Cellular interactions between social experience, alcohol sensitivity, and GABAergic inhibition in a crayfish neural circuit

Abstract

We report here that prior social experience modified the behavioral responses of adult crayfish to acute alcohol exposure. Animals housed individually for 1 wk before alcohol exposure were less sensitive to the intoxicating effects of alcohol than animals housed in groups, and these differences are based on changes in the nervous system rather than differences in alcohol uptake. To elucidate the underlying neural mechanisms, we investigated the neurophysiological responses of the lateral giant (LG) interneurons after alcohol exposure. Specifically, we measured the interactions between alcohol and different GABA_A-receptor antagonists and agonists in reduced crayfish preparations devoid of brain-derived tonic GABAergic inhibition. We found that alcohol significantly increased the postsynaptic potential of the LG neurons, but contrary to our behavioral observations, the results were similar for isolated and communal animals. The GABA_A-receptor antagonist picrotoxin, however, facilitated LG postsynaptic potentials more strongly in communal crayfish, which altered the neurocellular interactions with alcohol, whereas TPMPA [(1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid], an antagonist directed against GABA_A-receptors with ρ subunits, did not produce any effects. Muscimol, an agonist for GABA_A-receptors, blocked the stimulating effects of alcohol, but this was independent of prior social history. THIP [4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol], an agonist directed against GABA_A-receptors with δ subunits, which were not previously known to exist in the LG circuit, replicated the suppressing effects of muscimol. Together, our findings provide strong evidence that alcohol interacts with the crayfish GABAergic system, and the interplay between prior social experience and acute alcohol intoxication might be linked to changes in the expression and function of specific GABA_A-receptor subtypes.NEW & NOTEWORTHY The complex interactions between alcohol and prior social experience are still poorly understood. Our work demonstrates that socially isolated crayfish exhibit lower neurobehavioral sensitivity to acute ethanol compared with communally housed animals, and this socially mediated effect is based on changes in the nervous systems rather than on differences in uptake or metabolism. By combining intracellular neurophysiology and neuropharmacology, we investigated the role of the main inhibitory neurotransmitter GABA, and its receptor subtypes, in shaping this process.

Keywords: alcohol; crayfish; inhibition; neurons; social.

Graphical abstract

Figure 1.

A: circuit diagram of the preparation used for electrophysiology experiments. PSPs in the LG neuron were evoked with electric stimulation of the sensory nerves that contain the primary sensory afferents (SA) and recorded with an intracellular electrode in the axon of LG. A second extracellular electrode was placed between more rostral ganglia to monitor ascending LG action potentials. The SAs connect to LG directly via electrical synapses and to sensory interneurons (SI) via chemical synapses. Excitatory SIs connect to LG via electrical synapses, while unidentified interneurons provide delayed inhibition. The broken line shown in parallel to the LG axon denotes descending tonic inhibition, which was eliminated in this preparation of the isolated nerve cord. B: an example recording of LG PSP just below threshold (black trace) overlaid on an LG action potential (gray trace). The gray trace was taken 90 s after the black one. The action potential is truncated. The arrowhead points to the stimulus artifact, and the black arrows point to LG PSP time points that are 3 ms (“early”) and 5 ms (“late”) after stimulus onset. The Greek letters indicate the different phases of the LG PSP that are generated by electrical (α) as well as chemical (excitatory, β; mostly inhibitory, γ) inputs from sensory afferent (SA) and sensory interneurons (SI). LG, lateral giant; PSPs, postsynaptic potentials.

Figure 2.

Both the onset of tail-flipping behavior (TF) and the onset of supine position (SP) occurred later in adult crayfish that were individually isolated for 1 wk (ISOs) prior to acute 2 M EtOH exposure compared with communally housed crayfish (COMs). Means ± SD are shown. The differences are significant for both behaviors. *P ≤ 0.05. **P ≤ 0.01.

Figure 3.

Concentration of EtOH in the hemolymph of ISOs and COMs measured immediately after 30-min exposure to 2 M EtOH. Means ± SD are shown. Animals from both groups accumulated substantial amounts of EtOH in their hemolymph, but there was no significant difference in total EtOH concentration. COM, communally housed; HL, hemolymph; ISO, socially isolated.

Figure 4.

A: control data (Saline), aggregated across both social conditions. Circles are the linear mixed model’s estimates, and lines are its 95% confidence intervals. The x-axis is shaded based on the solution being superfused at each time. Time points correspond to the end of baseline (Ba), saline (Sa), EtOH (Et), and washout (Wa). *P ≤ 0.05; ***P ≤ 0.001. B: PSP differences observed between ISOs (black) and COMs (gray) in the Saline condition. The circles represent point estimates of the effect that communalization has during saline exposure, controlling for the main effects of saline exposure and phase. Lines are 95% confidence intervals. The x-axis is shaded based on the solution being superfused at each time. Time points correspond to the end of baseline (Ba), saline (Sa), EtOH (Et), and washout (Wa). *P ≤ 0.05. COM, communally housed; ISO, socially isolated; PSP, postsynaptic potential.

