Auditory brainstem responses are resistant to pharmacological modulation in Sprague Dawley wild-type and Neurexin1α knockout rats

Abstract

Sensory processing in the auditory brainstem can be studied with auditory brainstem responses (ABRs) across species. There is, however, a limited understanding of ABRs as tools to assess the effect of pharmacological interventions. Therefore, we set out to understand how pharmacological agents that target key transmitter systems of the auditory brainstem circuitry affect ABRs in rats. Given previous studies, demonstrating that Nrxn1α KO Sprague Dawley rats show substantial auditory processing deficits and altered sensitivity to GABAergic modulators, we used both Nrxn1α KO and wild-type littermates in our study. First, we probed how different commonly used anesthetics (isoflurane, ketamine/xylazine, medetomidine) affect ABRs. In the next step, we assessed the effects of different pharmacological compounds (diazepam, gaboxadol, retigabine, nicotine, baclofen, and bitopertin) either under isoflurane or medetomidine anesthesia. We found that under our experimental conditions, ABRs are largely unaffected by diverse pharmacological modulation. Significant modulation was observed with (i) nicotine, affecting the late ABRs components at 90 dB stimulus intensity under isoflurane anesthesia in both genotypes and (ii) retigabine, showing a slight decrease in late ABRs deflections at 80 dB stimulus intensity, mainly in isoflurane anesthetized Nrxn1α KO rats. Our study suggests that ABRs in anesthetized rats are resistant to a wide range of pharmacological modulators, which has important implications for the applicability of ABRs to study auditory brainstem physiology.

Keywords: Auditory brainstem responses; Neurexins; Neurophysiology; Non-invasive brain technology; Pharmacological modulations.

Fig. 1

Comparison of auditory brainstem responses between Nrxn1α KO Sprague Dawley and wild-type littermates rats. ABR waveforms across different stimulus intensities (90, 70, 50 dB) under A isoflurane, B ketamine/xylazine and C medetomidine anesthesia. Recordings from the WT are in blue (N = 12) and Nrxn1α KO in red (N = 12). Data displayed as mean ± SEM, was tested with unpaired CBPT. No robust significant differences were found between genotypes across anesthesia methods. Grey bars above the graphs indicate clusters of significant differences before CBPT-based correction for multiple comparisons, i.e., indicating statistical trends

Fig. 2

Auditory brainstem responses post pharmacological treatment in WT Sprague Dawley rats under isoflurane anesthesia. ABR waveforms across different stimulus intensities (90, 70, 50 dB) post intraperitoneal injection with diazepam (3 mg/kg) in magenta; (N = 14), gaboxadol (10 mg/kg) in teal; (N = 14), baclofen (5 mg/kg) in blue; (N = 14), bitopertin (10 mg/kg) in purple, retigabine (3 mg/kg) in red; (N = 18), nicotine (5 mg/kg) in yellow; (N = 14),; (N = 14), or vehicle solution in black (0.9% saline + 0.3% Tween). Within each experimental block, dosing was counterbalanced, and applied 15 min prior to the ABR recordings for all compounds, except for bitopertin (60 min pre-treatment time). The Black bars above the graphs indicate clusters of significant differences between conditions. The Gray bars indicate clusters that have not reached significance threshold post-permutations. Data displayed as mean ± SEM

Fig. 3

Auditory brainstem responses post pharmacological treatments in Nrxn1α Sprague Dawley rats under isoflurane anesthesia. ABR waveforms across different stimulus intensities (90, 70, 50 dB) post intraperitoneal injection with diazepam (3 mg/kg) in magenta; (N = 14), gaboxadol (10 mg/kg) in teal; (N = 14), baclofen (5 mg/kg) in blue; (N = 13), bitopertin (10 mg/kg) in purple; (N = 11), retigabine (3 mg/kg) in red; (N = 16), nicotine (5 mg/kg) in yellow; (N = 14), or vehicle solution in black (0.9% saline + 0.3% Tween). Within each experimental block, dosing was counterbalanced, and applied 15 min prior to the ABR recordings for all compounds, except for in bitopertin (60 min pre-treatment time). The Black bars above the graphs indicated CBPT clusters of significant differences within subjects, i.e., between conditions. The Gray bars indicate clusters that have not reached significance threshold post-permutations. Data displayed as mean ± SEM

Fig. 4

Auditory brainstem responses post pharmacological treatment in WT Sprague Dawley rats under medetomidine anesthesia. ABR waveforms across different stimulus intensities (90, 70, 50 dB) post intraperitoneal injection with diazepam (3 mg/kg) in magenta; (N = 12), bitopertin (10 mg/kg) in purple; (N = 12), retigabine (3 mg/kg) in red; (N = 12), or vehicle solution in black (0.9% saline + 0.3% Tween). Within each experimental block, dosing was counterbalanced, and applied 15 min prior to the ABR recordings for all compounds, except for in bitopertin (60 min pre-treatment time). Data displayed as mean ± SEM, was tested with unpaired CBPT. No robust significant differences were found between genotypes across anesthesia methods. Grey bars above the graphs indicate clusters of significant differences before CBPT-based correction for multiple comparisons, i.e., indicating statistical trends

Fig. 5

Auditory brainstem responses post pharmacological treatment in Nrxn1α KO Sprague Dawley rats under medetomidine anesthesia. ABR waveforms across different stimulus intensities (90, 70, 50 dB) post intraperitoneal injection with diazepam (3 mg/kg) in magenta; (N = 12), bitopertin (10 mg/kg) in purple; (N = 12), retigabine (3 mg/kg) in red; (N = 12), or vehicle solution in black (0.9% saline + 0.3% Tween). Within each experimental block, dosing was counterbalanced, and applied 15 min prior to the ABR recordings for all compounds, except for in bitopertin (60 min pre-treatment time). Data displayed as mean ± SEM, was tested with unpaired CBPT. No robust significant differences were found between genotypes across anesthesia methods. Grey bars above the graphs indicate clusters of significant differences before CBPT-based correction for multiple comparisons, i.e., indicating statistical trends