Electrical excitability of early neurons in the human cerebral cortex during the second trimester of gestation

Abstract

Information about development of the human cerebral cortex (proliferation, migration, and differentiation of neurons) is largely based on postmortem histology. Physiological properties of developing human cortical neurons are difficult to access experimentally and therefore remain largely unexplored. Animal studies have shown that information about the arousal of electrical activity in individual cells within fundamental cortical zones (subventricular zone [SVZ], intermediate zone, subplate [SP], and cortical plate [CP]) is necessary for understanding normal brain development. Here we ask where, in what cortical zone, and when, in what gestational week (gw), human neurons acquire the ability to generate nerve impulses (action potentials [APs]). We performed electrical recordings from individual cells in acute brain slices harvested postmortem from the human fetal cerebral cortex (16-22 gw). Tetrodotoxin-sensitive Na(+) current occurs more frequently among CP cells and with significantly greater peak amplitudes than in SVZ. As early as 16 gw, a relatively small population of CP neurons (27%) was able to generate sodium APs upon direct current injection. Neurons located in the SP exhibited the highest level of cellular differentiation, as judged by their ability to fire repetitive APs. At 19 gw, a fraction of human CP and SP neurons possess beta IV spectrin-positive axon initial segments populated with voltage-gated sodium channels (PanNav). These results yield the first physiological characterization of developing human fetal cortical neurons with preserved morphologies in intact surrounding brain tissue.

Figure 1.

Cellular and molecular composition of the human fetal cortex. (A) Hoechst staining of an acute cortical slice taken from the lateral telencephalic wall of the human fetal cortex. Scale bar: 100 μm. (B) Live infrared video microscopy of cortical cells in CP (top), IZ (middle), and SVZ (bottom). Scale bar: 20 μm. (C) Cells in the CP (top) and SVZ (bottom), labeled with GFAP (red) and MAP2 (green). Scale bar: 20 μm.

Figure 2.

Morphological and passive physiological properties of SVZ and CP cells. (A) Rhodamine-filled cells in CP (top) and SVZ (bottom). Arrows mark cellular processes. Scale bar: 5 μm. Actual current traces for these 4 cells are displayed in Supplementary Fig. 1. (B) SVZ cells (gray) typically had 1 or 2 long process, whereas CP cells (black) have 2 or more, shorter processes. n.d.a., no data available due to no dye filling or loss of cell prior to sufficient filling time. (C) CP cells, on average, have higher input resistance than SVZ cells. (D) Peak I_Na amplitudes in SVZ cells with 1–5 processes. “n” Values indicate number of cells recorded with zero sodium current at each process. For clarity, 0 process (n = 3) and 1 process (n = 4 at zero I_Na) were omitted from the graph. Note that SVZ cells with detectable I_Na, greater than 0, typically had 1 main process. (E) Peak sodium current amplitudes for CP cells with 1–5 primary processes. Note, CP cells endowed with an I_Na greater than 0 typically had 2–4 primary processes.

Figure 3.

Voltage-gated sodium and potassium current densities. (A) Sodium currents (I_Na, arrows) recorded in the human SVZ and CP can be roughly divided into 4 ranges: zero (0 pA), small (<200 pA), medium (200–500 pA), and large (>500 pA). Note that I_Na amplitudes greater than 200 pA were found in CP but not in the SVZ. (B, C) For each gw examined, we plotted the fraction of cells with detectable I_Na (white) with the total number of cells recorded (black) in the SVZ (B) and CP (C). (D) Average current densities (pA/pF) in the SVZ (gray) and CP (black). (E) Average I_Na density between the SVZ (gray) and CP (black) at different gestational ages. (F) Same as in (E) except I_K density is analyzed across gw. Values are expressed as mean ± standard error of the mean in (D), (E), and (F). Asterisks denote statistically significant differences between SVZ and CP (P < 0.05). Stars in (E) and (F) indicate significant difference between 16 and 22 gw in the CP only (P < 0.05).

