Nitric oxide synthase protein levels, not the mRNA, are downregulated in olfactory bulb interneurons of reeler mice

Homozygous mutations in the Reelin gene result in severe disruption of brain development. The histogenesis of layered regions, like the neocortex, hippocampus and the cerebellum, is most notably affected in mouse reeler mutants and similar traits are also present in mice lacking molecular components of the Reelin signalling pathway. Moreover, there is evidence for an additional role of Reelin in sustaining synaptic plasticity in adult networks. Nitric oxide is an important gaseous messenger that can modulate neuronal plasticity both in developing and mature synaptic networks and has been shown to facilitate synaptic changes in the hippocampus, cerebellum and olfactory bulb. We studied the distribution and content of neuronal nitric oxide synthase in the olfactory bulbs of reeler and wildtype mice. Immunocytochemistry reveals that Reelin and neuronal nitric oxide synthase containing interneurons are two distinct, non overlapping cell populations of the olfactory bulb. We show by in situ hybridization that both nitrergic and Reelin expressing cells represent only a subset of olfactory bulb GABAergic neurons. Immunoblots show that neuronal nitric oxide synthase protein content is decreased by two thirds in reeler mice causing a detectable loss of immunolabelled cells throughout the olfactory bulb of this strain. However, neuronal nitric oxide synthase mRNA levels, essayed by quantitative real-time RT-PCR, are unaffected in the reeler olfactory bulb. Thus, disruption of the Reelin signalling pathway may modify the turnover of neuronal nitric oxide synthase in the olfactory bulb and possibly affects nitric oxide functions in reeler mice.     

Reelin可增强海马神经元的多巴胺D1受体表达

目的研究Reelin对小鼠海马神经元多巴胺D1受体的调控及其是否依赖Reelin受体（ApoER2／VLDLR）发挥调控作用。方法原代分离培养新生C57BL／6J小鼠海马神经元,将培养5d后的神经元随机分为6组：空白对照组、CR．50组（CR．50）,EDTA组（EDTA）,Reelin组（Reelin）,Reelin拮抗组（Reelin＋CR50）和Reelin受体拮抗组（Reelin＋EDTA）。分别加入Reelin、CR．50、EDTA进行共培：养,其后利用免疫荧光的方法观察Reelin受体ApoER2、VLDLR和多巴胺Dl受体在海马神经元上的定位以及各组多巴胺Dl受体的变化。结果（1）Reelin受体ApoER2、VLDLR均与多巴胺D1受体在海马神经元上共定位;（2）Redin拮抗组的多巴胺D1受体荧光强度（0．215±O．09）较Reelin组（O．663±0．15）减弱（P〈0．01）;（3）Reelin受体拮抗组的多巴胺D1受体荧光强度（0．687±0．11）与Reelin组（0．666±0．15）无明显差异（P〉0．05）。结论Reelin可增强小鼠海马神经元上的多巴胺D1受体的表达,Reelin上调多巴胺Dl受体的作用并不是依赖Reelin的经典受体ADoER2、VLDLR．其调控机制有待进一步研究。     

Functional consequences of hippocampal neuronal ectopia in the apolipoprotein E receptor-2 knockout mouse

Little is known about the impact ectopically located neurons have on the functional connectivity of local circuits. The ApoER2 knockout mouse has subtle cytoarchitectural disruptions, altered prepulse inhibition, and memory abnormalities. We evaluated this mouse mutant as a model to study the role ectopic neurons play in the manifestation of symptoms associated with brain diseases. We found that ectopic CA1 pyramidal and inhibitory neurons in the ApoER2 knockout hippocampus are organized into two distinct stratum pyramidale layers. In vitro analyses found that ApoER2 is not required for neurons to reach maturity in regard to dendritic arborization and synaptic structure density, and electrophysiological testing determined that neurons in both strata pyramidale are integrated into the hippocampal network. However, the presence of these two layers alters the spatiotemporal pattern of hippocampal activity, which may explain why ApoER2 knockout mice have selective cognitive dysfunctions that are revealed only under challenging conditions.     

Ambiguus motoneurons innervating laryngeal and esophageal muscles are malpositioned in the Reelin-deficient mutant rat,Shaking Rat Kawasaki

Conclusions. The present study confirmed that ambiguus motoneurons innervating intrinsic laryngeal and esophageal muscles are radially malpositioned in the brainstem of the Shaking Rat Kawasaki (SRK), a reelin-deficient mutant rat. Objectives. Ambiguus motoneurons innervating the striated muscles of the larynx and esophagus take a long migration from their original birth plate in the floor of the fourth ventricle to their final settlement in the ventral margin of the medulla oblongata. To examine whether the migration of ambiguus nucleus neurons is affected in SRK, we studied localization of ambiguus motoneurons of postnatal day 21 (P21) normal and SRK rats. Materials and methods. To label ambiguus motoneurons retrogradely, horseradish peroxidase (HRP) was injected into some laryngeal muscles including cricothyroid, thyroarytenoid and posterior cricoarytenoid muscles, and the cervical and abdominal esophageal muscles of the SRK and normal controls 2 days before sacrifice. Results. In the P21 normal rat, HRP-positive laryngeal and esophageal motoneurons were found in the nucleus ambiguus, whereas in the P21 SRK, they were scattered from the base of the fourth ventricle to the ventro-lateral margin of the medulla, suggesting that radial migration of ambiguus motoneurons from their birthplace to their final settlement is guided by Reelin protein.     

Transient Downregulation of Dab1 Protein Levels during Development Leads to Behavioral and Structural Deficits: Relevance for Psychiatric Disorders

Psychiatric disorders have been hypothesized to originate during development, with genetic and environmental factors interacting in the etiology of disease. Therefore, developmentally regulated genes have received attention as risk modulators in psychiatric diseases. Reelin is an extracellular protein essential for neuronal migration and maturation during development, and its expression levels are reduced in psychiatric disorders. Interestingly, several perinatal insults that increase the risk of behavioral deficits alter Reelin signaling. However, it is not known whether a dysfunction in Reelin signaling during perinatal stages increases the risk of psychiatric disorders. Here we used a floxed dab1 allele to study whether a transient decrease in Dab1, a key component of the Reelin pathway, is sufficient to induce behavioral deficits related to psychiatric disorders. We found that transient Dab1 downregulation during perinatal stages leads to permanent abnormalities of structural layering in the neocortex and hippocampus. In contrast, conditional inactivation of the dab1 gene in the adult brain does not result in additional layering abnormalities. Furthermore, perinatal Dab1 downregulation causes behavior impairments in adult mice, such as deficits in memory, maternal care, pre-pulse inhibition, and response to cocaine. Some of these deficits were also found to be present in adolescence. We also show that D-cycloserine rescues the cognitive deficits observed in floxed dab1 mice with layering alterations in the hippocampus and neocortex. Our results indicate a causal relation between the downregulation of Dab1 protein levels during development and the structural and behavioral deficits associated with psychiatric diseases in the adult.     

Effect of Reelin signaling on pre-plate splitting during cerebral cortex development

The development of the 6-layered cerebral neocortex is one of the most important events during nervous system development, and disturbances could result in various malformations, causing clinically intractable diseases, such as epilepsy and cerebral palsy. Pre-plate splitting is the first developmental step of the cortical plate formation. Without correct pre-plate splitting, normal cerebral cortex structures are disturbed. The Reelin-Dab1 molecular pathway plays a critical role during cerebral cortex development, and deficiencies in this pathway result in failed pre-plate splitting and an inverted cortical plate. This paper summarizes findings involving Reelin and pre-piate splitting and further explores the precise role of Reelin during pre-plate splitting.     

Prenatal and early life diesel exhaust exposure disrupts cortical lamina organization: Evidence for a reelin-related pathogenic pathway induced by interleukin-6

