Navjot Kaur and Mohan VemuriLife Technologies, 7335 Executive Way, Frederick, MD 21704 USA.

Neurobasal® Electro and B-27® Electro: Optimized media for Electrophysiology

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RESULTSFigure 1. Measurement of electrical activity Figure 4. Neurotransmitter receptors expression

ABSTRACT Neurons are specialized for integration and propagation of electricalevents. It is through such electrical activity that neurons communicatewith each other as well as with muscle and other end organs.Electrophysiology is the fundamental technique to assess suchelectrical activities to study the functions and dysfunctions in culturedneurons. Neurons are usually cultured in serum-free systems which

FEATURES AND BENEFITS

1. The B-27® Electrophysiology Kit is specifically designed to enhanceelectrophysiological spike rates in neural cell cultures when comparedwith other media and supplement combinations.

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Fig 1: Multielectrode array to measure spontaneous spike rates:Hippocampal neurons cultured in NEUROBASAL® Electro medium

neurons. Neurons are usually cultured in serum free systems whichinclude basal medium such as Neurobasal® supplemented with B-27®

and GlutaMAX-ITM. Neurons cultured in such media show lowelectrophysiological spike rates relative to media supplemented withserum. We have developed an optimized medium Neurobasal® Electroand a supplement B-27® Electro which promote higher spike rates by amechanism involving greater synaptogenesis that is reflected byincreased immunocytochemical marker expression of pre-synapticSynaptophysin and post synaptic PSD-95. The immunoreactive GABAand NMDA puncta also increased 2.8- fold and 1.6-fold respectively(n=3, p<0.05) over the period of three week. The rate of cell survivalwas indistinguishable at day 4 between Neurobasal®/B-27® and

2. The complete medium is prepared by supplementing Neurobasal®Electro with 2% B-27® Electro and 0.5mM GlutaMAXTM-I.

3. B-27® Supplement, Electro, when used as a supplement to Neurobasal®Medium, Electro, promotes optimal viability and long-term survival of ratembryonic hippocampal and cortical neurons.

4. Hippocampal and cortical neurons cultured in this new formulation showincreased density of synapses, a marker for information processing.

5. Neurons cultured in this system improve the efficiency of multielectrode

Neurobasal® Electro/B-27® ElectroNeurobasal®/B-27®

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supplemented with B-27® Electro produced higher spike rates ascompared to neurons cultured in NEUROBASAL® medium supplementedwith 2% B-27®. Day 7=4.2, day 14=3.4 fold and day 21= 78 fold increase;n=2, ** denotes p < 0.001, *** denotes p<0.0001.

Fig 4: Rat cortical neuron cultures were immunostained with anti-NMDA-R, (green) and anti-GABA-R (red) antibodies. Neurons cultured inNEUROBASAL® Electro medium/B-27® Electro produced 1.6-, 1.5-, 1.2-fold increase in NMDA receptor and 2.8-, 3.5-, 2-fold increase in GABAreceptor immunoreactivity at day 7, 14, and 21 respectively. n=2, *denotes p < 0.05, ** p < 0.001, *** p<0.0001.

was indistinguishable at day 4 between Neurobasal /B 27 andNeurobasal Electro®/B-27® Electro assessed by Live/Dead® cellviability assay (n=3, 98.0 ± 0.8 vs. 97.2 ± 1.6). These results showthat this media system is an improvement to Neurobasal®/B-27® forcellular networks in culture with an increased density of synapses andtransmitter receptors which produce higher spike rates in neuralnetworks. Neurobasal® Electro and B-27® Electro will render betterelectrophysiological studies, besides enabling normal functionalstudies.

MATERIALS AND METHODS

arrays used in neurotoxicology and neuropharmacology studies.

REFERENCES

1. Brewer, G.J. et al. (1993) Optimized survival of hippocampal neurons inB27-supplemented Neurobasal, a new serum-free medium combination.J Neurosci Res 35: 567-576.

