Mary E. Blue

The formation and maturation of synapses in the visual cortex of the rat. II. Quantitative analysis

SummarySynapse formation and maturation were examined in the visual cortex of albino rats from birth to maturity. During the first few days of postnatal life, synapses were sparsely scattered in the subplate zone and in layer I. They appeared immature as judged by the irregular shapes of the presynaptic and postsynaptic profiles, the relatively poorly defined membrane specializations and the presence of only a few synaptic vesicles in the presynaptic structures. As the neuropil matured, synapses were observed throughout the cortex, showing increased thickening of the membrane specializations and more vesicles. However, it was not until the end of the fourth postnatal week that they appeared qualitatively indistinguishable from synapses identified in the adult material.A feature characteristic of the developing visual cortex was the presence of vacant membrane specializations that resembled type I postsynaptic densities. These specializations, which were located either opposite extracellular space or opposite another neuronal process, were only evident during the initial stages of synaptogenesis and their frequency decreased as the number of synapses increased. In addition, transitional forms between these densities and true type I synapses were identified during the first two postnatal weeks. Structures that resembled vacant postsynaptic densities typical of type II synapses were not observed. The earliest identified forms of type II synaptic contacts identified consisted of two profiles that exhibited symmetrical membrane specializations and cleft material. Based on these observations, a scheme has been proposed for the formation of type I and type II synapses in the visual cortex of the rat.

Apoptosis has a prolonged role in the neurodegeneration after hypoxic ischemia in the newborn rat.

Birth asphyxia can cause moderate to severe brain injury. It is unclear to what degree apoptotic or necrotic mechanisms of cell death account for damage after neonatal hypoxia–ischemia (HI). In a 7-d-old rat HI model, we determined the contributions of apoptosis and necrosis to neuronal injury in adjacent Nissl-stained, hematoxylin and eosin-stained, and terminal deoxynucleotidyl transferase-mediated UTP nick end-labeled sections. We found an apoptotic–necrotic continuum in the morphology of injured neurons in all regions examined. Eosinophilic necrotic neurons, typical in adult models, were rarely observed in neonatal HI. Electron microscopic analysis showed “classic” apoptotic and necrotic neurons and “hybrid” cells with intermediate characteristics. The time course of apoptotic injury varied regionally. In CA3, dentate gyrus, medial habenula, and laterodorsal thalamus, the density of apoptotic cells was highest at 24–72 hr after HI and then declined. In contrast, densities remained elevated from 12 hr to 7 d after HI in most cortical areas and in the basal ganglia. Temporal and regional patterns of neuronal death were compared with expression of caspase-3, a cysteine protease involved in the execution phase of apoptosis. Immunocytochemical and Western blot analyses showed increased caspase-3 expression in damaged hemispheres 24 hr to 7 d after HI. A p17 peptide fragment, which results from the proteolytic activation of the caspase-3 precursor, was detected in hippocampus, thalamus, and striatum but not in cerebral cortex. The continued expression of activated caspase-3 and the persistence of cells with an apoptotic morphology for days after HI suggests a prolonged role for apoptosis in neonatal hypoxic ischemic brain injury.

Postmortem brain abnormalities of the glutamate neurotransmitter system in autism

Background: Studies examining the brains of individuals with autism have identified anatomic and pathologic changes in regions such as the cerebellum and hippocampus. Little, if anything, is known, however, about the molecules that are involved in the pathogenesis of this disorder. Objective: To identify genes with abnormal expression levels in the cerebella of subjects with autism. Method: Brain samples from a total of 10 individuals with autism and 23 matched controls were collected, mainly from the cerebellum. Two cDNA microarray technologies were used to identify genes that were significantly up- or downregulated in autism. The abnormal mRNA or protein levels of several genes identified by microarray analysis were investigated using PCR with reverse transcription and Western blotting. α-Amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid (AMPA)- and NMDA-type glutamate receptor densities were examined with receptor autoradiography in the cerebellum, caudate-putamen, and prefrontal cortex. Results: The mRNA levels of several genes were significantly increased in autism, including excitatory amino acid transporter 1 and glutamate receptor AMPA 1, two members of the glutamate system. Abnormalities in the protein or mRNA levels of several additional molecules in the glutamate system were identified on further analysis, including glutamate receptor binding proteins. AMPA-type glutamate receptor density was decreased in the cerebellum of individuals with autism (p < 0.05). Conclusions: Subjects with autism may have specific abnormalities in the AMPA-type glutamate receptors and glutamate transporters in the cerebellum. These abnormalities may be directly involved in the pathogenesis of the disorder.

Correspondence between 5-HT2 receptors and serotonergic axons in rat neocortex

The anatomic relationship between serotonergic (5-HT) axons and 5-HT2 receptors in the rat forebrain was determined by a combined analysis of transmitter immunocytochemistry and receptor autoradiography. High densities of 5-HT2 receptors, localized by the ligand N1-methyl-2-125I-LSD (125I-MIL), are found in neocortex and striatum; these regions also receive a dense serotonergic innervation. Regional variations in the density of 5-HT2 receptors and 5-HT axons correspond closely in most, but not all, areas of the forebrain. In somatosensory cortex (SI), the laminar distribution of 5-HT2 receptors closely matches that of 5-HT axons: in particular, a dense band of 5-HT2 receptors in layer Va of SI is in precise register with a dense plexus of fine 5-HT axons. We have also observed a close spatial relationship between 5-HT2 receptors and fine axons in other areas of the forebrain, suggesting that 5-HT2 receptors may be selectively linked to a particular type of 5-HT axon terminal. Since fine axons of this type have been reported to arise from the dorsal raphe nucleus, it appears likely that 5-HT2 receptors may mediate the effects of dorsal but not median raphe projections.

The immunophilins, FK506 binding protein and cyclophilin, are discretely localized in the brain : Relationship to calcineurin

The immunosuppressant drugs cyclosporin A and FK506 bind to small, predominantly soluble proteins cyclophilin and FK506 binding protein, respectively, to mediate their pharmacological actions. The immunosuppressant actions of these drugs occur through binding of cyclophilin-cyclosporin A and FK506 binding protein-FK506 complexes to the calcium-calmodulin-dependent protein phosphatase, calcineurin, inhibiting phosphatase activity. Utilizing immunohistochemistry, in situ hybridization and autoradiography, we have localized protein and messenger RNA for FK506 binding protein, cyclophilin and calcineurin. All three proteins and/or messages exhibit a heterogenous distribution through the brain and spinal cord, with the majority of the localizations being neuronal. We observe a striking co-localization of FK506 binding protein and calcineurin in most brain regions and a close similarity between calcineurin and cyclophilin. FK506 binding protein and cyclophilin localizations largely correspond to those of calcineurin, although cyclophilin is enriched in some brain areas that lack calcineurin. The dramatic similarities in localization of FK506 binding proteins and cyclophilins with calcineurin suggest related functions.

Neurobiology of Rett syndrome: a genetic disorder of synapse development

Rett syndrome is a developmental disorder that restricts brain growth beginning in the first year of life and evidence from neuropathology and neuroimaging indicates that axonodendritic connections are especially vulnerable. In a study of amino acid neurotransmitter receptors using receptor autoradiography in tissue slices of frontal cortex and the basal ganglia, we found a biphasic age-related pattern with relatively high receptor densities in young RS girls and lower densities at later time. Using microarray analysis of gene expression in frontal cortex, we found that some of the most prominent alterations occurred in gene products related to synapses, including the NMDA receptor NR1 subunit, the cytoskeletal protein MAP-2 and synaptic vesicle proteins. Using a new antibody that recognizes MeCP2, the transcription factor mutated in RS, we established that most neurons in the rodent brain express this transcription factor. We hypothesize that a major effect of mutations in the MeCP2 protein is to cause age-related disruption of synaptic proliferation and pruning in the first decade of life.

Amyloid pathology is associated with progressive monoaminergic neurodegeneration in a transgenic mouse model of Alzheimer's disease.

β-Amyloid (Aβ) pathology is an essential pathogenic component in Alzheimers disease (AD). However, the significance of Aβ pathology, including Aβ deposits/oligomers and glial reactions, to neurodegeneration is unclear. In particular, despite the Aβ neurotoxicity indicated by in vitro studies, mouse models with significant Aβ deposition lack robust and progressive loss of forebrain neurons. Such results have fueled the view that Aβ pathology is insufficient for neurodegeneration in vivo. In this study, because monoaminergic (MAergic) neurons show degenerative changes at early stages of AD, we examined whether the APPswe/PS1ΔE9 mouse model recapitulates progressive MAergic neurodegeneration occurring in AD cases. We show that the progression forebrain Aβ deposition in the APPswe/PS1ΔE9 model is associated with progressive losses of the forebrain MAergic afferents. Significantly, axonal degeneration is associated with significant atrophy of cell bodies and eventually leads to robust loss (∼50%) of subcortical MAergic neurons. Degeneration of these neurons occurs without obvious local Aβ or tau pathology at the subcortical sites and precedes the onset of anxiety-associated behavior in the mice. Our results show that a transgenic mouse model of Aβ pathology develops progressive MAergic neurodegeneration occurring in AD cases.

