The Effects of Electrical and Optical Stimulation of Midbrain Dopaminergic Neurons on Rat 50-kHz Ultrasonic Vocalizations

Abstract

Rationale: Adult rats emit ultrasonic vocalizations (USVs) at around 50-kHz; these commonly occur in contexts that putatively engender positive affect. While several reports indicate that dopaminergic (DAergic) transmission plays a role in the emission of 50-kHz calls, the pharmacological evidence is mixed. Different modes of dopamine (DA) release (i.e., tonic and phasic) could potentially explain this discrepancy.

Objective: To investigate the potential role of phasic DA release in 50-kHz call emission.

Methods: In Experiment 1, USVs were recorded in adult male rats following unexpected electrical stimulation of the medial forebrain bundle (MFB). In parallel, phasic DA release in the nucleus accumbens (NAcc) was recorded using fast-scan cyclic voltammetry. In Experiment 2, USVs were recorded following response-contingent or non-contingent optogenetic stimulation of midbrain DAergic neurons. Four 20-s schedules of optogenetic stimulation were used: fixed-interval, fixed-time, variable-interval, and variable-time.

Results: Brief electrical stimulation of the MFB increased both 50-kHz call rate and phasic DA release in the NAcc. During optogenetic stimulation sessions, rats initially called at a high rate comparable to that observed following reinforcers such as psychostimulants. Although optogenetic stimulation maintained reinforced responding throughout the 2-h session, the call rate declined to near zero within the first 30 min. The trill call subtype predominated following both electrical and optical stimulation.

Conclusion: The occurrence of electrically-evoked 50-kHz calls, time-locked to phasic DA (Experiment 1), provides correlational evidence supporting a role for phasic DA in USV production. However, in Experiment 2, the temporal dissociation between calling and optogenetic stimulation of midbrain DAergic neurons suggests that phasic mesolimbic DA release is not sufficient to produce 50-kHz calls. The emission of the trill subtype of 50-kHz calls potentially provides a marker distinguishing positive affect from positive reinforcement.

Keywords: fast-scan cyclic voltammetry; midbrain dopaminergic neurons; nucleus accumbens; optogenetics; phasic dopamine; ultrasonic vocalizations.

Figure 1

Electrical stimulation of the medial forebrain bundle (MFB) and 50-kHz call rate (Experiment 1). Each rat (n = 3) was tested under each stimulation condition. Calls after MFB stimulation (post, 0–55 s following stimulation onset) were significantly greater than calls before MFB stimulation (pre, 0–55 s before stimulation). Each stimulation train was spaced 5–6 min apart. ^***p < 0.002 pre vs. post (Sign test, n = 12 i.e., 4 stimulation parameters × 3 rats).

Figure 2

Time course of 50-kHz call emission and phasic DA release following electrical stimulation of the medial forebrain bundle (MFB). Stimulated DA release in the nucleus accumbens (NAcc) was timed-locked to MFB stimulation (24 biphasic 120 μA 60 Hz pulses, each pulse comprising a pair of 2-ms phases) and the onset of USV emission. (A) shows an increase in call rate following MFB stimulation. The 1-s stimulation started at 0 s. (B) is a false-color plot from a representative rat, showing changes in DA current (green color) in relation to applied potential (y-axis) and time (x-axis), with onset of MFB stimulation occurring at t = 0 s, at the oxidation potential of dopamine (red arrow), i.e., ~0.65 V (vs. Ag/AgCl reference). (C) shows the corresponding background-subtracted cyclic voltammogram from the same rat at the point of peak DA current seen in (B).

Figure 3

Mean peak DA current (log) for each electrode (n = 5 electrodes) across various stimulation parameters. At both frequencies tested [30 Hz–(A); 60 Hz–(B)], peak DA current increased with current (respectively: Wilcoxon, p < 0.05; Friedman, p = 0.002).

Figure 4

Cumulative lever responses for each rat (n = 4) as a function of time since the last optogenetic stimulation. (A) shows lever responses increasing as the next stimulation opportunity approached (at 20 s). (B) shows lever pressing at a near-constant rate during the 10 s prior to the onset of the next stimulation opportunity (times randomly selected from lagged exponential distribution with mean 20 s).

Figure 5

Mean lever presses and mean calls for each schedule (n = 4 rats). (A,B) show that lever press rates under both schedules remained stable across successive 5-min time bins within the first 30 min of the session. (C) shows that response rates tended to increase across the 2-h session. (D–F,H) show mean calls across all four reinforcement schedules, decreasing over the first 30 min of the for each rat. (G) shows that the median call rate decreased over the first 30 min for all schedules, with rats pooled. There was also a significant increase in call number when the optogenetic stimulation was non-contingent (VT and FT schedules) vs. contingent on a lever-press (FI and VI schedules). FI, fixed interval; VI, variable interval; FT, fixed interval; VT, variable interval. All schedules are 20 s. Sign-test ^*p < 0.02 (n = 8 i.e., 4 rats × 2 schedules).

Figure 6

Percentage of total calls and call subtypes under each reinforcement schedule for all rats (n = 4), as a function of time from stimulation. Time bin “1” represents the time at which the midbrain optogenetic stimulation occurred. Pie charts show percentages of calls (14 subtypes and 2 categories) before (–10 to –1) and after (2–10) the stimulation. The most common calls are labeled (for details of call subtypes see Supplementary Table 5). FI, fixed interval; FT, fixed time; VI, variable interval; VT, variable time. All schedules are 20 s.