Deep brain stimulation results in local glutamate and adenosine release: investigation into the role of astrocytes

Abstract

Background: Several neurological disorders are treated with deep brain stimulation; however, the mechanism underlying its ability to abolish oscillatory phenomena associated with diseases as diverse as Parkinson's disease and epilepsy remain largely unknown.

Objective: To investigate the role of specific neurotransmitters in deep brain stimulation and determine the role of non-neuronal cells in its mechanism of action.

Methods: We used the ferret thalamic slice preparation in vitro, which exhibits spontaneous spindle oscillations, to determine the effect of high-frequency stimulation on neurotransmitter release. We then performed experiments using an in vitro astrocyte culture to investigate the role of glial transmitter release in high-frequency stimulation-mediated abolishment of spindle oscillations.

Results: In this series of experiments, we demonstrated that glutamate and adenosine release in ferret slices was able to abolish spontaneous spindle oscillations. The glutamate release was still evoked in the presence of the Na channel blocker tetrodotoxin, but was eliminated with the vesicular H-ATPase inhibitor bafilomycin and the calcium chelator 2-bis(2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetrakis acetoxymethyl ester. Furthermore, electrical stimulation of purified primary astrocytic cultures was able to evoke intracellular calcium transients and glutamate release, and bath application of 2-bis (2-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetrakis acetoxymethyl ester inhibited glutamate release in this setting.

Conclusion: Vesicular astrocytic neurotransmitter release may be an important mechanism by which deep brain stimulation is able to achieve clinical benefits.

Figure 1

High Frequency Stimulation (HFS) abolishes network oscillations in the ferret thalamus (A) Spontaneous spindle oscillations in a ferret thalamic slice preparation. Boxed areas indicate segments during which HFS was applied (a,b). Enlargements of segments from the top trace during HFS (duration ∼3s, frequency 100Hz, intensity 300μA, pulse width 100μs) showing absence of spindle wave following HFS. Spontaneous spindle waves return 20-25s following HFS. (B) Schema of thalamic slice preparation demonstrating placement of stimulating and extracellular recording electrodes in the ferret lateral geniculate nucleus (LGN), within ∼100μm of each other.

Figure 2

HFS in the ferret thalamic slice results in glutamate release that is not blocked by the classic neuronal exocytosis inhibitor tetrodotoxin. HFS of the LGN (100Hz, 100μs pulse width, 300μA) for 10s in the in vitro ferret thalamic slice abolished spindles waves in the post-stimulation period (A, upper trace) and resulted in a concurrent increase in extracellular glutamate as measured by an enzyme-linked glutamate sensor (A, lower trace). After tetrodotoxin (TTX, 2μM) was applied to the bath, neuronal activity was abolished (B, upper trace); however, an increase in extracellular glutamate was still recorded by the glutamate sensor (B, lower trace). The stimulating electrode and the glutamate sensor electrode were positioned within ∼100μm of each other.

Figure 3

HFS induces adenosine efflux in the in vitro ferret thalamic slice. (A) Voltage waveform for measuring adenosine. (B) The pseudo-color plot shows adenosine efflux by HFS (125Hz, 200μA, for 5s, 100μs pulse width), detected with WINCS-based FSCV at a CFM. The axes and color gradient indicate the time, the potential waveform applied at the CFM, and the currents detected from the CFM, respectively. Adenosine exhibits unique voltammetric oxidation peaks; the green oval surrounded by the purple ring (at approximately +1.5V) and the purple oval (at approximately +1.0V) represent the 1^st and 2^nd oxidation peaks of adenosine, respectively. (C) Black and green dotted horizontal lines in (B) indicate 1^st and 2^nd adenosine oxidation peak currents versus time. This current-time plot clearly shows adenosine efflux induced by HFS. (D) Black and red solid vertical lines in (B) refer to the relationship between background charging currents (before and after HFS, respectively) and applied voltages. A large background current is present at the CFM (Black solid line, see also inset). HFS increased this background current only slightly (red solid line, see also inset). Black-lined box indicates the source of the data shown in the expanded area. (E) A cyclic-voltammogram was obtained by background subtraction (subtracted black line-indicated currents from red line-indicated currents in (D)). A representative cyclic voltammogram shows the 1^st and 2^nd oxidation peaks at +1.5 and +1.0V, respectively. The green line indicates the differential oxidation peak obtained by forward-going potential from -0.4 to +1.5V, and black line by reverse-going potential from +1.5 to -0.4V.

Figure 4

Glutamate measurements made in the in vitro ferret thalamic slice demonstrate that HFS-induced glutamate release is vesicular and calcium-dependent. (A) Tetrodotoxin (TTX) treatment has no effect on glutamate release. (B) HFS-induced glutamate release is suppressed by treatment with the calcium chelator, BAPTA-AM (50μM, 1h; n=3). (C) Bath application of Bafilomycin (1μM, 45min; n=9), an inhibitor of vacuolar type H⁺-ATPase, in addition to TTX, resulted in inhibition of glutamate release. (D) The anion-channel blocker NPPB (100μM, 10min), does not inhibit HFS-induced glutamate release in the thalamic slice. The stimulating electrode and the glutamate sensor electrode were positioned within ∼100μm of each other.

Figure 5

HFS induces glutamate release in primary astrocyte cultures. (A) Immunocytochemistry of a primary astrocytic culture revealed that > 98% of the cells were astrocytes as shown by GFAP staining. (B) High frequency stimulation (HFS) of the astrocytic culture (100Hz, 100μs pulse width, 300μA) for 10 seconds resulted in an increase in extracellular glutamate as measured by an enzyme-linked glutamate sensor. Astrocytic glutamate release was both frequency (C) and intensity (D) dependent. The stimulating electrode and the glutamate sensor electrode were positioned within ∼100μm of each other.

Figure 6

High frequency stimulation induces Ca²⁺ release from cultures astrocytes. (A) High frequency stimulation induced calcium increases as indicated by the fluorescent dye, fluo-4. (B) The fluorescence intensity for several individual cells, designated in (A), was monitored following HFS and demonstrates transient increases in a unique cell-specific pattern. (C) Mean calcium increase monitored from multiple cells. Note HFS was applied for 10s beginning at 15s with the following parameters: 100Hz, 100μs pulse width, 300μA. (D) HFS-induced glutamate release was significantly decreased (**P<0.01 vs. control) following chelation of calcium with BAPTA-AM (50μM, 45min) suggesting a calcium-dependent mechanism of release.