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. 2016 Nov 2;3(5):ENEURO.0115-16.2016.
doi: 10.1523/ENEURO.0115-16.2016. eCollection Sep-Oct 2016.

Subsecond Sensory Modulation of Serotonin Levels in a Primary Sensory Area and Its Relation to Ongoing Communication Behavior in a Weakly Electric Fish

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Free PMC article

Subsecond Sensory Modulation of Serotonin Levels in a Primary Sensory Area and Its Relation to Ongoing Communication Behavior in a Weakly Electric Fish

Haleh Fotowat et al. eNeuro. .
Free PMC article

Abstract

Serotonergic neurons of the raphe nuclei of vertebrates project to most regions of the brain and are known to significantly affect sensory processing. The subsecond dynamics of sensory modulation of serotonin levels and its relation to behavior, however, remain unknown. We used fast-scan cyclic voltammetry to measure serotonin release in the electrosensory system of weakly electric fish, Apteronotus leptorhynchus. These fish use an electric organ to generate a quasi-sinusoidal electric field for communicating with conspecifics. In response to conspecific signals, they frequently produce signal modulations called chirps. We measured changes in serotonin concentration in the hindbrain electrosensory lobe (ELL) with a resolution of 0.1 s concurrently with chirping behavior evoked by mimics of conspecific electric signals. We show that serotonin release can occur phase locked to stimulus onset as well as spontaneously in the ELL region responsible for processing these signals. Intense auditory stimuli, on the other hand, do not modulate serotonin levels in this region, suggesting modality specificity. We found no significant correlation between serotonin release and chirp production on a trial-by-trial basis. However, on average, in the trials where the fish chirped, there was a reduction in serotonin release in response to stimuli mimicking similar-sized same-sex conspecifics. We hypothesize that the serotonergic system is part of an intricate sensory-motor loop: serotonin release in a sensory area is triggered by sensory input, giving rise to motor output, which can in turn affect serotonin release at the timescale of the ongoing sensory experience and in a context-dependent manner.

Keywords: 5-HT; communication behavior; electrosensory; fast-scan cyclic voltammetry; serotonin; weakly electric fish.

