Effects of furosemide on cochlear neural activity, central hyperactivity and behavioural tinnitus after cochlear trauma in guinea pig

Abstract

Cochlear trauma causes increased spontaneous activity (hyperactivity) to develop in central auditory structures, and this has been suggested as a neural substrate for tinnitus. Using a guinea pig model we have previously demonstrated that for some time after cochlear trauma, central hyperactivity is dependent on peripheral afferent drive and only later becomes generated intrinsically within central structures. Furosemide, a loop diuretic, reduces spontaneous firing of auditory afferents. We investigated in our guinea pig model the efficacy of furosemide in reducing 1) spontaneous firing of auditory afferents, using the spectrum of neural noise (SNN) from round window recording, 2) hyperactivity in inferior colliculus, using extracellular single neuron recordings and 3) tinnitus at early time-points after cochlear trauma. Tinnitus was assessed using gap prepulse inhibition of acoustic startle (GPIAS). Intraperitoneal furosemide, but not saline, caused a marked decrease in both SNN and central hyperactivity. Intracochlear perfusion with furosemide similarly reversed central hyperactivity. In animals in which GPIAS measurements suggested the presence of tinnitus (reduced GPIAS), this could be reversed with an intraperitoneal injection with furosemide but not saline. The results are consistent with furosemide reducing central hyperactivity and behavioural signs of tinnitus by acting peripherally to decrease spontaneous firing of auditory afferents. The data support the notion that hyperactivity may be involved in the generation of tinnitus and further suggest that there may be a therapeutic window after cochlear trauma using drug treatments that target peripheral spontaneous activity.

Figure 1

A: Overview of all animals used in the present study as allocated to the different groups. B: Schematic representation of the experimental design of behavioural experiments.

Figure 2. Data from 7 animals that underwent acoustic trauma and that did not develop a GPIAS deficit for a period of 10 weeks A: Histogram showing mean % GPIAS.

Shown is % GPIAS measured with 8 and 14 kHz background noise before (black bars) and 10 weeks after trauma (white bars). B: CAP thresholds (mean ± SEM). Left cochlea before acoustic trauma (open circles), immediately after acoustic trauma (black diamonds) and after recovery from acoustic trauma (open triangles). Contralateral control cochlea after recovery (black circles). Statistical significance of differences between pre and post acoustic trauma data: #p<0.001. C: Threshold loss for individual animals at 10 weeks after acoustic trauma.

Figure 3. Data from animals that developed GPIAS deficits between 3 and 6 weeks post-trauma Histograms in A and B show % GPIAS before acoustic trauma (AT), after AT and after treatment.

A: Data from 4 animals given furosemide (black bars) and 3 animals given saline (white bars). All animals shown in A showed significant PPI. B: Data from 4 animals with significant PPI which were given furosemide (black bars, same animals as in panel A) and 3 animals without significant PPI which were also given furosemide (white bars). Significance is shown from repeated measure two-way ANOVA and post-tests. Statistical significance: *p<0.05; **p<0.01. ***p<0.001. C: CAP thresholds from animals shown in A (mean ± SEM). Left cochlea before acoustic trauma (open circles), immediately after acoustic trauma (black diamonds) and after recovery from acoustic trauma (open triangles). Contralateral control cochlea after recovery (black circles). Statistical significance of differences between pre and post acoustic trauma data: #p<0.001. D: Threshold loss for individual animals shown in A. Black symbols are animals given furosemide and open symbols animals given saline.

Figure 4. A: Spectrum of neural noise (SNN) recorded from the round window in 8 animals plotted as percentage of original value before and after an i.p. injection with furosemide (black line with filled circles n = 4) or saline (black line with open circle n = 4).

Time of injection indicated by dotted line. B: Mean spontaneous firing rate of CNIC neurons recorded before and after i.p. furosemide and before and after saline (n = 4 for each group; mean ± SEM). C: CAP thresholds (mean ± SEM) at different frequencies recorded from the left cochlea before acoustic trauma (open circles), immediately after acoustic trauma (black diamonds) and after recovery from acoustic trauma (open triangles), as well as from the contralateral control cochlea after recovery (black circles). *p<0.05; **p<0.01; #p<0.001 statistical significance as compared to before trauma data. D and E: Spontaneous firing rate of 2 individual neurons from 2 different animals plotted over time before, during and after intracochlear perfusion of furosemide (black circles). Black bar indicates timing of intracochlear perfusion. Open circles indicate measurement of the neuron’s threshold to CF tones. Neuronal CF is 10 kHz and 12.4 kHz, in D and E, respectively.