Figure 4.

CP cells fire sodium APs. (A) Voltage waveforms in the human cerebral cortex during the second trimester of gestation. Labels indicate cortical zones where selected voltage waveforms (passive, abortive, full-size, or repetitive AP) were observed. Note that all cells in SVZ showed passive responses upon current depolarization. (B) First derivative (dV/dt) of the voltage waveforms. Cells with a nonlinear voltage waveform (active membrane response, spike) showed a large negative component in dV/dt (arrow), more negative than −2 V/s. Cells with a passive membrane response had very small or zero negative component. (C) Amplitude of the negative component of dV/dt is plotted versus the peak sodium current (I_Na) obtained in the same CP neuron (n = 107), on a semilogarithmic scale. Horizontal line indicates cutoff (−2 V/s) for passive response (Table 2). (D) Same data as in (C) plotted on a linear scale. Horizontal dashed line depicts the cutoff amplitude of I_Na (−222 pA). Cells exhibiting an I_Na amplitude greater than −222 pA have a nonlinear membrane response to direct depolarization (abortive or full-size AP) and, therefore, can be identified as neuronal progeny. All data in (C) and (D) are negative values; minuses before labels for x- and y-axes are omitted for clarity.

Figure 5.

Biophysical properties of voltage-gated currents. (A) Isolated sodium current (I_Na) from a human CP neuron at 20 gw. Inset, current (I)–voltage (V) relationship (average of 6 cells). (B) Activation and inactivation curves for I_Na fitted with a Boltzmann equation. The half-activation voltage (V_½Act) was −41.3 mV and V_½Inact = −38 mV. (C) Isolated potassium current (TTX subtracted) from a cell in the CP capable of firing a sodium AP. (D) Activation plot of potassium current from cells in the SVZ with zero sodium current (squares) and cells in the CP with large sodium current amplitudes (circles). V_½Act SVZ = −6 mV and V_½Act CP = +17 mV.

Figure 6.

AIS in the second trimester of gestation. (A) The highest density of βIV spectrin–positive AIS (green) was located in the CP with a fraction of these AIS also positive for PanNav (sodium channels, red). (B) A small number of AIS were found in the SP. In contrast to CP, SP AIS were radially oriented and often copopulated with voltage-gated sodium channels (PanNav, arrows). (C) Axon initial segments were not present in the SVZ based on the absence of βIV spectrin and voltage-gated sodium channels. (D) Higher magnification of the colocalization of βIV spectrin (green) and PanNav channels (red) at AIS. Scale bars in (A–C): 10 μm and (D): 5 μm. Blue, Hoechst staining of cell nuclei.

Figure 7.

Human cortical development—an electrophysiological view. Infrared differential interference contrast video microscopy of acute human fetal cortical slices reveals 3 distinct zones: 1) dense cellular layer near the pial surface (CP); 2) scarce cellular area filled with radial fibers (the IZ and SP); and 3) a second dense cellular area near the lateral ventricle (VZ and SVZ). Upon direct current injection, SVZ cells were unable to generate an AP showing only a passive response (passive). From the SVZ, young neurons migrate through the IZ and SP to their final destination in the CP. Cells in the IZ were mostly bipolar (blue inset). The majority of IZ cells are unable to generate full-size APs. The most mature physiological cells recorded in this study were found in the SP, positioned just below the CP, and above the IZ. Here young neurons were capable of firing repetitive APs and had radially oriented AIS (green) copopulated with voltage-gated sodium channels (red dots). The greatest diversity of physiological properties existed in the CP. Cells of the CP had passive (no AP), abortive, full, or repetitive AP firing (top boxes). CP cells endowed with AP generation (abortive or full) exhibited multipolar morphology (blue inset). Some but not all CP cells posses AIS. A fraction of AIS in CP were copopulated with voltage-gated sodium channels. Unlike in the SP, in the CP, AIS appeared randomly oriented.