Figures

Figure 1.
Figure 1.
Nissl-stained transverse section of ELL, and the schematics of in vitro and in vivo experimental setups. A, The stimulating bipolar electrode and the CFME were placed in the ELL pyramidal cell layer. A triangular waveform (top right) sweeping between −0.1 and +1 V at 1000 V/s was applied to the CFME once every 100 ms. The voltage was kept at 0.2 V between the scans. The background-subtracted CV (bottom right, a representative CV from slice recordings) was calculated for each scan. o, Oxidation peak; r, reduction peak. Arrows point to the temporal order of the points in the CV. B, Sketch of the in vivo setup: Esense-stim was presented to the fish with a carrier frequency (f) equal to the EODf of the fish plus Df. ref, Reference electrode; rec, EOD-recording electrode. Curved gray lines schematize the existence of a global electric field but do not represent the actual, more intricate orientation of the electric field lines.
Figure 2.
Figure 2.
In vitro electrical stimulation in ELL-PCL evokes 5-HT release in the LS and MS, but not CMS. A, Representative average color plot for electrical stimulation trials in LS (left panel, ntrials = 3, nfish = 1) and CMS (right panel, ntrials = 3, same fish). Each trial for electrical stimulation consisted of the application of 24, 0.1-ms-wide, 20 V voltage pulses at 60 Hz. Vertical red bars depict the onset and duration of electrical stimulation. Horizontal and vertical dashed lines correspond to the peak oxidation voltage and time of the peak current, respectively. B, Time-course of the average (±SEM) current at the peak oxidation voltage for stimulation and recording in LS (blue) and CMS (black). Same fish as in A, ntrials = 3 for each region. Oxidation peak 0.76 V (SD = 0.01 V), reduction peak 0.17 V (SD = 0.01 V). Inset depicts the average (±SEM) CV for LS measured at peak current for these trials. C, Representative average color plot for electrical stimulation trials in MS (ntrials = 3, nfish = 1). Each trial for electrical stimulation consisted of the application of 24, 0.1-ms-wide, 60 V voltage pulses at 60 Hz. D, Time-course of the average (±SEM) current at the peak oxidation voltage for stimulation and recording in MS. The locations of the redox peaks were similar to those found in LS [oxidation peak, 0.77 V (SD, 0.02 V); reduction peak, 0.14 V (SD, 0.01 V)]. Inset depicts the average (±SEM) CV. E, Average normalized current measured at peak oxidation voltage before (ACSF control, black) and after incubation in citalopram (Cital., red; nfish = 3, ntrials = 3 per condition). All currents were normalized to the average peak amplitude under control conditions for each fish. Dashed lines show the SEM.
Figure 3.
Figure 3.
In vivo, 5-HT is released spontaneously as well as in response to Esense-stim. A, An example color plot of the time-course of the current response elicited by the presentation of a 20-s-long Esense-stim with Df = 60 Hz. B, C, Example recordings in the same fish in the absence of stimulation (blank trial) without and with spontaneous release, respectively. The color scale bar on the right applies to all color plots.
Figure 4.
Figure 4.
Electrosensory stimuli modulate 5-HT levels in vivo in LS, and not in CMS. A, Representative average time-courses of redox currents in LS (left, ntrials = 7, nfish = 1) and CMS (right, ntrials = 7, same fish) in response to Esense-stim with the same set of Df values. The recordings in CMS were acquired before moving the same electrode to LS. Horizontal and vertical dashed lines correspond to the peak oxidation voltage and time of the peak current, respectively. The thick horizontal bars on top depict the timing and duration of the stimulus. B, Average current at peak oxidation voltage in LS (red) and CMS (black). Dashed lines show the SEM. Inset, Average CV at the time of the peak current in LS [red curve, oxidation peak at 0.69 V (SD, 0.06 V); reduction peak, 0.167 V (SD = 0.03)]. Time zero corresponds to stimulus onset. The pink shaded box corresponds to the duration of the stimulus.
Figure 5.
Figure 5.
Sensory modulation of 5-HT levels in LS is phase locked to Esense-stim. A, Time-course of the normalized 5-HT oxidation peak currents elicited by Esense-stim (black curve, nfish = 9, ntrials = 142) and those that occurred spontaneously in the same group of fish (gray curve, nfish = 9, ntrials = 158). Dashed lines show SEM. Insets on top show the CV at the two peaks indicated by the arrows. Pink shaded box corresponds to the duration of the stimulus. Colored curves show the average (dashed lines, SEM) of the current in response to stimuli with various values of Df, with the color code shown by the color bar. B, Individual blank trials (gray curves) and their average (black curve) in a representative fish. Time zero corresponds to the reference time for background subtraction. C, PDF plot of the AUC for Esense-stim (red filled bars) and blank trials (gray filled bars). Gaussian fits to the distributions: orange: Esense-stim [μ = 3.3 s (SE = 0.39 s), σ = 4.7 s (SE = 0.28 s)]; black: blank [μ =-0.24 s (SE = 0.44 s), σ = 5.6 s (SE = 0.32 s)]. D, PDF plot for the response latency for Esense-stim trials. NR, proportion of the trials with no response. Trials used for generating C and D are the same as those depicted in A.
Figure 6.
Figure 6.
Aud-stim did not modulate 5-HT levels in LS. A, Average normalized peak current (nfish = 4) in response to Esense-stim (red, ntrials = 48) and Aud-stim (blue, ntrials = 18), and during blank trials (black, ntrials = 54). B, The AUC for Aud-stim trials was not significantly different from those of blank trials and was significantly smaller than those for Esense-stim trials. The median is shown with the middle red line; the lower and upper edges of the box plots correspond to the 25th and 75th percentiles; and the whiskers mark the extent of the data points. Outliers are shown as red crosses.
Figure 7.
Figure 7.
Relationship between chirp production and 5-HT response. A, Pie chart depicting the proportion of trials that showed chirp and/or 5-HT responses (nfish = 8, ntrials = 131). 5-HT release was observed equally frequently together with chirp production as without chirp production (39% vs. 43%). Trials without 5-HT release occurred equally often with chirps as without (8% vs. 10%). B, Chirp latency and 5-HT response latency calculated relative to stimulus onset. The first chirp was equally likely to happen before or after 5-HT threshold crossing. The blue diagonal line depicts the unity line. Inset shows the PDF of the delays observed between 5-HT threshold crossing and the first chirp on a trial-by-trial basis (nfish = 8, ntrials = 51). Positive delays correspond to the first chirp occurring before the 5-HT threshold crossing. The mean of the distribution was not significantly different from zero. C, There was a small positive correlation between AUC and |Df| in trials in which the fish chirped (red asterisks, ρ = 0.25, p = 0.04, nfish = 8, ntrials = 64), but not in those in which the fish did not chirp (black circles, ρ = −0.007, p = 0.95, nfish = 8, ntrials = 67). Red line, Linear fit to the AUC of chirp trials: slope = 0.036 s2, intercept = 1.24 s, R 2 = 0.06). Black line, Linear fit to the AUC of no-chirp trials: slope = −0.0009 s2, intercept = 3.62 s, R 2 = 4.5 × 10−5). D, We observed a small, but significant correlation between the number of chirps produced and the AUC of the 5-HT current for stimuli with a |Df| of <20 Hz (red asterisks, ρ = −0.34, p = 0.02, nfish = 8, ntrials = 47), but not for stimuli with a |Df| of ≥20 Hz (black circles, ρ = 0.12, p = 0.27, nfish = 7, ntrials = 84). Red line, Linear fit to the AUC of trials with a |Df| of <20 Hz: slope = −4.37 s, intercept = 3.63 s, R 2 = 0.12). Black line, Linear fit to AUC of no-chirp trials: slope = 1.87 s, intercept = 3.41 s, R 2 = 0.014).
Figure 8.
Figure 8.
The effect of behavioral context on 5-HT response. A, For trials with a |Df| of <20 Hz, the AUC of 5-HT current was significantly smaller in trials in which the fish produced chirps (nfish = 6; chirp trials, ntrials = 14; no-chirp trials, ntrials = 14; pKWT = 0.02). B, Chirping did not affect the 5-HT response to stimuli with large |Df| values (nfish = 4; chirp trials, ntrials = 41; no-chirp trials, ntrials = 34; pKWT = 0.72). C, KWT p value estimates for comparisons made between the AUC calculated over 5-s-long, nonoverlapping windows between chirp and no-chirp trials. Red, |Df| <20 Hz; blue, |Df| ≥20 Hz. The difference in AUC between chirp and no-chirp trials for |Df| values <20 Hz gains significance within the second 5 s window after stimulus onset. There was no significant difference between chirp and no-chirp trials for |Df values ≥20 Hz. The horizontal dashed line corresponds to pKWT = 0.05.

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