Selective and Context-Dependent Social and Behavioral Effects of Δ9-Tetrahydrocannabinol in Weakly Electric Fish

Abstract

Cannabinoid (CB) receptors are widespread in the nervous system and influence a variety of behaviors. Weakly electric fish have been a useful model system in the study of the neural basis of behavior, but we know nothing of the role played by the CB system. Here, we determine the overall behavioral effect of a nonselective CB receptor agonist, namely Δ9-tetrahydrocannabinol (THC), in the weakly electric fish Apte-ronotus leptorhynchus. Using various behavioral paradigms involving social stimuli, we show that THC decreases locomotor behavior, as in many species, and influences communication and social behavior. Across the different experiments, we found that the propensity to emit communication signals (chirps) and seek social interactions was affected in a context-dependent manner. We explicitly tested this hypothesis by comparing the behavioral effects of THC injection in fish placed in a novel versus a familiar social and physical environment. THC-injected fish were less likely to chirp than control fish in familiar situations but not in novel ones. The tendency to be in close proximity to other fish was affected only in novel environments, with control fish clustering more than THC-injected ones. By identifying behaviors affected by CB agonists, our study can guide further comparative and neurophysiological studies of the role of the CB system using a weakly electric fish as a model.

Keywords: Apteronotus leptorhynchus; Cannabinoid; Communication; Electric fish; Neuromodulator; Neurotransmitter; Social behavior; THC (Δ9-tetrahydrocannabinol).

Figure 1:. Disruption of the EOD at high dose of THC.

A. Spectrogram of the disrupted EOD of a single fish with insets of EOD waveform excerpts. The excerpt (i.e., time 0 on the spectrogram) starts 10 min after injection. The main EOD frequency at any given moment can be seen as the dark red-brown line. A normal EOD would be visualized on the spectrogram as a single flat line. Here the EOD frequency decreases, fluctuates, has multiple peaks of frequency power, and is overall very unstable. Four insets (top and bottom) taken at different points on the spectrogram show the details of the time varying EOD waveform, with a fifth inset (right) providing a more detailed view of an example EOD being unstable. In the insets, black scale bars in the upper right corner of each inset represent 10 ms and 1 mV. B. Propensity of EOD disruption as a function of THC dose. We quantified both the proportion of fish that displayed any disruption and the proportion of the time the EOD is affected in these fish during the 20 min following injection (n=8 for each dose <100 mg/kg and n=4 for 100mg/kg).

Figure 2:. Chirp Chamber stimulation and recording visualization.

A. Illustration of the recording set-up. The fish is placed in a mesh tube wired with a recording dipole to capture the fish’s EOD (depicted by purple shades electric field lines). A stimulation dipole is placed on one side to mimic a nearby conspecific (red-green dipole and field lines). B. Recordings are displayed as spectrogram (showing intensity as a function of frequency and time). In this example (stimulus frequency: beat of −10 Hz) chirps are highlighted with grey stars and the increase in baseline frequency (JAR) with the grey arrow.

Figure 3:. Selective effect of THC on responses to communication signals.

A. Baseline JAR response prior to THC injection as a function of stimulus frequency. Stimuli were simple beats (sinusoidal AM) created by presenting an artificial EOD at the indicated frequency above or below the test fish’s frequency. The average (± s.e., n=28) across the fish used in B is shown here. B. Change in JAR response displayed in A after THC injections (post- pre) at different doses (average ± s.e., n=8 for each dose <100 mg/kg and n=4 for 100mg/kg). C. Chirping rate in response to chirp stimuli of different beat frequencies. The average (± s.e., n=28) across the fish used in D is shown here. D. Change in chirping response displayed in C after THC injections (post- pre) at different doses (average ± s.e., n=8 for each dose <100 mg/kg and n=4 for 100mg/kg).

Figure 4:. THC decreases attack behavior in agonistic situations.

A. Agonistic situations are created by placing the fish in a confined space. Here only half the square tank is used, and access to the other half containing an air bubbler and water recycling inputs/outputs is restricted with a mesh barrier. A stimulation electrode is placed in the middle of the test compartment (red-green dipole creating an electric field) and recording electrode at each extremity (black circles). Movement of the fish is recorded with an IR camera while a stimulus with a low frequency beat and chirps is played. B. Decrease in attack lunges with increased THC dose. For each of the 10 bouts of stimulation during an experiment, the number of attack lunges produced by the fish is quantified. The mean (± s.e.) is displayed for experiment sets with different THC treatments (n=8 each for no injection and 33mg/kg; n=10 for 0 mg/kg and 66 mg/kg).

Figure 5:. THC selectively affect movement and social behaviors in the first hour post injection.

Four aspects of the behavior (means ± s.e.; n=8 for each group) during the agonistic situation described in Fig. 4 are quantified during a bout stimulation performed after a variable acclimatization period following THC injection. A. JAR response (as illustrated in Fig, 2). B. Average movement speed during stimulation. C. Attack lunges produced by the fish. D. Chirps emitted by the fish in response to the stimulation. Stars indicate statistically significant differences (**: p<0.01; *: p<0.05)

Figure 6:. Characterizing the effect of THC on social interactions in a small group of fish.

A. A large compartmentalized tank is used to allow the fish to explore and cluster in individual compartments. Each compartment contains a hiding tube and is connected to others by small openings. A pair of recording electrodes (black circles) is placed in each compartment which are also equipped with a water input/output (grey dots) providing cleaned, warmed and oxygenated water. Four fish are initially placed in the four corners of the tank. A 3 hr recording session is started after injection. Two types of trials were performed: in the novel environment trials, fish were placed in the tank immediately after injection and with unknown tankmates whereas, in the familiar environment trials, fish used are long-term tankmates and are placed in the testing tank 24 hr prior to injection. B. Recordings from each compartment were displayed as spectrograms (NB: when needed to resolve ambiguities, we also looked at power spectra where the time dimension is collapsed). Fish EODs show up as dark lines in the 0.6–1 kHz range and the 1^st harmonic of their EOD in the 1.2–2 kHz range. Based either on the baseline frequency or 1^st harmonic trace, the position of the 4 fish at the beginning of each 1 s time frame was determined (white arrow) and chirps where marked (white star) to be counted.

Figure 7:. THC has different effects on fish in familiar versus novel socio-physical environments.

Using the experimental paradigm described in Fig.6, we quantified several aspects of the fish’s behavior at different times during the 3 hr trial (i.e., 30 min windows starting at 20 min post injection, 75 min or 145 min). Ten experiments (single 4 fish) were performed for each treatment group (novel/ familiar, THC/control). Averages across fish (± s.e.; n=24 fish) are shown either as an average across the three analysis windows (A,C,D) or separately (B,E). A,B. Position variability decreases with THC injection independently of the socio-physical environment conditions. Position variability was quantified via the change in composition of each compartment (see methods) and reflects the tendency to move and explore the environment. C. Clustering is decreased by THC but only in novel environments. The proportion of time spend in a compartment by itself (cluster size 1; filled bars) or with other fish (clusters 2–4; patterned of empty bars) is displayed for the different treatment groups. D, E. Chirping is decreased by THC but only in familiar environments. Chirp rate per individual fish is displayed for each treatment group.