Cerebrospinal fluid of brain trauma patients inhibits in vitro neuronal network function via NMDA receptors.

Neurological diseases frequently induce pathological changes of cerebrospinal fluid (CSF) that might secondarily influence brain activity, as the CSF-brain barrier is partially permeable. However, functional effects of CSF on neuronal network activity have not been specified to date. Here, we report that CSF specimens from patients with reduced Glasgow Coma Scale values caused by severe traumatic brain injury suppress synchronous activity of in vitro-generated neuronal networks in comparison with controls. We present evidence that underlying mechanisms include increased N-methyl-D-aspartate receptor activity mediated by a CSF fraction containing elevated amino acid concentrations. These proof-of-principle data suggest that determining effects of CSF specimens on neuronal network activity might be of diagnostic value.

Cryopreserved rat cortical cells develop functional neuronal networks on microelectrode arrays.

Neurons growing on microelectrode arrays (MEAs) are promising tools to investigate principal neuronal network mechanisms and network responses to pharmaceutical substances. However, broad application of these tools, e.g. in pharmaceutical substance screening, requires neuronal cells that provide stable activity on MEAs. Cryopreserved cortical neurons (CCx) from embryonic rats were cultured on MEAs and their immunocytochemical and electrophysiological properties were compared with acutely dissociated neurons (Cx). Both cell types formed neuritic networks and expressed the neuron-specific markers microtubule associated protein 2, synaptophysin, neurofilament and gamma-aminobutyric acid (GABA). Spontaneous spike activity (SSA) was recorded after 9 up to 74 days in vitro (DIV) in CCx and from 5 to 30 DIV in Cx, respectively. Cx and CCx exhibited synchronized burst activity with similar spiking characteristics. Tetrodotoxin (TTX) abolished the SSA of both cell types reversibly. In CCx SSA-inhibition occurred with an IC50 of 1.1 nM for TTX, 161 microM for magnesium, 18 microM for D,L-2-amino-5-phosphonovaleric acid (APV) and 1 microM for GABA. CCx cells were easy to handle and developed long living, stable and active neuronal networks on MEAs with similar characteristics as Cx. Thus, these neurochips seem to be suitable for studying neuronal network properties and screening in pharmaceutical research.

Neuronal network properties of human teratocarcinoma cell line-derived neurons.

Understanding the structural and functional development of neurons in networks has a high impact to estimate the potentials for restorative therapies. Neurons derived from the human NT2 cell line (hNT) formed networks with a clustered neuritic architecture in vitro, whereas primary dissociated embryonic rat cortical neurons (Cx) displayed a more homogenous cell assembly. Spontaneous spikes of both cell types were recorded on microelectrode arrays within 2 weeks after seeding, but hNT showed a mostly uncorrelated firing pattern in contrast to Cx with highly synchronized bursting. hNT neurons were less sensitive to TTX (IC50 = 5.7 +/- 0.1 nM vs. IC50 = 1.1 +/- 0.2 nM), magnesium (IC50 = 1.83 +/- 0.01 mM vs. IC50 = 0.161 +/- 0.023 mM), and APV (IC50 > 100 microM vs. IC50 = 18 microM). We conclude that embryonic cortical neurons and hNT neurons have different network properties. This should be carefully considered before hNT neurons are used in therapeutic approaches, e g., central nervous system (CNS) grafting.

Implications for hyperhomocysteinemia: not homocysteine but its oxidized forms strongly inhibit neuronal network activity.

Severe hyperhomocysteinemia (50-200 microM) often presents itself with acute neuronal dysfunction including seizures and psychosis. Its moderate form (15-50 microM) is associated with cognitive impairment and dementia. We investigated the neuropharmacological effects of homocysteine and its oxidized forms, homocysteinesulfinic acid (HCSA) and homocysteic acid (HCA), on neuronal network function utilizing dissociated cortical neurons from embryonic Wistar rats on microelectrode arrays. All substances inhibited dose-dependently and reversibly spontaneous neuronal network activity within seconds: L-HCSA and L-HCA blocked spontaneous spike rate (SSR) significantly at very low concentrations, with an IC50 of 1.9 and 1.3 microM, respectively; whereas the dose-response curve of D,L-homocysteine revealed an IC50 of 401 microM. These effects were antagonized by 2-amino-5-phosphonovaleric acid (APV) pointing to the NMDA receptor as mediator of this fast and reversible inhibition of network activity. We conclude that a neuronal dysfunction observed in hyperhomocysteinemia is likely due to HCSA and HCA since effective concentrations of homocysteine are not reached in patients.