In Vivo Ambient Serotonin Measurements at Carbon-Fiber Microelectrodes

Abstract

The mechanisms that control extracellular serotonin levels in vivo are not well-defined. This shortcoming makes it very challenging to diagnose and treat the many psychiatric disorders in which serotonin is implicated. Fast-scan cyclic voltammetry (FSCV) can measure rapid serotonin release and reuptake events but cannot report critically important ambient serotonin levels. In this Article, we use fast-scan controlled adsorption voltammetry (FSCAV), to measure serotonin's steady-state, extracellular chemistry. We characterize the "Jackson" voltammetric waveform for FSCAV and show highly stable, selective, and sensitive ambient serotonin measurements in vitro. In vivo, we report basal serotonin levels in the CA2 region of the hippocampus as 64.9 ± 2.3 nM (n = 15 mice, weighted average ± standard error). We electrochemically and pharmacologically verify the selectivity of the serotonin signal. Finally, we develop a statistical model that incorporates the uncertainty in in vivo measurements, in addition to electrode variability, to more critically analyze the time course of pharmacological data. Our novel method is a uniquely powerful analysis tool that can provide deeper insights into the mechanisms that control serotonin's extracellular levels.

Figure 1

(A) Representative FSCV (i) and FSCAV (ii) color plots of 100 nM serotonin in vitro. (B) Cyclic voltammograms extracted from the vertical dashed lines in A(i) and A(ii) after normalization (current/maximum current). Vertical orange dashed lines represent integration limits.

Figure 2

Repeated FSCAV measurements over 120 min in 100 nM serotonin (n = 4 electrodes ± SEM).

Figure 3

In vitro CVs for HA (1 μM), adenosine (1 μM), DOPAC (2 μM), NE (1 μM), UA (1 μM), DA (100 nM), AA (200 μM), H₂O₂ (1 mM), and 5-HIAA (10 μM). Vertical dashed lines represent integration limits utilized for serotonin analysis.

Figure 4

Serotonin selectivity curve (n = 4 electrodes ± SEM). The inset shows linear serotonin range (orange markers), the green stars represent the addition of 5-HIAA to serotonin. All blue markers represent serotonin/5-HIAA mixture with 5-HIAA being a 100 times the serotonin concentration. All inset calibrations are n = 4 electrodes ±SEM.

Figure 5

(A) Representative FSCAV color plots of serotonin in vivo (i) and in vitro (ii). (B) CVs extracted from the 3rd scan indicated by vertical dashed lines in A(i) and A(ii). The inset shows ambient serotonin measurements in CA2 region of mouse hippocampus. Gray markers represent individual mice and orange marker represents weighted averaged response (n = 15 mice ± standard error).

Figure 6

Faint blue markers represent individual mouse responses to i.p. Pargyline (75 mg kg⁻¹) and faint red markers represent individual mice responses to i.p. GBR 12909 (15 mg kg⁻¹). Files were collected 60 min before and after drug administration. Dark blue dots represent averaged Pargyline response (n = 5 mice ± SEM) and dark red dots represent averaged GBR 12909 response (n = 5 mice ± SEM). Yellow bar at 0 min is injection time. Representative FSCV color plots and CVs before and after FSCAV file collection are inset (top, pargyline; bottom, GBR 12909, α = predrug and β = postdrug ). White bars at bottom of color plot denotes stimulation (2 s). Inset center shows [serotonin] vs time traces taken from color plots). Red bars below [serotonin] vs time is the stimulation. (Asterisks above solid blue markers indicate post hoc test: *p < 0.05 and ****p < 0.0001.)

Figure 7

Faint green markers represent individual mice responses and dark green dots represent averaged response to i.p. Desipramine (15 mg kg⁻¹) (n = 5 mice ± SEM). Files were collected 60 min before and after drug administration. Yellow bar at 0 min is injection time. Representative FSCV color plots and CVs before and after FSCAV file collection are inset (α = predrug and β = postdrug). White bars at bottom of color plot denotes time of stimulation (2 s). Inset center are representative [serotonin] vs time traces of evoked serotonin response before (black) and after (green) drug administration. Red bars below [serotonin] versus time is stimulation period (2 s).

Figure 8

Files were collected 60 min before and after (A) pargyline, (B) GBR12909, and (C) desipramine administration. Circles represent averaged serotonin response (n = 5 mice ±95% CI). Vertical gray lines represent 95% confidence intervals, and the blue line is the fitted model. Red vertical line in A represent point of change after drug administration, that is, 2.60 min.