2. Brewer, G.J. Isolation and culture of adult rat hippocampal neurons.(1997) J. Neurosci. Methods 71 14-155.

Figure 2. Comparison of cell survival

Figure 5. Synaptogenesis

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Neurobasal®/B-27® Neurobasal® Electro/B-27®Electro

Fig 2: Live/Dead® cell viability assay: Neurons cultured in NEUROBASAL®

Electro medium supplemented with B-27® Electro maintain equivalent cellsurvival rate as compared to neurons cultured in NEUROBASAL® mediumsupplemented with 2% B-27®. Neuronal survival rate was comparable evenat day 21 cultures(n=5, 77± 6 vs.78 ±7).

Primary Neuronal Cultures: Rat E18 freshly isolated or cryopreservedcortical or hippocampal neurons were cultured on poly-D-lysine coatedsurfaces. Cells were maintained in NEUROBASAL® mediumsupplemented with 2% B-27® or NEUROBASAL® Electro mediumsupplemented with B-27® Electro. Both media were furthersupplemented with 0.5 mM GlutaMAXTM-I for 4 to 21 days in standard5% CO2 conditions.

Measurement of electrical activities: Rat hippocampal neurons wereplated at 1000 cells/mm2 onto a multi electrode array Axion

3. Brewer, G.J. et al. (2008) NbACtiv4 medium improvement toNeurobasal/B27 increases neuron synapse densities and network spikerates on multielectrode array.

4. Brewer, G.J. et al. (2009) Neuron network activity scales exponentiallywith synapse density. J. Neural Eng. 6: 014001

PRODUCT INFORMATIONB-27® Electrophysiology Kit A13794-01 1kit Figure 3. Neurite Outgrowth

Neurobasal®/B-27® Neurobasal® Electro/B-27®Electro

Anti-PSD-95/Alexa-488Anti-Synaptophysin/Alexa-594Merge : Yellow

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*Neurobasal® Electro/B-27® ElectroNeurobasal®/B-27®

Biosystems MEA system ( MM Muse 01) and spike rates werecalculated using accompanying software. Cells were allowed tocultured in each media and readings were acquired on day 7, 14, and21. Statistical analyses used student t- test or ANOVA with rejection ofthe null hypothesis at p<0.05.

Cell survival assessment: Cells were stained using Calcein-AM andEthD1 to evaluate the survival rate of neurons using LIVE/DEAD cellviability /cytotoxicity kit. Live (green) and dead (red) cells were countedusing ImageJ and cell survival was calculated as percentage of livecells over total cell population. Statistical analyses used student t- test

p y gy

B-27® Supplement, Electro (50X) A13585SA 10mL

Neurobasal® Medium, Electro (1X) A13586DJ 500mL

TRADEMARKS/LICENSING

The trademarks mentioned herein are the property of Life TechnologiesCorporation or their respective owners.

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Neurobasal® Electro/B-27® ElectroNeurobasal®/B-27®

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Fig 5: Neurons cultured in NEUROBASAL® Electro medium/B-27® Electroshowed increased density of synapses. Rat cortical neurons wereimmunostained for pre-synaptic vesicle marker Synaptophysin (red) andpost-synaptic marker PSD-95 (green). Integrated density showed 1.5-foldincrease in receptors expression measured from merged images (yellow).n=5 fields, * denotes p < 0.05.

or ANOVA with rejection of the null hypothesis at p<0.05.

Immunocytochemistry: Cells were cultured for 4 to 21 days and fixed in4% paraformaldehyde, permeabilized with 0.3% Triton® -X, andblocked with 5% goat serum. The cells were incubated with primaryantibodies for overnight and with secondary antibodies for one hour atroom temperature. Antibodies details are provided in figure legends.

B-27 is a registered trademark of Southern Illinois University. Triton is aregistered trademark of Union Carbide Corporation .

For Research Use Only. Not intended for any animal or human therapeuticor diagnostic use.

Life Technologies • 7335 Executive Way• Frederick, MD 21704• www.lifetechnologies.com

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Fig 3: Comparison of neuronal branching in NEUROBASAL® Electromedium supplemented with B-27® Electro vs. NEUROBASAL® /B-27® : Ratcortical neurons showed 1.7 fold increase in number of neurites per cell,and 1.6 fold increase in dendrite length The means of longest axon lengthwere not significantly different. (n=10 fields, ** denotes p < 0.001) .