NbActiv4 medium improvement to Neurobasal/B27 increases neuron synapse densities and network spike rates on multielectrode arrays

Abstract

The most interesting property of neurons is their long-distance propagation of signals as spiking action potentials. Since 1993, Neurobasal/B27 has been used as a serum-free medium optimized for hippocampal neuron survival. Neurons on microelectrode arrays (MEA) were used as an assay system to increase spontaneous spike rates in media of different compositions. We find spike rates of 0.5 s(-1) (Hz) for rat embryonic hippocampal neurons cultured in Neurobasal/B27, lower than cultures in serum-based media and offering an opportunity for improvement. NbActiv4 was formulated by addition of creatine, cholesterol and estrogen to Neurobasal/B27 that synergistically produced an eightfold increase in spontaneous spike activity. The increased activity with NbActiv4 correlated with a twofold increase in immunoreactive synaptophysin bright puncta and GluR1 total puncta. Characteristic of synaptic scaling, immunoreactive GABAAbeta puncta also increased 1.5-fold and NMDA-R1 puncta increased 1.8-fold. Neuron survival in NbActiv4 equaled that in Neurobasal/B27, but with slightly higher astroglia. Resting respiratory demand was decreased and demand capacity was increased in NbActiv4, indicating less stress and higher efficiency. These results show that NbActiv4 is an improvement to Neurobasal/B27 for cultured networks with an increased density of synapses and transmitter receptors which produces higher spontaneous spike rates in neuron networks.

Fig. 1

Comparison of neuronal cultures in Neurobasal/B27 to NbActiv4 on both arrays and glass cover slips for differences in electrical activity and immunocytology. (A) 5 cm square MEA with gold leads and attached ring to contain culture medium. (B) 60 electrodes in the central 1 mm² sense spike activity. (C) and (D) Similar phase contrast images of Neurobasal/B27 and NbActiv4 cultures after three weeks in vitro. Dark circular electrodes are 10 μm diameter electrodes on 200 μm spacing. (E) and (F) Immunofluorescent images (tau-red, MAP2-green, bisbenzamide-blue) to compare neurite branching. (G) Tau neurite branching per cell is higher in NbActiv4 (n=16 fields from 2 cultures).

Fig. 2

NbActiv4 increases spontaneous spike rates and bursting compared to neurons cultured in Neurobasal/B27. Activity (μV) is compared over the indicated times.

Fig. 3

NbActiv4 increases electrical activity, the percentage of active electrodes, but not astroglial density compared to cultures in Neurobasal/B27. (A) Mean spontaneous spike rates of electrodes with activity above 0.03 Hz (n=6-7 cultures). (B) Percentage active electrodes with activity above 0.03 Hz (n=6-7 cultures). C) Mean astroglia counts are not significantly different (n = 12 fields of 0.145 mm² from 2 cultures).

Fig. 4

NbActiv4 increases total GABA puncta 40% and bright NR1 puncta 100% compared to neurons cultured in Neurobasal/B27. (A) and (B) Immunoreactive red GABA and green NR1 synaptic puncta. (C) and (D) Bright and dim segmentation thresholds for GABA and NR1 show effect of medium on stained puncta (n = 16 fields of 0.016 mm² from 2 cultures).

Fig. 5

NbActiv4 increases bright synaptophysin puncta 2-fold and total Glu-R1 puncta 2-fold compared to neurons cultured in Neurobasal/B27. (A) and (B) Immunoreactive red Glu-R1 and green synaptophysin puncta. (C) and (D) Bright and dim segmentation thresholds for synaptophysin and Glu-R1 show effect of medium on puncta (n = 16 fields of 0.016 mm² from 2 cultures).

Fig. 6

Under resting conditions, neurons consume oxygen at a significantly lower rate in NbActiv4 than in Neurobasal/B27, yet remain capable of higher respiration after uncoupling with FCCP (n=6-7 cultures). This suggests greater mitochondrial efficiency of neurons in NbActiv4.