Microtubule-associated protein 6 mediates neuronal connectivity through Semaphorin 3E-dependent signalling for axonal growth.

Structural microtubule associated proteins (MAPs) stabilize microtubules, a property that was thought to be essential for development, maintenance and function of neuronal circuits. However, deletion of the structural MAPs in mice does not lead to major neurodevelopment defects. Here we demonstrate a role for MAP6 in brain wiring that is independent of microtubule binding. We find that MAP6 deletion disrupts brain connectivity and is associated with a lack of post-commissural fornix fibres. MAP6 contributes to fornix development by regulating axonal elongation induced by Semaphorin 3E. We show that MAP6 acts downstream of receptor activation through a mechanism that requires a proline-rich domain distinct from its microtubule-stabilizing domains. We also show that MAP6 directly binds to SH3 domain proteins known to be involved in neurite extension and semaphorin function. We conclude that MAP6 is critical to interface guidance molecules with intracellular signalling effectors during the development of cerebral axon tracts.

Automated immunohistochemical method to quantify neuronal density in brain sections: application to neuronal loss after status epilepticus.

BACKGROUND: To study neurotoxic processes, it is necessary to quantify the number of neurons in a given brain structure and estimate neuronal loss. Neuronal densities can be estimated by immunohistochemical quantitation of a neuronal marker such as the protein NeuN. However, NeuN expression may vary, depending on certain pathophysiological conditions and bias such quantifications. NEW METHOD: We have developed a simple automatic quantification of neuronal densities in brain sections stained with DAPI and antibody to NeuN. This method determines the number of DAPI-positive nuclei also positive for NeuN in at least two adjacent sections within a Z-stack of optical sections. RESULTS: We tested this method in animals with induced status epilepticus (SE) a state of intractable persistent seizure that produces extensive neuronal injury. We found that SE significantly reduced neuronal density in the piriform cortex, the amygdala, the dorsal thalamus, the CA3 area of the hippocampus, the dentate gyrus and the hilus, but not in the somatosensory cortex or the CA1 area. SE resulted in increases in the total density of cellular nuclei within these brain structures, suggesting gliosis. COMPARISON WITH EXISTING METHODS: This automated method was more accurate than simply estimating the overall NeuN fluorescence intensity in the brain section, and as accurate, but less time-consuming, than manual cell counts. CONCLUSION: This method simplifies and accelerates the unbiased quantification of neuronal density. It can be easily applied to other models of brain injury and neurodegeneration, or used to screen the efficacy of neuroprotective treatments.

In vivo D-serine hetero-exchange through alanine-serine-cysteine (ASC) transporters detected by microelectrode biosensors.

D-serine, a co-agonist of N-methyl D-aspartate (NMDA) receptors, has been implicated in neurological and psychiatric disorders such as cerebral ischemia, lateral amyotrophic sclerosis, or schizophrenia. D-serine signaling represents an important pharmacological target for treating these diseases; however, the biochemical mechanisms controlling extracellular D-serine levels in vivo are still unclear. D-serine heteroexchange through small neutral amino acid transporters has been shown in cell cultures and brain slices and could provide a biochemical mechanism for the control of D-serine extracellular concentration in vivo. Alternatively, exocytotic D-serine release has also been proposed. In this study, the dynamics of D-serine release and clearance were explored in vivo on a second-by-second time scale using microelectrode biosensors. The rate of D-serine clearance in the rat frontal cortex after a microionophoretic injection revealed a transporter-mediated uptake mechanism. D-serine uptake was blocked by small neutral l-amino acids, implicating alanine-serine-cysteine (ASC) transporters, in particular high affinity Asc-1 and low affinity ASCT2 transporters. Interestingly, changes in alanine, serine, or threonine levels resulted in D-serine release through ASC transporters. Asc-1, but not ASCT2, appeared to release D-serine in response to changes in amino acid concentrations. Finally, neuronal silencing by tetrodotoxin increased D-serine extracellular concentration by an ASC-transporter-dependent mechanism. Together, these results indicate that D-serine heteroexchange through ASC transporters is present in vivo and may constitute a key component in the regulation of D-serine extracellular concentration.

