Human Cerebrospinal Fluid Induces Neuronal Excitability Changes in Resected Human Neocortical and Hippocampal Brain Slices

Abstract

Human cerebrospinal fluid (hCSF) has proven advantageous over conventional medium for culturing both rodent and human brain tissue. In addition, increased activity and synchrony, closer to the dynamic states exclusively recorded in vivo, were reported in rodent slices and cell cultures switching from artificial cerebrospinal fluid (aCSF) to hCSF. This indicates that hCSF possesses properties that are not matched by the aCSF, which is generally used for most electrophysiological recordings. To evaluate the possible significance of using hCSF as an electrophysiological recording medium, also for human brain tissue, we compared the network and single-cell firing properties of human brain slice cultures during perfusion with hCSF and aCSF. For measuring the overall activity from a majority of neurons within neocortical and hippocampal human slices, we used a microelectrode array (MEA) recording technique with 252 electrodes covering an area of 3.2 × 3.2 mm². A second CMOS-based MEA with 4225 sensors on a 2 × 2 mm² area was used for detailed mapping of action potential waveforms and cell identification. We found that hCSF increased the number of active electrodes and neurons and the firing rate of the neurons in the slices and induced an increase in the numbers of single channel and population bursts. Interestingly, not only an increase in the overall activity in the slices was observed, but a reconfiguration of the network could also be detected with specific activation and inactivation of subpopulations of neuronal ensembles. In conclusion, hCSF is an important component to consider for future human brain slice studies, especially for experiments designed to mimic parts of physiology and disease observed in vivo.

Keywords: CMOS-MEA; cortex; hippocampus; human cerebrospinal fluid; human tissue; microelectrode array; organotypic slices.

FIGURE 1

Experimental and overview of activity. (A) The brain tissue is surgically resected en bloc and the tissue block (top) is cut into 250-μm slices (middle) using a vibratome. The slices are incubated on membrane insets in six-well plates (bottom) with hCSF as medium for up to 2 weeks. (B) Pictures of the 256-MEA chip (top), the CMOS-MEA chip (middle), and the spacing of the CMOS-MEA electrodes (bottom). (C) Experimental protocol. Slices were perfused with aCSF for an acclimation period of 30 min, followed by 60-min aCSF (blue bar), 60-min hCSF (red bar), and again 60-min aCSF (blue bar) perfusion. Recording was performed continuously for 30 min in aCSF, hCSF, and aCSF, as indicated. Underneath, a typical human slice culture is shown with all cortical layers (L1–L6) and overlay of the MEA on the slice. (D) Qualitative overview over all 12 slices recorded using 256-MEA. When hCSF was washed in, the general activity of slices increased, with new active areas emerging and with the already active areas showing a further increase in number of spikes presented here as heat maps. Each electrode is represented by a small square and darker color indicates higher activity (spike rate). Activity was normalized for each slice to the maximum spike rate per electrode in aCSF.

FIGURE 2

hCSF induces a general increase in neuronal activity and specific activation and inactivation of subpopulations of neurons. (A) The general activity measured by the number of active electrodes and spike rate, with an average of each hippocampus slice represented by a circle and each cortex slice as a triangle, increased when hCSF was washed in and then decreased again when hCSF was washed out. The average number of electrodes and spike rate in one cortical slice did not increase but decreased instead, this can also be seen in the corresponding heat map in Figure 1D (cortex slice 3). (B) Overview presenting identified electrodes recording activity only in hCSF (dark green), electrodes recording increased spike rate in hCSF (green), recording a decreased spike rate in hCSF (light red), electrodes only active in aCSF (red), and electrodes recording stable activity (for which spike rate increased or decreased by less than 50%). The majority of the electrodes only recorded spikes in hCSF or measured an increased spike rate in hCSF (522 and 186 electrodes, respectively, of a total of 1250 electrodes). Interestingly, 105 electrodes only recorded activity in aCSF and 141 electrodes recorded a decreased spike rate in hCSF compared to aCSF. Examples of electrode recording revealing (C) increased activity and (D) decreased activity when hCSF was washed in. Changed excitability was also detected in extracellular recordings using the CMOS-MEA. (E) Overview of identified single-unit activity (n = 73 cells in three slices) with extracellular voltage recorded by a selected sensor showing the increased excitability of one cell in hCSF compared to aCSF. Selected extracellular waveforms of cells active in (F) both conditions and (G) in hCSF or in aCSF only. (H) The waveform width was not different between the spikes recorded from neurons only active in aCSF, only active in hCSF, or active in both (stable); significance is indicated by *p < 0.05; **p < 0.01.

FIGURE 3

Human CSF promotes synchronized activity. (A) The number of bursts detected in single electrodes increased when hCSF was washed in and decreased back toward aCSF baseline levels when hCSF was washed out again (each hippocampus slice represented by a circle and each cortex slice as a triangle). Burst duration did not change when hCSF was washed in. The number of population bursts increased in all cortical slices (triangles) and in two of the hippocampal slices (circles) following hCSF wash in and then decreased in all slices but one (hippocampal) during the washout. The number of cells (measured by number of active electrodes) participating in population bursts increased in all slices during hCSF and then decreased back toward baseline levels during washout. Significance is indicated by ^∗p < 0.05; ^∗∗p < 0.01; ^∗∗∗p < 0.001. (B–D) Representation of different activity patterns recorded in aCSF (left) and hCSF (right) (B) Activity pattern from hippocampus slice 5 (see heat-map in Figure 1D). Upper panels: Summarized spike rate trace. Lower panels: Raster plots showing the activity on all 252 recording channels. Comparison of the raster plots (aCSF vs hCSF) reveals additional active channels in hCSF, which record continuous but not bursting activity. (C) Activity patterns from hippocampus slice 1 (heat-map in Figure 1D). Comparison of the raster plots reveals additional channels in hCSF, which record bursting activity in a new area of the slice (channel number 1–100). (D) Activity patterns from cortex slice 4 (heat-map in Figure 1D). Comparison of the raster plots shows how non-synchronized spiking activity recorded in aCSF becomes highly synchronized in hCSF across the recorded slice area. (E) Activity patterns from cortex slice 3 (heat-map in Figure 1D). Comparison of the raster plots shows a synchronization shift in hCSF with most spikes recorded during the population burst.