Nucleus Accumbens Chemogenetic Inhibition Suppresses Amphetamine-Induced Ultrasonic Vocalizations in Male and Female Rats

Abstract

Adult rats emit ultrasonic vocalizations (USVs) related to their affective states, potentially providing information about their subjective experiences during behavioral neuroscience experiments. If so, USVs might provide an important link between invasive animal preclinical studies and human studies in which subjective states can be readily queried. Here, we induced USVs in male and female Long Evans rats using acute amphetamine (2 mg/kg), and asked how reversibly inhibiting nucleus accumbens neurons using designer receptors exclusively activated by designer drugs (DREADDs) impacts USV production. We analyzed USV characteristics using "Deepsqueak" software, and manually categorized detected calls into four previously defined subtypes. We found that systemic administration of the DREADD agonist clozapine-n-oxide, relative to vehicle in the same rats, suppressed the number of frequency-modulated and trill-containing USVs without impacting high frequency, unmodulated (flat) USVs, nor the small number of low-frequency USVs observed. Using chemogenetics, these results thus confirm that nucleus accumbens neurons are essential for production of amphetamine-induced frequency-modulated USVs. They also support the premise of further investigating the characteristics and subcategories of these calls as a window into the subjective effects of neural manipulations, with potential future clinical applications.

Keywords: 22 kHz vocalizations; 50 kHz vocalizations; UMAP; amphetamine; chemogenetics; clozapine-n-oxide; females; males; nucleus accumbens.

Figure 1

Nucleus Accumbens DREADD Expression: (A) Expression of the AAV2-hSyn-hM4Di-mCherry vector is depicted in a typical rat, shown in a coronal plane. mCherry and hM4Di are co-expressed in NAc neurons (red stain), and DAPI (4′,6-diamidino-2-phenylindole, in blue) defines anatomical landmarks, including anterior commissure (ac). (B) Viral expression localization is shown in each of the 16 tested animals in 3 coronal views of rat brain [48], with numbers indicating the approximate bregma-relative anterior coordinate.

Figure 2

Nucleus Accumbens Inhibition Suppresses the Number of Frequency Modulated and Trill USVs: (A) USVs of each call type are depicted in 10 min bins in the post-vehicle, but pre-amphetamine baseline (left), and for 1 h after amphetamine (right; amphetamine injection occurred at the dotted vertical line). Pie charts show relative prevalence of each call subtype shown for pre-amphetamine baseline (left) and for 1 h after amphetamine (right). (B) CNO day data is shown in the same manner as in panel A. (C) Summary data are shown for the entire 1 h post-amphetamine session for the vehicle day (black) and CNO day (red), depicting main effects of this treatment. Dots depict data from individual animals. * p < 0.05. (D) Post-amphetamine summary data is shown in the same manner as in panel C, but separately in females (left) and males (right). Statistically similar CNO-suppression of the number of FM and Trill, but not Flat, USVs is seen in both sexes.

Figure 3

Characteristics of Quantified USVs: All USVs emitted on the vehicle + amphetamine test day were categorized by an observer into one of four categories: Low-frequency (LF), Flat, non-trill-containing frequency-modulated (FM), and trill-containing (Trill). Parameters (principal frequency, frequency modulation, duration, and sinuosity) for each emitted vocalization were quantified by Deepsqueak, and per-animal averages computed for each call type. (A) The mean and SEM of these per-rat average values for each call type are shown for each parameter, with dots indicating the mean value for each tested rat. (B) Examples of each of the 4 call types are shown. Scale bar indicates 20 ms. (C) UMAP representation of how calls relate to one another on the 4 measured parameters is depicted. Each emitted call is represented by a dot, which was color-coded based on manually assigned call type (LF = orange, Flat = yellow, FM = blue, Trill = green). Calls with similar characteristics in the 4 dimensions are represented within a cluster. To illustrate the proportion of call types present within visually determined clusters (circled), pie charts show the percentage of calls in each cluster that were assigned to each call type. Though each cluster differs in the types of calls contained within them, clusters do not neatly parse calls into our 4 pre-defined categories, which were based on the literature. This suggests that the 4-category system we used to define calls into subcategories may not fully capture underlying call subtypes present in the data, which could have functional significance. (D) Per-rat averages on each USV parameter, collapsed across call types, is shown broken down by the emitting animal’s sex. No significant sex differences were found, suggesting that rats do not differ based on sex in the fundamental features of emitted USVs. (E) Further demonstrating this point, the same UMAP plot is shown, with calls color-coded by sex rather than call type, demonstrating that both sexes emit calls throughout the entirety of 4-dimensional space, and in each of the data-derived clusters depicted.

Figure 4

Nucleus Accumbens Inhibition Does Not Affect the Characteristics of Amphetamine-Induced USVs: Relative to vehicle day (black), USVs on CNO day (red) did not differ in principal frequency, frequency modulation, duration, or sinuosity for either (A) frequency-modulated non-trill, or (B) trill-containing USVs. Average values for each rat are shown with dots within bars.