Immobilization method to preserve enzyme specificity in biosensors: consequences for brain glutamate detection.

Microelectrode biosensors are a promising technique to probe the brain interstitial fluid and estimate the extracellular concentration of neurotransmitters like glutamate. Their selectivity is largely based on maintaining high substrate specificity for the enzymes immobilized on microelectrodes. However, the effect of enzyme immobilization on substrate specificity is poorly understood. Furthermore, the accuracy of biosensor measurements for brain biological extracts has not been reliably established in comparison with conventional analytical techniques. In this study, microelectrode biosensors were prepared using different enzyme immobilization methods, including glutaraldehyde, a conventional cross-linker, and poly(ethylene glycol) diglycidyl ether (PEGDE), a milder immobilization reagent. Glutaraldehyde, but not PEGDE, significantly decreased the apparent substrate specificity of glutamate and glucose oxidase. For glutaraldehyde prepared biosensors, detection of secondary substrates by glutamate oxidase increased, resulting in a significant overestimate of glutamate levels. This effect was not observed with PEGDE-based biosensors, and when brain microdialysates were analyzed, the levels of glutamate detected by biosensors were consistent with those detected by capillary electrophoresis. In addition, basal concentrations of glutamate detected in vivo were approximately 10-fold lower than the levels detected with glutaraldehyde-based biosensors (e.g., 1.2 µM vs 16 µM, respectively). Overall, enzyme immobilization can significantly impact substrate specificity, and PEGDE is well-suited for the preparation of stable and selective biosensors. This development questions some of the previous biosensor studies aimed at detecting glutamate in the brain and opens new possibilities for specific neurotransmitter detection.

d-Serine diffusion through the blood-brain barrier: effect on d-serine compartmentalization and storage.

d-Serine is a co-agonist of N-methyl-d-aspartate (NMDA) receptors. It has been implicated in the etiology of schizophrenia and has shown efficacy as an adjuvant to reduce positive and negative symptoms of schizophrenia. In addition, d-serine can modulate cognition in animals when administered alone. However, the neurochemical effects of exogenous d-serine on extra- and intra-cellular d-serine brain levels are poorly understood. In this study, we used both high performance liquid chromatography (HPLC) and enzyme-based microelectrode biosensors to quantify d-serine in the rat brain. We demonstrated levels of 2.3-2.8µM in the extracellular medium, 4µM in plasma and 188pmol/mg in brain tissue samples. An intraperitoneal (i.p.) d-serine injection (1g/kg) produced a slow increase in extracellular d-serine concentration in the cortex despite a surge in d-serine up to 13mM in the plasma, indicating poor diffusion through the blood-brain barrier. Using the respective volume fractions of blood, extracellular and intracellular spaces published in the literature, we estimated that d-serine intracellular stores represented more than 99% of total d-serine. These intracellular stores almost doubled 3h after d-serine administration. Overall, our data indicate that d-serine administration increases brain extra- and intra-cellular concentrations despite weak diffusion through the blood-brain barrier. These results pave the way for a better understanding of the neurochemical mechanisms by which d-serine administration modulates cognition.

Covalent enzyme immobilization by poly(ethylene glycol) diglycidyl ether (PEGDE) for microelectrode biosensor preparation.

Poly(ethylene glycol) diglycidyl ether (PEGDE) is widely used as an additive for cross-linking polymers bearing amine, hydroxyl, or carboxyl groups. However, the idea of using PEGDE alone for immobilizing proteins on biosensors has never been thoroughly explored. We report the successful fabrication of microelectrode biosensors based on glucose oxidase, d-amino acid oxidase, and glutamate oxidase immobilized using PEGDE. We found that biosensors made with PEGDE exhibited high sensitivity and a response time on the order of seconds, which is sufficient for observing biological processes in vivo. The enzymatic activity on these biosensors was highly stable over several months when they were stored at 4 °C, and over at least 3d at 37 °C. Glucose microelectrode biosensors implanted in the central nervous system of anesthetized rats reliably monitored changes in brain glucose levels induced by sequential administration of insulin and glucose. PEGDE provides a simple, low cost, non-toxic alternative for the preparation of in vivo microelectrode biosensors.