Figure 5.

A: effects of PTX aggregated across social conditions. Circles are the linear mixed model’s estimates, and lines are its 95% confidence interval. Black circles and lines are the estimates and confidence intervals for the saline condition, presented here for comparison. Significance brackets are broken to emphasize that comparisons are between the changes seen in drug and saline control conditions, rather than directly between phases. The x-axis is shaded based on the solution being superfused at each time. Time points correspond to the end of baseline (Ba), picrotoxin (Pi), EtOH (Et), and washout (Wa). **P ≤ 0.01, ***P ≤ 0.001. B: PSP differences observed between ISOs (black) and COMs (gray) in the PTX condition. The circles represent point estimates of the effect of communalization, controlling for the main effects of PTX exposure and phase. Lines are 95% confidence intervals. Significance brackets are broken to emphasize that comparisons are between the changes seen in PTX and saline control conditions, rather than directly between phases. The x-axis is shaded based on the solution being superfused at each time. Time points correspond to the end of baseline (Ba), picrotoxin (Pi), EtOH (Et), and washout (Wa). *P ≤ 0.05; **P ≤ 0.01. COM, communally housed; ISO, socially isolated; PSP, postsynaptic potential; PTX, picrotoxin.

Figure 6.

A: effects of TPMPA aggregated across social conditions. Circles are the linear mixed model’s estimates, and lines are its 95% confidence interval. Black circles and lines are the estimates and confidence intervals for the saline condition, presented here for comparison. Significance brackets are broken to emphasize that comparisons are between the changes seen in drug and saline control conditions, rather than directly between phases. The x-axis is shaded based on the solution being superfused at each time. Time points correspond to the end of baseline (Ba), TPMPA (TP), EtOH (Et), and washout (Wa). B: PSP differences observed between ISOs (black) and COMs (gray) in the TPMPA condition. The circles represent point estimates of the effect of communalization, controlling for the main effects of TPMPA exposure and phase. Lines are 95% confidence intervals. The x-axis is shaded based on the solution being superfused at each time. Time points correspond to the end of baseline (Ba), TPMPA (TP), EtOH (Et), and washout (Wa). COM, communally housed; ISO, socially isolated; PSP, postsynaptic potential; TPMPA, (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid.

Figure 7.

A: effects of muscimol aggregated across social conditions. Circles are the linear mixed model’s estimates, and lines are its 95% confidence interval. Black circles and lines are the estimates and confidence intervals for the saline condition, presented here for comparison. Significance brackets are broken to emphasize that comparisons are between the changes seen in drug and saline conditions, rather than directly between phases. The x-axis is shaded based on the solution being superfused at each time. Time points correspond to the end of baseline (Ba), muscimol (Mu), EtOH (Et), and washout (Wa).*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. B: PSP differences observed between ISOs (black) and COMs (gray) in the muscimol condition. The circles represent point estimates of the effect of communalization, controlling for the main effects of muscimol exposure and phase. Lines are 95% confidence intervals. The x-axis is shaded based on the solution being superfused at each time. Time points correspond to the end of baseline (Ba), muscimol (Mu), EtOH (Et), and washout (Wa). *= P ≤ 0.05. COM, communally housed; ISO, socially isolated; PSP, postsynaptic potential.

Figure 8.

A: effects of THIP aggregated across social conditions. Circles are the linear mixed model’s estimates, and lines are its 95% confidence interval. Black circles and lines are the estimates and confidence intervals for the saline condition, presented here for comparison. Significance brackets are broken to emphasize that comparisons are between the changes seen in drug and saline conditions, rather than directly between phases. The x-axis is shaded based on the solution being superfused at each time. Time points correspond to the end of baseline (Ba), THIP (TH), EtOH (Et), and washout (Wa). **P ≤ 0.01, ***P ≤ 0.001. B: PSP differences observed between ISOs (black) and COMs (gray) in the THIP condition. The circles represent point estimates of the effect of communalization, controlling for the main effects of THIP exposure and phase. Lines are 95% confidence intervals. The x-axis is shaded based on the solution being superfused at each time. Time points correspond to the end of baseline (Ba), THIP (TH), EtOH (Et), and washout (Wa). COM, communally housed; ISO, socially isolated; PSP, postsynaptic potential; THIP, 4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol.