Analysis of quantification methods used for cell viability, cell morphology, and synaptic formation in modeling HIV associated dementia in primary neuronal cultures.

Change is inevitable, changes in neuronal function occur in physiologic and pathologic processes. The ability to reliably analyze and quantify those changes in neuronal morphology and function has been an important part of technical developments in Neuroscience. A key innovation in the Neuroscience was the development of primary neuronal cultures. Primary neuronal cultures allow neurons to be dissociated and studied as individual components. The study of specific pathologic processes associated with neurodegeneration have benefited greatly from the development and characterization of dissociated primary neuronal cultures. Human Immunodeficiency Virus can lead to a neurodegenerative process. Establishing a consistent model for studying the effects of HIV infection in the brain has provided a unique challenge. The use of analysis of quantification of neuronal changes in dissociated primary neurons modeling HIV dementia has proven useful. As the study of this disorder continues the characterization of the model system will become increasing important. This review will focus on analysis of specific techniques used to quantify specific changes in neurons in this model system. As this field moves forward it will be important to specifically focus on techniques involved in cell viability, morphologic changes, and synaptic formatio

Axonal regeneration in hippocampal and spinal cord organotypic slice cultures

Under normal conditions, axonal regeneration after lesions is not possible in mature CNS but can occur in embryonic and early postnatal nervous systems. In recent years, a number of possible strategies to enhance axonal regeneration and eventually treat spinal cord and brain injuries have been identified, some of which have been used successfully in animal experiments, but till now there is still no successful treatment available for patients. This problem is partly due to the complexity of the animal experiments which makes it difficult to compare different treatment strategies. In this project, we have used organotypic slice culture models to test the effectiveness of pharmacological compounds that interfere with various signal transduction mechanisms, to promote axonal regeneration. We used the entorhino hippocampal slice cultures to assess regeneration of entorhinal fibers projecting to the dentate gyrus after mechanical lesions and treatment. It was previously shown (Prang et. al., 2001) that there is a marked decrease in regenerating fibers when a lesion is made at 6 7 days in vitro or later in slices derived from postnatal day 5 6 mice. We took this as a control model where there is little spontaneous axonal regeneration, added treatments on the day of lesion and later traced for entorhinal axons with biotinylated dextran amine (BDA). In this study it was shown that compounds acting on the cAMP, PKC and G proteins can promote regeneration. Furthermore, we have identified the inhibition of the PI3 kinase pathway and the IP 3 receptor as potential drug targets that promote axonal regeneration. In order to study axonal growth in a spinal cord environment we have developed a spinal cord longitudinal organotypic slice culture model which allowed us to follow axons along the rostro caudal extension of the spinal cord. Slices of cervical spinal cord were cut in the sagittal plane from early postnatal mice and were maintained in culture for various time periods up to 4 weeks. Histological and immunohistochemical stainings of the cultures have shown that these slice cultures maintain the ventro dorsal polarity of the spinal cord and that an intrinsic fibre projection develops which runs along the rostro caudal extension of the spinal cord slice culture. After mechanical lesion, these fibres have the ability to regenerate spontaneously demonstrating the intrinsic ability of the spinal cord for repair, but this ability is decreased with increasing time in culture. During the culture period the axons became myelinated and expressed synaptic markers. These cultures could thus serve also as a model for myelin formation and synaptogenesis. We have analyzed the potential of axons from longitudinal spinal cord cultures to grow into an adjacent slice of cerebellar tissue. We could show that spinal cord axons do enter the cerebellar slice in particular when early postnatal spinal cord is combined with postnatal cerebellum. Pharmacological treatments were used to enhance axonal growth. Similarly to our findings in the entorhino hippocampal model, cAMP activators and PKC inhibitors promoted axonal growth from the spinal cord to the cerebellum. In cocultures of longitudinal spinal cord slices with cortical slices we have shown that fibers from the cortical slices grew extensively into the spinal cord slice and extended caudally for substantial distances. Our results demonstrate that organotypic slice cultures can be a useful tool to study axonal growth and regeneration. Intrinsic spinal cord axons have a considerable potential for spontaneous regeneration in the early postnatal period and are able to grow both through a mechanical lesion and into another tissue. Moreover, compounds interfering with signal transduction mechanisms, particularly cAMP, PKC, PI3 Kinase, G proteins and IP3 receptors, were able to promote axonal growth and regeneration in diverse slice culture models making them interesting drug candidates for the promotion of axonal regeneration

Enlarged infarct volume and loss of BDNF mRNA induction following brain ischemia in mice lacking FGF-2

FGF-2, a potent multifunctional and neurotrophic growth factor, is widely expressed in the brain and upregulated in cerebral ischemia. Previous studies have shown that intraventricularly or systemically administered FGF-2 reduces the size of cerebral infarcts. Whether endogenous FGF-2 is beneficial for the outcome of cerebral ischemia has not been investigated. We have used mice with a null mutation of the fgf2 gene to explore the relevance of endogenous FGF-2 in brain ischemia. Focal cerebral ischemia was produced by occlusion of the middle cerebral artery (MCAO). We found a 75% increase in infarct volume in fgf2 knock-out mice versus wild type littermates (P < 0.05). This difference in the extent of ischemic damage was observed after 24 h, and correlated with decreased viability in fgf2 mutant mice following MCA occlusion. Increased infarct volume in fgf2 null mice was associated with a loss of induction in hippocampal BDNF and trkB mRNA expression. These findings indicate that signaling through trkB may contribute to ameliorating brain damage following ischemia and that bdnf and trkB may be target genes of FGF-2. Together, our data provide the first evidence that endogenous FGF-2 is important in coping with ischemic brain damage suggesting fgf2 as one crucial target gene for new therapeutic strategies in brain ischemia

Structure-stiffness relation of live mouse brain tissue determined by depth-controlled indentation mapping

The mechanical properties of brain tissue play a pivotal role in neurodevelopment and neurological disorders. Yet, at present, there is no consensus on how the different structural parts of the tissue contribute to its stiffness variations. Here, we have gathered depth-controlled indentation viscoelasticity maps of the hippocampus of isolated horizontal live mouse brain sections. Our results confirm the highly viscoelestic nature of the material and clearly show that the mechanical properties correlate with the different morphological layers of the samples investigated. Interestingly, the relative cell nuclei area seems to negatively correlate with the stiffness observed

The Combined Impact Of IgLON Family Proteins Lsamp And Neurotrimin On Developing Neurons And Behavioral Profiles In Mouse

Cell surface neural adhesion proteins are critical components in the complex orchestration of cell proliferation, apoptosis, and neuritogenesis essential for proper brain construction and behavior. We focused on the impact of two plasticity-associated IgLON family neural adhesion molecules, Neurotrimin (Ntm) and Limbic system associated membrane protein (Lsamp), on mouse behavior and its underlying neural development. Phenotyping neurons derived from the hippocampi of Lsamp−/−, Ntm−/− and Lsamp−/−Ntm−/− mice was performed in parallel with behavioral testing. While the anatomy of mutant brains revealed no gross changes, the Ntm−/− hippocampal neurons exhibited premature sprouting of neurites and manifested accelerated neurite elongation and branching. We propose that Ntm exerts an inhibitory impact on neurite outgrowth, whereas Lsamp appears to be an enhancer of the said process as premature neuritogenesis in Ntm−/− neurons is apparent only in the presence of Lsamp. We also show interplay between Lsamp and Ntm in regulating tissue homeostasis: the impact of Ntm on cellular proliferation was dependent on Lsamp, and Lsamp appeared to be a positive regulator of apoptosis in the presence of Ntm. Behavioral phenotyping indicated test-specific interactions between Lsamp and Ntm. The phenotypes of single mutant lines, such as reduced swimming speed in Morris water maze and increased activity in the elevated plus maze, were magnified in Lsamp−/−Ntm−/− mice. Altogether, evidence both from behavioral experiments and cultured hippocampal cells show combined and differential interactions between Ntm and Lsamp in the formation of hippocampal circuits and behavioral profiles. We demonstrate that mutual interactions between IgLON molecules regulate the initiation of neurite sprouting at very early ages, and even cell-autonomously, independent of their regulation of cell-cell adhesion

The ASD Living Biology: from cell proliferation to clinical phenotype.

Autism spectrum disorder (ASD) has captured the attention of scientists, clinicians and the lay public because of its uncertain origins and striking and unexplained clinical heterogeneity. Here we review genetic, genomic, cellular, postmortem, animal model, and cell model evidence that shows ASD begins in the womb. This evidence leads to a new theory that ASD is a multistage, progressive disorder of brain development, spanning nearly all of prenatal life. ASD can begin as early as the 1st and 2nd trimester with disruption of cell proliferation and differentiation. It continues with disruption of neural migration, laminar disorganization, altered neuron maturation and neurite outgrowth, disruption of synaptogenesis and reduced neural network functioning. Among the most commonly reported high-confidence ASD (hcASD) genes, 94% express during prenatal life and affect these fetal processes in neocortex, amygdala, hippocampus, striatum and cerebellum. A majority of hcASD genes are pleiotropic, and affect proliferation/differentiation and/or synapse development. Proliferation and subsequent fetal stages can also be disrupted by maternal immune activation in the 1st trimester. Commonly implicated pathways, PI3K/AKT and RAS/ERK, are also pleiotropic and affect multiple fetal processes from proliferation through synapse and neural functional development. In different ASD individuals, variation in how and when these pleiotropic pathways are dysregulated, will lead to different, even opposing effects, producing prenatal as well as later neural and clinical heterogeneity. Thus, the pathogenesis of ASD is not set at one point in time and does not reside in one process, but rather is a cascade of prenatal pathogenic processes in the vast majority of ASD toddlers. Despite this new knowledge and theory that ASD biology begins in the womb, current research methods have not provided individualized information: What are the fetal processes and early-age molecular and cellular differences that underlie ASD in each individual child? Without such individualized knowledge, rapid advances in biological-based diagnostic, prognostic, and precision medicine treatments cannot occur. Missing, therefore, is what we call ASD Living Biology. This is a conceptual and paradigm shift towards a focus on the abnormal prenatal processes underlying ASD within each living individual. The concept emphasizes the specific need for foundational knowledge of a living child's development from abnormal prenatal beginnings to early clinical stages. The ASD Living Biology paradigm seeks this knowledge by linking genetic and in vitro prenatal molecular, cellular and neural measurements with in vivo post-natal molecular, neural and clinical presentation and progression in each ASD child. We review the first such study, which confirms the multistage fetal nature of ASD and provides the first in vitro fetal-stage explanation for in vivo early brain overgrowth. Within-child ASD Living Biology is a novel research concept we coin here that advocates the integration of in vitro prenatal and in vivo early post-natal information to generate individualized and group-level explanations, clinically useful prognoses, and precision medicine approaches that are truly beneficial for the individual infant and toddler with ASD

Dependence receptor involvement in subtilisin-induced long-term depression and in long-term potentiation

The serine protease subtilisin induces a form of long-term depression (LTD) which is accompanied by a reduced expression of the axo-dendritic guidance molecule Unco-ordinated-5C (Unc-5C). One objective of the present work was to determine whether a loss of Unc-5C function contributed to subtilisin-induced LTD by using Unc-5C antibodies in combination with the pore-forming agents Triton X-100 (0.005%) or streptolysin O in rat hippocampal slices. In addition we have assessed the effect of subtilisin on the related dependence receptor Deleted in Colorectal Cancer (DCC) and used antibodies to this protein for functional studies. Field excitatory postsynaptic potentials (fEPSPs) were analysed in rat hippocampal slices and protein extracts were used for Western blotting. Subtilisin produced a greater loss of DCC than of Unc-5C, but the antibodies had no effect on resting excitability or fEPSPs and did not modify subtilisin-induced LTD. However, antibodies to DCC but not Unc-5C did reduce the amplitude of theta-burst long-term potentiation (LTP). In addition, two inhibitors of endocytosis – dynasore and tat-gluR2(3Y) – were tested and, although the former compound had no effect on neurophysiological responses, tat-gluR2(3Y) did reduce the amplitude of subtilisin-induced LTD without affecting the expression of DCC or Unc-5C but with some loss of PostSynaptic Density Protein-95. The results support the view that the dependence receptor DCC may be involved in LTP and suggest that the endocytotic removal of a membrane protein or proteins may contribute to subtilisin-induced LTD, although it appears that neither Unc-5C nor DCC are involved in this process. (220)

The SNARE Protein Syntaxin 3 Confers Specificity for Polarized Axonal Trafficking in Neurons.

Cell polarity and precise subcellular protein localization are pivotal to neuronal function. The SNARE machinery underlies intracellular membrane fusion events, but its role in neuronal polarity and selective protein targeting remain unclear. Here we report that syntaxin 3 is involved in orchestrating polarized trafficking in cultured rat hippocampal neurons. We show that syntaxin 3 localizes to the axonal plasma membrane, particularly to axonal tips, whereas syntaxin 4 localizes to the somatodendritic plasma membrane. Disruption of a conserved N-terminal targeting motif, which causes mislocalization of syntaxin 3, results in coincident mistargeting of the axonal cargos neuron-glia cell adhesion molecule (NgCAM) and neurexin, but not transferrin receptor, a somatodendritic cargo. Similarly, RNAi-mediated knockdown of endogenous syntaxin 3 leads to partial mistargeting of NgCAM, demonstrating that syntaxin 3 plays an important role in its targeting. Additionally, overexpression of syntaxin 3 results in increased axonal growth. Our findings suggest an important role for syntaxin 3 in maintaining neuronal polarity and in the critical task of selective trafficking of membrane protein to axons

Loss of AP-3 function affects spontaneous and evoked release at hippocampal mossy fiber synapses

Synaptic vesicle (SV) exocytosis mediating neurotransmitter release occurs spontaneously at low intraterminal calcium concentrations and is stimulated by a rise in intracellular calcium. Exocytosis is compensated for by the reformation of vesicles at plasma membrane and endosomes. Although the adaptor complex AP-3 was proposed to be involved in the formation of SVs from endosomes, whether its function has an indirect effect on exocytosis remains unknown. Using mocha mice, which are deficient in functional AP-3, we identify an AP-3-dependent tetanus neurotoxin-resistant asynchronous release that can be evoked at hippocampal mossy fiber (MF) synapses. Presynaptic targeting of the tetanus neurotoxin-resistant vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) is lost in mocha hippocampal MF terminals, whereas the localization of synaptobrevin 2 is unaffected. In addition, quantal release in mocha cultures is more frequent and more sensitive to sucrose. We conclude that lack of AP-3 results in more constitutive secretion and loss of an asynchronous evoked release component, suggesting an important function of AP-3 in regulating SV exocytosis at MF terminals