Electrical Stimulation Evokes Rotational Behavior In Tandem with Exocytotic-like Increases in Dopamine Measured by In Vivo Intracerebral Microdialysis

Abstract

Background: Electrical Stimulation is a traditional tool in neuroscience and is commonly used in vivo to evoke behavior and in vitro to study neural mechanisms. In vivo intracerebral microdialysis, also a traditional technique, is used to assay neurotransmitter release. However, the combination of these techniques is highly limited to studies using anesthetized animals; therefore, evoking and measuring exocytotic neurotransmitter release in awake models is lacking. Combining these techniques in an awake animal preparation is presented here with evidence to support the mechanistic action of electrical stimulation in vivo.

New methods: This report presents converging evidence to validate the combination of intracerebral electrical stimulation with microdialysis as a novel procedure to study exocytotic-like dopamine release in behaving animals.

Results: It is shown that electrical stimulation of the medial forebrain bundle can be used to evoke frequency- and intensity-dependent exocytotic-like dopamine overflow and rotational behavior that are sensitive to Na⁺ channel blockade and Ca⁺⁺ availability.

Comparison with existing methods: Studies using modern techniques to evoke neurotransmitter release, combined with in vivo intracerebral microdialysis, and measured behavioral output are scarce. In contrast, commonly used pharmacological methods often are less precise and inefficient to evoke exocytotic dopamine release and behavior. Here we demonstrate, the combination of in vivo intracerebral microdialysis with electrical stimulation as a simple approach to simultaneously assess physiologically relevant neurotransmitter 'release' and behavior.

Conclusions: Research that aims to understand how dopamine neurotransmission is altered in behavioral disorders can utilize this innovative combination of electrical stimulation with in vivo intracerebral microdialysis.

Keywords: Electrical stimulation; calcium; exocytosis; nigrostriatal dopamine; rotational behavior; sodium channels.

Figure 1.

Experimental timeline. See text for full description.

Figure 2.

Integration of Electrical and Fluid Commutators to Prevent Tangling During Movement. The image depicts (A) the plexiglas swivel flange (Plastics One, Model FSM-1) placed immediately above the testing chamber that integrates (B) the fluid commutator (Instech, Model 375/D/22QM) used to deliver Ringers for IVMCD and (C) electrical commutator (Plastics One, Model SL2C) used for ES.

Figure 3.

Histological Verification of Accurately Placed Microdialysis Probes, Cannulae and Electrode Implants. Schematics along the coronal plane, mm AP to bregma (Paxinos & Watson, 2006), illustrate successful placement of A) dialysis probes, B) infusion cannulae, and C) electrodes, in 8 out of 9 animals from experiment 1B. Animals with inaccurate placements were excluded from the study and not depicted in this figure.

Figure 4.

The Linear Relationship of ESRB with Increasing Intensity of ES and Na⁺ Channel Dependence. A) ESRB was measured in quarter turns evoked by increasing intensity (10-sec trains, 2-min ISI) and averaged across five days (± SEM); see Methods, a priori criterion (n=22). ESRB contraversive to the stimulated hemisphere increased as intensity was increased from 50–300 μA in 50 μA intervals (r² = 0.99, p < 0.0001). B) Similar to Figure 4A, ESRB (Mean ± SEM); was measured following infusions of 6 μl of saline (●, n=11) or 4% lidocaine (○, n=11). Na⁺ channel blockade significantly decreased ESRB at 200, 250 and 300 μAs (∗, p’s< 0.05) compared to saline control.

Figure 5.

Na+ Channel Dependence of Electrically Evoked DA and ESRB. A) Extracellular levels of DA (fmol/min, Mean ± SEM) were collected across the following timeline (see X-axis): during baseline (B), a 10-min collection interval during which ES (|||||; 5 X 10-sec trains, 2-min ISI) was applied (1), and two additional 10-min intervals to establish a response timeline (2,3). Animals (n=9, within subjects) were tested under two treatment conditions: during control conditions (Before Lidocaine, ●) and following treatment (After Lidocaine, ○). During baseline measures (B), lidocaine decreased spontaneous levels of extracellular DA compared to control conditions (∗, p< 0.05). ES evoked a significant increase in extracellular DA from the control baseline condition (, p< 0.05) that returned to baseline levels immediately upon termination of ES. In the lidocaine condition, there were no significant differences across time. Na⁺ channel blockade significantly attenuated ES-evoked extracellular DA compared to control conditions (∗, p< 0.05). B) ESRB (Mean ± SEM) was measured in quarter turns during each 10-sec train of ES applied during the 10-min dialysis interval immediately following baseline measures (see Interval 1 of X-Axis in Fig. 5A). Prior to lidocaine (●) all animals demonstrated vigorous contraversive turning in response to ES. Lidocaine (○) significantly reduced ESRB compared to control conditions (∗, p< 0.05). Note that the rate of turning was sustained across the 5 trains of ES in both conditions.