Preclinical research in Rett syndrome: Setting the foundation for translational success

In September of 2011, the National Institute of Neurological Disorders and Stroke (NINDS), the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD), the International Rett Syndrome Foundation (IRSF) and the Rett Syndrome Research Trust (RSRT) convened a workshop involving a broad cross-section of basic scientists, clinicians and representatives from the National Institutes of Health (NIH), the US Food and Drug Administration (FDA), the pharmaceutical industry and private foundations to assess the state of the art in animal studies of Rett syndrome (RTT). The aim of the workshop was to identify crucial knowledge gaps and to suggest scientific priorities and best practices for the use of animal models in preclinical evaluation of potential new RTT therapeutics. This review summarizes outcomes from the workshop and extensive follow-up discussions among participants, and includes: (1) a comprehensive summary of the physiological and behavioral phenotypes of RTT mouse models to date, and areas in which further phenotypic analyses are required to enhance the utility of these models for translational studies; (2) discussion of the impact of genetic differences among mouse models, and methodological differences among laboratories, on the expression and analysis, respectively, of phenotypic traits; and (3) definitions of the standards that the community of RTT researchers can implement for rigorous preclinical study design and transparent reporting to ensure that decisions to initiate costly clinical trials are grounded in reliable preclinical data.

DEVELOPMENTAL EXPRESSION OF METHYL-CpG BINDING PROTEIN 2 IS DYNAMICALLY REGULATED IN THE RODENT BRAIN

The gene encoding methyl-CpG binding protein 2 (MeCP2) is mutated in the large majority of girls that have Rett Syndrome (RTT), an X-linked neurodevelopmental disorder. To better understand the developmental role of MeCP2, we studied the ontogeny of MeCP2 expression in rat brain using MeCP2 immunostaining and Western blots. MeCP2 positive neurons were present throughout the brain at all ages examined, although expression varied by region and age. At early postnatal ages, regions having neurons that were generated early and more mature had the strongest MeCP2 expression. Late developing structures including cortex, hippocampus and cerebellum exhibited the most significant changes in MeCP2 expression. Of these regions, the cerebellum showed the most striking cell-specific changes in MeCP2 expression. For example, the early-generated Purkinje cells became MeCP2 positive by P6, while the late-generated granule cells did not express MeCP2 until the fourth postnatal week. The timing of MeCP2 expression in the granule cell layer is coincident with the onset of granule cell synapse formation. Although more subtle, the degree of MeCP2 expression in cortex and hippocampus was most closely correlated with synaptogenesis in both regions. Our finding that MeCP2 expression is correlated with synaptogenesis is consistent with the hypothesis that Rett Syndrome is caused by defects in the formation or maintenance of synapses.

Altered Development of Glutamate and GABA Receptors in the Basal Ganglia of Girls with Rett Syndrome

Rett syndrome (RS), a genetic disorder found almost exclusively in females, is associated with psychomotor regression and stereotyped hand movements. To determine whether a defect in basal ganglia amino acid neurotransmission plays a role in RS, NMDA-, AMPA-, kainate (KA)-, and metabotropic (mGluR)-type glutamate receptors (GluRs) and GABA receptors were labeled autoradiographically in the caudate, putamen, and globus pallidus of postmortem brain slices from 9 RS girls and 10 age-related controls. The cases were divided into younger (8 years or younger) and older age groups to study age-related changes in receptor binding density. We found significant reductions in AMPA and NMDA receptor density in the putamen and in KA receptor density in the caudate of older RS cases compared to controls. In contrast, mGluR density in the basal ganglia of RS patients was not altered significantly. The density of GluRs in control subjects generally showed more limited changes with age than in RS cases. In contrast to ionotropic GluRs, GABA receptor density was significantly increased in the caudate of young RS patients. The effects on GluR density in the putamen, which serves a primary motor function, were consistent with the motor deficits observed in RS, while those on amino acid transmitter receptors in the caudate may account for some cognitive features. Our studies demonstrate regional, receptor-subtype, and age-specific alterations in amino acid neurotransmitter receptors in the basal ganglia of RS girls. These changes may correlate with age-related clinical stages observed in RS.