Pilot study of iPS-derived neural cells to examine biologic effects of alcohol on human neurons in vitro.

Neural Differentiation Timeline(A) Timeline of neural differentiation includes culturing iPS cell colonies (B) to confluency for 7 days, followed by culture in suspension for 4 days to allow for the formation of embryoid bodies, followed by incubation in neural induction media and growth on a laminin substrate to trigger the formation of neuroepithelial cells displaying neural tube-like rosette structures, which are expanded in suspension (C) culture for several days prior to plating dissociated cells on coverslips. Dissociated cells are cultured in a neural differentiation media containing the growth factors BDNF, GDNF, and IGF to promote the differentiation of neural cells (D, arrows indicate neural cell bodies, red arrow indicates recording electrode for performing electrophysiology). Dissociated neural cells were then cultured for 12–18 weeks in neural differentiation media. At 10-weeks post-plating neural cultures displayed neuronal lineage marker beta III-tubulin and glial cell lineage s100β positive cells (E), indicating that iPS cells differentiated into cultures containing a mixed culture of neurons and glia. Focal synapsin-1 immunostaining along beta III tubulin (F) positive processes were observed indicating the formation of synaptic specializations within the culture.

Electrophysiological Properties of iPS-Derived NeuronsWhole-cell patch recordings from 12-week iPS-derived neurons from a non-alcohol dependent subject demonstrate functional neuronal properties, including generation of action potentials, spontaneous synaptic activity, and ionotropic receptor currents. (A) Neurons generate trains of action potentials in response to current injection. (B) Spontaneous synaptic activity (Inset) close-up of a spontaneous event demonstrates fast and slow kinetic components of the event. (C) GABA receptor current response to a 200 ms puff of 50μM GABA indicates that functional GABA receptors are present (arrow indicates when GABA was applied). (D) Depolarization response to a 200 ms puff of 50μM glutamate in the presence of Mg2+ indicates that functional AMPA receptors are expressed. Response was completely abolished when the AMPA antagonist DNQX (10µM) was perfused into the recording chamber (arrows indicate when glutamate was applied). (E) NMDA channel mediated depolarization in response to a 200 ms puff of 50μM glutamate and 10 μM glycine in a magnesium-free recording solution that contained the AMPA receptor antagonist DNQX (10µM). The response was completely abolished when NMDA receptor antagonist CPP (6µM) was perfused into the recording chamber (Dashed arrows indicate when glutamate plus glycine solution was applied).

Acute Alcohol Attenuation of NMDA Response is Not Observed After 7-Days of Alcohol ExposureWhole-cell patch clamp recordings from neurons derived from a non-alcohol dependent subject. (A) Example of the time course illustrating the inhibition of the NMDA response by acute alcohol exposure (50 mM) in a naïve neuron. (B) Example of the lack of effect of alcohol on NMDA receptors following 7-day exposure to 50 mM alcohol. (C) Group data for the effect of acute alcohol which significantly reduced the amplitude of the NMDA response (n=8, paired t-test, p= .036). (D) Group data for 7-day alcohol pre-treated cells showing no significant effect of bath applied alcohol on NMDA response (n=10, paired t-test, p= .77). Differences in the mean amplitude of the baseline depolarization between naïve and chronically treated cells (panels C and D) relate in large measure to variation in the distance of the puffing pipette from the patched cells examined.

NMDA Receptor Subunits are Upregulated After Chronic Alcohol Exposure in Neural Cells Derived from AlcoholicsNeural cells (age 12–15 weeks) derived from 7 different subjects, 3 non-alcoholics and 4 alcoholics, were treated daily with 50 mM alcohol for 7 days and levels of GRIN1, GRIN2A, GRIN2B, and GRIN2D mRNA expression was analyzed using RT-PCR. No significant changes were observed in the expression of these NMDA receptor subunit genes after 7-day alcohol exposure (relative to the sham condition) in neural cells derived from the group of non-alcoholic donor subjects (left group of data panels A–D). In contrast significant increases in mRNA levels compared to the sham condition were seen for neural cells derived from the alcoholic subject group for NMDA subunit genes (A) GRIN1, (B) GRIN2A, and (D) GRIN2D, while a trend towards significance was observed for (C) GRIN2B (p=0.06). Scatter plot symbols represent results from individual culture wells together with mean and SEM (significance level compared with sham condition * < 0.05, ** <0.01, *** <0.001).

Variability Between Subjects in NMDA Subunit mRNA changes following Alcohol Exposure and WithdrawalIndividual subject data normalized to each subject’s average sham NMDA receptor gene expression normalized to GUSB. iPS-derived neural cells from non-alcohol dependent (Non-AD) subjects are indicated on the left side of the x-axis, while neural cell generated from alcohol-dependent (AD) subjects are indicated on the right. Only one non-AD subject demonstrated an increase in NMDA mRNA expression following 7 days of alcohol exposure. In contrast, under these conditions, all four AD subjects demonstrated increased expression of GRIN1, which returned to baseline levels in three of the four subjects. Three AD subjects demonstrated increased GRIN2A expression, which continued to increase after 24 hours of withdrawal in all three subjects. Two AD subjects showed increases in GRIN2B and GRIN2D expression; with the exception of GRIN2B in one AD subject, these changes returned to baseline after withdrawal. (Symbols: open bars − sham condition; solid bars − 7-day of alcohol exposure; gray bars − 7-day alcohol + 24-hour withdrawal; error bars represent SEM).