Figure 6.

Na⁺ Channel Blockade Enhances ES-evoked Monoamine Metabolite Overflow. Extracellular levels of monoamine metabolites (fmol/min, Mean ± SEM) were measured during baseline (B), a 10-min collection interval during which ES (|||||; 5 X 10-sec trains, 2-min ISI) was applied (1), and two additional 10-min intervals to establish a response timeline (2,3). Animals were tested under two treatment conditions: during control conditions (Before Lidocaine, ●) and following treatment (After Lidocaine, ○). During baseline measures (B), lidocaine did not alter spontaneous levels of extracellular metabolites compared to control conditions. A) DOPAC: Regardless of treatment condition, ES increased overall DOPAC overflow (average of samples 1, 2, 3) compared to baseline levels (planned T tests, p’s ≤ 0.053; data not shown, see Results). Extracellular DOPAC levels were enhanced by lidocaine during and after ES compared to the control condition (Sidak, ∗, p’s< 0.05, pairwise comparison within each time interval). B) HVA: In the control condition, HVA levels increased significantly during the last post-ES sample (3) compared to sample 1 during which ES was applied (, p<0.05). After lidocaine, ES increased overall HVA overflow (average of samples 1, 2, 3) compared to baseline levels (planned T test, p < 0.05; data not shown, see Results). Extracellular HVA levels were enhanced by lidocaine during and after ES compared to the control condition (Sidak, ∗, p’s< 0.05, pairwise comparison within each time interval). C) 5HIAA: In the control condition, 5HIAA levels did not change across repeated sampling. In contrast, after lidocaine, ES increased overall 5HIAA overflow (average of samples 1, 2, 3) compared to baseline levels (planned T test, p < 0.05; data not shown, see Results). These statistical analyses reveal a main effect by lidocaine and an interaction effect by lidocaine and ES to enhance extracellular 5HIAA levels compared to the control condition (Sidak, ∗, p’s< 0.05, pairwise comparison within each time interval).

Figure 7.

ES-evoked Intensity- and Frequency-Dependent DA Overflow and ESRB. DA overflow and ESRB were simultaneously measured during two 10-min dialysis collection intervals in which ES was differentially applied (5 X 10-sec trains, 2-min ISI). Extracellular DA (Panels A, C) and ESRB (Panels B, D) were converted to percent of spontaneous baseline levels (Mean ± SEM. A & B) To assess the effects of intensity upon ES-induced DA overflow (Panel A) and ESRB (Panel B), frequency was held constant at 50 Hz and intensity was set to 150 μA and subsequently to 300 μA. There was a significant intensity-dependent increase in DA overflow and ESRB (∗, p’s < 0.05, n=6). C & D) To assess the effects of frequency upon ES-induced DA overflow (Panel C) and ESRB (Panel D), intensity was held constant at 300 μA and frequency was set to 25 Hz and subsequently to 50 Hz. There was a significant frequency-dependent increase in DA overflow and ESRB (∗, p’s < 0.05, n=6).

Figure 8.

Ca⁺⁺ Dependence of Electrically Evoked DA and ESRB. DA overflow and ESRB were simultaneously measured during two 10-min dialysis collection intervals in which ES was applied (5 X 10-sec trains, 2-min ISI). Extracellular DA (A) and ESRB (B) were converted to percent of spontaneous baseline levels (Mean ± SEM). A) To test for Ca++ dependence of ES-evoked DA, ES was applied during control conditions (0.0 mM EGTA) and again with 13.5 mM EGTA. There was a significant decrease in DA overflow after Ca⁺⁺ availability was depleted (∗,.p < 0.05, n=5). B) To test for Ca⁺⁺ dependence of ESRB, ES was applied during control conditions (0.0 mM EGTA) and again with 13.5 mM EGTA. There was a significant decrease in ESRB after Ca⁺⁺ availability was depleted (∗,.p < 0.05, n=5).