the KOs, excessive basal phosphorylation of ERK1/2 disrupts its interactions with cortactin, thereby blocking the latter protein's use of actomyosin transport systems. These impairments are predicted to compromise the response of the subsynaptic cytoskeleton to learning-related afferent activity, both locally and at distant sites.     

Hippocampal pyramidal cells in adult Fmr1 knockout mice exhibit an immature-appearing profile of dendritic spines

Fragile X syndrome (FXS) is a common form of mental retardation caused by the absence of functional fragile X mental retardation protein (FMRP). FXS is associated with elevated density and length of dendritic spines, as well as an immature-appearing distribution profile of spine morphologies in the neocortex. Mice that lack FMRP (Fmr1 knockout mice) exhibit a similar phenotype in the neocortex, suggesting that FMRP is important for dendritic spine maturation and pruning. Examination of Golgi-stained pyramidal cells in hippocampal subfield CA1 of adult Fmr1 knockout mice reveals longer spines than controls and a morphology profile that, while essentially opposite of that described in the Fmr1 knockout neocortex, appears similarly immature. This finding strongly suggests that FMRP is required for the processes of spine maturation and pruning in multiple brain regions and that the specific pathology depends on the cellular context.     

Long-term effects of cytokine treatment on cognitive behavioral recovery and neuronal regeneration in soman-poisoned mice

Increasing numbers of reports have substantiated to date, a beneficial influence of cytokine treatment on neurogenesis processes in damaged rodent brains. Most of these investigations further revealed that cytokine treatment induces either partial or full recovery of cognitive behavior impaired by cerebral lesions.Hence, we investigated the effects of a cytokine treatment on neuronal regeneration and cognitive behavior in mice subjected to nerve agent exposure. Subcutaneous injection of a mixture of 40 μg/kg fibroblast growth factor-2 (FGF-2) and epidermal growth factor (EGF) was administered daily over 8 days to soman-poisoned mice (1.2 LD50 soman). Memory performances (T-maze and Morris water maze) and emotional behavior (elevated plus maze; auditory and contextual response in a fear conditioning task) were assessed on post-soman days 30 and 90. Brains were collected on post-soman days 9, 30 and 90 so as to perform NeuN-immunohistochemistry in the hippocampus and amygdala (neuronal regeneration quantification).Following soman-induced brain lesions, a spontaneous neuronal regeneration occurred in both the hippocampus and amygdala. Cytokine treatment enhanced neuronal regeneration in the hippocampus however not in the amygdala. Soman poisoning fostered altogether memory impairments as well as anxiety or fear-like behavioral disturbances in mice. A spontaneous recovery of standard emotional behavior occurred overtime. Such a recovery displayed significantly enhanced speed under cytokine treatment. Unfortunately, no memory performance recovery was evidenced in soman-intoxicated mice whether treated or not with cytokines.     

Altered fear circuits in 5-HT(1A) receptor KO mice.

The study of genetically altered mice has been used successfully to determine the influence of different neurotransmitter receptors on fear and anxiety. Mice with a genetic deletion of the serotonin 1A receptor (5-HT(1A)R knockout [KO]) have been shown to be more fearful in a number of behavioral conflict tests, confirming the important role of this receptor in modulating anxiety. Factor analysis of the behavior of WT and 5-HT(1A)R KO mice in the open field test shows that locomotion and anxiety measures segregate independently, supporting the idea that the anxious behavior of the KO mice is not the result of altered locomotion. KO mice also show increased anxiety in the novelty-suppressed feeding task, which differs from the other conflict tests in the motivational drive of the animals. In response to a discrete aversive stimulus, foot shock, the KO mice show increased freezing and increased tachycardia. However, activation of the hypothalamic-pituitary-adrenal axis in response to stress appears to be slightly blunted in the KO animals. Together, these data support the idea that the 5-HT(1A)R modulates an important fear circuit in the brain. The dual function of the 5-HT(1A)R as both a presynaptic autoreceptor, negatively regulating serotonin activity, and a postsynaptic heteroreceptor, inhibiting the activity of nonserotonergic neurons in forebrain structures, has complicated interpretation of the anxious phenotype of these KO mice. A more complete understanding of the function of the 5-HT(1A)R awaits further study of its role in behaving animals using tissue-specific antagonists and novel transgenic mice with tissue-specific expression of the receptor.     

