Altered Sodium and Potassium, but not Calcium Currents in Cerebellar Granule Cells in an In Vitro Model of Neuronal Injury

Abstract

Acute injury of central nervous system (CNS) starts a cascade of morphological, molecular, and functional changes including formation of a fibrotic scar, expression of transforming growth factor beta 1 (TGF-β1), and expression of extracellular matrix proteins leading to arrested neurite outgrowth and failed regeneration. We assessed alteration of electrophysiological properties of cerebellar granule cells (CGCs) in two in vitro models of neuronal injury: (i) model of fibrotic scar created from coculture of meningeal fibroblasts and cerebral astrocytes with addition of TGF-β1; (ii) a simplified model based on administration of TGF-β1 to CGCs culture. Both models reproduced suppression of neurite outgrowth caused by neuronal injury, which was equally restored by chondroitinase ABC (ChABC), a key disruptor of fibrotic scar formation. Voltage-dependent calcium current was not affected in either injury model. However, intracellular calcium concentration could be altered as an expression of inositol trisphosphate receptor type 1 was suppressed by TGF-β1 and restored by ChABC. Voltage-dependent sodium current was significantly suppressed in CGCs cultured on a model of fibrotic scar and was only partly restored by ChABC. Administration of TGF-β1 significantly shifted current-voltage relation of sodium current toward more positive membrane potential without change to maximal current amplitude. Both transient and sustained potassium currents were significantly suppressed on a fibrotic scar and restored by ChABC to their control amplitudes. In contrast, TGF-β1 itself significantly upregulated transient and did not change sustained potassium current. Observed changes of voltage-dependent ion currents may contribute to known morphological and functional changes in injured CNS.

Keywords: Calcium current; Cerebellar granule cells; Chondroitinase ABC; Fibrotic scar; Potassium current; Sodium current; TGF-β1.

Fig. 1

Altered CGCs neurite length in in vitro models of neuronal injury. a Examples of CGCs cultured under the control conditions (left panel), on an in vitro model of fibrotic scar (middle panel), and on an in vitro model of fibrotic scar with ChABC added (right panel). In the middle panel, formed scar is visible. It disappeared in the presence of ChABC. Scale bar represents 200 µm. b An average length of neurite cultured under the control conditions (n = 5 preparations, 30 neurons per preparation), on an in vitro model of fibrotic scar (n = 5 preparations, 30/29/29/30/30 neurons in individual preparations), and on an in vitro model of fibrotic scar with ChABC added (n = 5 preparations, 27/29/25/30/30 neurons in individual preparations), as marked. ***p < 0.001; F = 157.2. c Examples of CGCs cultured under the control conditions (left panel), in the presence of TGF-β1 (middle panel), and in the presence of TGF-β1 with ChABC (right panel). Scale bar represents 200 µm. d An average length of neurite cultured under the control conditions (n = 5 preparations, 30/30/29/29/29 neurons in individual preparations), in the presence of TGF-β1 (n = 5 preparations, 25/30/29/26/27 neurons in individual preparations), and the presence of TGF-β1 with ChABC (n = 3 preparations, 30 neurons in each preparation), as marked. ***p < 0.001, F = 69.2

Fig. 2

Sodium currents measured in both in vitro models of neuronal injury. a I–V relations for I _Na measured from CGCs cultured under the control conditions (open circle, n = 10), on an in vitro model of fibrotic scar (closed circle, n = 11), and on an in vitro model of fibrotic scar with ChABC added (closed triangle, n = 11). *p < 0.05; F = 5.2. Examples of current traces measured by depolarization pulse to 0 mV are shown below the graph. b I–V relations for I _Na measured from CGCs cultured under the control conditions (open circle, n = 14), and after 72 h exposure to TGF-β1 (closed circle, n = 14). **p < 0.01, ***p < 0.001 compared to the control. Examples of current traces measured by depolarization pulse to 0 mV are shown below the graph

Fig. 3

Potassium currents measured on an in vitro fibrotic scar model. a Representative examples of potassium currents measured from CGCs seeded in control conditions, on an in vitro fibrotic scar model, and on an in vitro fibrotic scar model with ChABC added, as marked. Arrows indicate at which time point a peak current amplitude and a steady-state current amplitude were measured. b I–V relations for a peak current amplitudes measured from CGCs cultured under the control conditions (open circle, n = 21), on an in vitro model of fibrotic scar (closed circle, n = 18), and on an in vitro model of fibrotic scar with ChABC added (closed triangle, n = 19). ***p < 0.001; F = 16.6 at +80 mV. c I–V relations for a steady-state current amplitudes evaluated from the same CGCs as in the panel b. **p < 0.01; F = 7.6 at +80 mV

Fig. 4

Potassium currents measured after a TGF-β1 treatment. a Representative examples of potassium currents measured from CGCs cultured under the control conditions, and CGCs to which TGF-β1 was added for 72 h, as marked. Peak current amplitude and a steady-state current amplitude were measured at a time point indicated by an arrow. b I–V relations for a peak current amplitudes measured from CGCs cultured under the control conditions (open circle, n = 15), and in the presence of TGF-β1 (closed circle, n = 14). **p < 0.01; F = 5.9 c I–V relations for a steady-state current amplitude evaluated from the same CGCs as in the panel (b)

Fig. 5

Calcium currents measured in both in vitro models of neuronal injury. a I–V relations for I _Ca measured from CGCs cultured under the control conditions (open circle, n = 10), on an in vitro model of fibrotic scar (closed circle, n = 10), and on an in vitro model of fibrotic scar with ChABC added (closed triangle, n = 11). Examples of current traces measured by depolarization pulse to 0 mV are shown on the right. b I–V relations for I _Ca measured from CGCs cultured under the control conditions (open circle, n = 14), and after 72 h exposure to TGF-β1 (closed circle, n = 18). Examples of current traces measured by depolarization pulse to 0 mV are shown on the right

Fig. 6

Relative comparison of mRNA expression levels for IP₃R1 evaluated in respect to mRNA expression level for housekeeper gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) under the control conditions, after 72 h exposure to TGF-β1, and TGF-β1 + ChABC, as marked. For each column number of independent preparations averaged was n = 3. ** p < 0.01; F = 16.5 versus control. Representative example of a gel is shown on the top. All PCR products were analyzed on 2 % agarose gels. Optical density (od/mm²) of individual bands was measured by Kodak camera and analyzed by PCBAS 2.08e software (Düsseldorf, Germany). Relative quantification was done relatively to housekeeper GAPDH. Positions in a gene for GAPDH (GAPDH1 795–814 and GAPDH2 506–525, overall size 309 bp) and IP₃R1 (IP₃R1A 70–90 and, IP₃R1B 573–593, overall size 535 bp)