Several epidemiological studies have shown associations between developmental exposure to traffic-related air pollution and increased risk for autism spectrum disorders (ASD), a spectrum of neurodevelopmental disorders with increasing prevalence rate in the United States. Though animal studies have provided support for these associations, little is known regarding possible underlying mechanisms. In a previous study we found that exposure of C57BL/6J mice of both sexes to environmentally relevant levels (250–300 µg/m³) of diesel exhaust (DE) from embryonic day 0 to postnatal day 21 (E0 to PND21) caused significant changes in all three characteristic behavioral domains of ASD in the offspring. In the present study we investigated a potential mechanistic pathway that may be of relevance for ASD-like changes associated with developmental DE exposure. Using the same DE exposure protocol (250–300 µg/m³ DE from E0 to PND21) several molecular markers were examined in the brains of male and female mice at PND3, 21, and 60. Exposure to DE as above increased levels of interleukin-6 (IL-6) in placenta and in neonatal brain. The JAK2/STAT3 pathway, a target for IL-6, was activated by STAT3 phosphorylation, and the expression of DNA methyltransferase 1 (DNMT1), a STAT3 target gene, was increased in DE-exposed neonatal brain. DNMT1 has been reported to down-regulate expression of reelin (RELN), an extracellular matrix glycoprotein important in regulating the processes of neuronal migration. RELN is considered an important modulator for ASD, since there are several polymorphisms in this gene linked to the disease, and since lower levels of RELN have been reported in brains of ASD patients. We observed decreased RELN expression in brains of the DE-exposed mice at PND3. Since disorganized patches in the prefrontal cortex have been reported in ASD patients and disrupted cortical organization has been found in RELN-deficient mice, we also assessed cortical organization, by labeling cells expressing the lamina-specific-markers RELN and calretinin. In DE-exposed mice we found increased cell density in deeper cortex (lamina layers VI–IV) for cells expressing either RELN or calretinin. These findings demonstrate that developmental DE exposure is associated with subtle disorganization of the cerebral cortex at PND60, and suggest a pathway involving IL-6, STAT3, and DNMT1 leading to downregulation of RELN expression that could be contributing to this long-lasting disruption in cortical laminar organization.     

Dietary-induced obesity disrupts trace fear conditioning and decreases hippocampal reelin expression

Both obesity and over-consumption of palatable high fat/high sugar “cafeteria” diets in rats has been shown to induce cognitive deficits in executive function, attention and spatial memory. Adult male Sprague–Dawley rats were fed a diet that supplemented standard lab chow with a range of palatable foods eaten by people for 8 weeks, or regular lab chow. Memory was assessed using a trace fear conditioning procedure, whereby a conditioned stimulus (CS) is presented for 10 s and then 30 s after its termination a foot shock (US) is delivered. We assessed freezing to the CS (flashing light) in a neutral context, and freezing in the context associated with footshock.A dissociation was observed between levels of freezing in the context and to the CS associated with footshock. Cafeteria diet fed rats froze less than control chow fed rats in the context associated with footshock (P < 0.01), indicating that encoding of a hippocampus-dependent context representation was impaired in these rats. Conversely, cafeteria diet fed rats froze more (P < 0.05) to the CS than chow fed rats, suggesting that when hippocampal function was compromised the cue was the best predictor of footshock, as contextual information was not encoded.Dorsal hippocampal mRNA expression of inflammatory and neuroplasticity markers was analysed at the end of the experiment, 10 weeks of diet. Of these, mRNA expression of reelin, which is known to be important in long term potentiation and neuronal plasticity, was significantly reduced in cafeteria diet fed rats (P = 0.003). This implicates reductions in hippocampal plasticity in the contextual fear memory deficits seen in the cafeteria diet fed rats.     

Regional brain variations of cytochrome oxidase activity in Relnrl-orl mutant mice

Cell malpositioning has been described in laminated structures of the spontaneous mutation, reeler, including the cerebellum, the hippocampus, and the neocortex. Despite the ectopic positions of different neuronal populations, the specificity of synaptic connections is maintained. The metabolic consequences of this form of neuropathology were examined in Reln(rl) mutant mice by quantitative measures of cytochrome oxidase (CO) activity, a mitochondrial enzyme essential for oxidative metabolism in neurons. Despite severe tissue disorganization but in line with the intact synaptic organization, the reeler mutation did not affect global metabolic activity of the laminated structures of the brain. CO activity, however, was altered in specific subregions of the cerebellum, hippocampus, and neocortex, as well as in septum and various brainstem (medial pontine, paramedial reticular, paragigantocellular reticular) regions anatomically related to these structures, attesting to large functional alterations in Reln(rl-orl) brain. Metabolic activity variations were also detected in the ventral tegmental area and ventral neostriatum of the mesolimbic dopaminergic pathway. The results are discussed and compared to the regional CO variations found in other ataxic mice, in regard to the structural defects, the integrity of the connections, and the mutation-specific effects.