Temporal neuropathologic and behavioral phenotype of 6neo/6neo Pompe disease mice

Pompe disease (glycogen storage disease II) is caused by mutations in the acid alpha-glucosidase gene. The most common form is rapidly progressive with glycogen storage, particularly in muscle, which leads to profound weakness, cardiac failure, and death by the age of 2 years. Although usually considered a muscle disease, glycogen storage also occurs in the CNS. We evaluated the progression of neuropathologic and behavioral abnormalities in a Pompe disease mouse model (6neo/6neo) that displays many features of the human disease. Homozygous mutant mice store excess glycogen within large neurons of hindbrain, spinal cord, and sensory ganglia by the age of 1 month; accumulations then spread progressively within many CNS cell types. "Silver degeneration" and Fluoro-Jade C stains revealed severe degeneration in axon terminals of primary sensory neurons at 3 to 9 months. These abnormalities were accompanied by progressive behavioral impairment on rotorod, wire hanging, and foot fault tests. The extensive neuropathologic alterations in this model suggest that therapy of skeletal and cardiac muscle disorders by systemic enzyme replacement therapy may not be sufficient to reverse functional deficits due to CNS glycogen storage, particularly early-onset, rapidly progressive disease. A better understanding of the basis for clinical manifestations is needed to correlate CNS pathology with Pompe disease manifestations.     

Multiple failures in the lutenising hormone surge generating system in GABAB1KO female mice

Female mice lacking GABAB receptors, GABAB1KO, show disrupted oestrous cycles, reduced pregnancies and increased hypothalamic Gnrh1 mRNA expression, whereas anteroventral periventricular/periventricular preoptic nucleus (AVPV/PeN) Kiss1 mRNA was not affected. In the present study, we characterise the important components of the gonadotrophic preovulatory surge, aiming to unravel the origin of this reproductive impairment. In GABAB1KO and wild‐type (WT) females, we determined: (i) hypothalamic oestrogen receptor (ER)α and β and aromatase mRNA and protein expression; (ii) ovulation index and oestrus serum follicle‐stimulating hormone (FSH) and pituitary Gnrh1r expression; (iii) in ovariectomised‐oestradiol valerate‐treated mice, we evaluated ex vivo hypothalamic gonadotrophin‐releasing hormone (GnRH) pulsatility in the presence/absence of kisspeptin (Kiss‐10, constant or pulsatile) and oestradiol (constant); and (iv) in ovariectomised‐oestradiol silastic capsule‐treated mice (proestrous‐like environment), we evaluated morning and evening kisspeptin neurone activation (c‐Fos+) and serum luteinising homrone (LH). In the medial basal hypothalamus of oestrus GABAB1KOs, aromatase and ERα mRNA and protein were increased, whereas ERβ was decreased. In GABAB1KOs, the ovulation index was decreased together with decreased first oestrus serum FSH and increased pituitary Gnrh1r mRNA. Under constant Kiss‐10 stimulation, hypothalamic GnRH pulse frequency did not vary, although GnRH mass/pulse was increased in GABAB1KOs. In WTs, pulsatile Kiss‐10 together with constant oestradiol significantly increased GnRH pulsatility, whereas, in GABAB1KOs, oestradiol alone increased GnRH pulsatility and this was reversed by pulsatile Kiss‐10 addition. In GABAB1KOs AVPV/PeN kisspeptin neurones were similarly activated (c‐Fos+) in the morning and evening, whereas WTs showed the expected, marked evening stimulation. LH correlated with activated kisspeptin cells in WT mice, whereas GABAB1KO mice showed high, similar LH levels both in the morning and evening. Taken together, all of these alterations point to impairment in the trigger of the preovulatory GnRH surge that entails the reproductive alterations described.