Gap-elicited pre-pulse inhibition of the acoustic startle

Since I observed that the electrophysiological gap detection paradigm is sensitive to differences in myelination levels, I was interested in implementing a behavioral correlate of the electrophysiological measure of gap detection, to understand how temporal acuity defects in the cortex affect sound perception. For this, I used gap-pre-pulse inhibition of the acoustic startle reflex, a paradigm that is widely used for assessing the detection of gaps at the behavioral level in different animal models (Dehmel et al., 2012; Friedman et al., 2004;

Moreno-Paublete et al., 2017).

Pre-pulse inhibition of the ASR is a mechanism of sensorimotor gating. The acoustic startle response (ASR) is a fast motor response, observed in all mammals, to an intense sudden sound stimulus. This usually includes contraction of facial and body muscles and eye closure, heart rate acceleration and pause of ongoing behaviors (Koch, 1999). When another non-startling sound (pre-pulse) is presented just before the starling sound, the startling reflex is partially inhibited in a process known as the pre-pulse inhibition (PPI) of the startle reflex, which is the quantitative measure of sensorimotor gating. In the CNS, this neural process helps the filtering of sensory information to help the processing at higher order cortical brain areas (Koch, 1999). Sensorimotor gating is affected in schizophrenic patients (Engel et al.,

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2015; Haß et al., 2017; Mena et al., 2016), and in general, a variety of neuropsychological pathologies have been associated with a reduced ability of gaiting information (for review, see (Koch, 1999). This reflex probably has an important behavioral advantage to prepare and protect an animal from an aversive stimulus. This behavior can be modulated (either enhanced of attenuated) by a series of external and internal aspects, thereby it provides a reliable measure of individual sensorimotor gating. In mammals, the ASR is believed to be mediated by the brainstem. It has been proposed that the ASR circuit relays in three interconnected structures: the cochlear nucleus, the neurons in the caudal pontine reticular formation and cranial and spinal motor neurons (Koch, 1999; Lee et al., 1996; Yeomans and Frankland, 1995). Silent gaps appearing in a continuous background, just before the startling sound, can also work as a pre-pulse and induce PPI of the ASR. The gap-elicited inhibition of the ASR has been widely used for testing temporal acuity in rodent animal models (Clark et al., 2000; Dehmel et al., 2012; Moreno-Paublete et al., 2017; Popelář et al., 2017; Walton et al., 1997). Measurements were performed as explained in Materials and Methods (section II.IX.I).

In Figure 35-A one can observe a brief scheme of the sounds played for this task. A background sound that consisted of a 70 dB (SPL) broad band noise was followed by gaps of different lengths (e.g. 5 or 25 ms). After each gap, a period of 50 ms of the background BBN was played and then followed by a 40 ms startle noise at ~105 dB. In Figure 35-B, consistent with previous reports, that there was a strong effect of the length of a silent gap placed before the startle pulse. Short gaps were more difficult to be detected and elicited less inhibition, compared to longer gaps (Popelář et al., 2017). Independent controls were used for each mutant line and measured together to reduce variability of the testing day. Nevertheless, since there were no differences between all the control animals of the different lines tested, I pulled together all controls of the MBP^neo (n=7), MBP^shi (n=2), and MBP^emx (n=6) lines (ANOVA, F(1,63)=1.7, p<0.19; F(1,99)=0.2, p<0.65; F(1,54)=2.16, p<0.14, respectively).

MBP^neo mice show a significant decrease in ASR inhibition in the presence of different gap lengths when compared to control animals. An ANOVA shows a significant effect of gap in the percentage of ASR inhibition (F(8,171)=43.53, p<0.0001), together with a strong effect of group (F(1,171)=20.3, p<0.0001), nevertheless, an interaction between gap and group was not present (F(8,171)=1.14, p=0.34).

91 Interestingly, heterozygote animals mice from the MBP^shi line, which have a loss of 50% MBP (MBP^+/-), do not show a deficit in PPI of the ASR (ANOVA, F(1,171)=2.21, p<0.13).

It seems that a minimum amount of myelin loss is needed to impair gap detection at the behavioral level.

In addition, some studies have related intra-cortical processes that are necessary for gap detection (Weible et al., 2014a, 2014b). One question that arose was whether

cortical-Figure 35. A paradigm of pre-pulse inhibition of the acoustic startle reflex using gaps reveals that MBP^neo mice have a behavioral impairment of temporal acuity.

A) Shows a schematic of the sounds used. A background broad band noise at 70dB was played, followed by silent gaps of different durations, presented in a pseudorandom order. From the gap offset to the startle pulse there was a period of 50 ms of background sound. The startle pulse consisted of a BBN at 105 dB and 40 ms duration. B) Shows the percentage of pre-pulse inhibition elicited by the different gaps. There is a strong relationship between the length of the gap and the increase in inhibition, since longer gaps are more noticeable. Only MBP^neo mice showed impaired PPI%. C) MBP^neo mice have a significant increase in gap-detection threshold compared to control animals (p=0.0035). D) MBP^neo mice showed a strong reduction of the startle-only amplitude (p=0.0012), probably due to a reduced functionality of the cerebellar projection neurons for the startle reflex.

A

B C

D

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specific loss of myelin would have similar effects in the behavioral detection of gaps. I used the MBP^emx mice to test this behavior. In this case, I also did not see any difference between control and MBP^emx mice for the PPI (ANOVA, F(1, 171)=1.36, p=0.24). I then obtained the specific values of gap detection for all the animals, and observed a very similar effect.

Comparing control animals with MBP^neo, we can observe a significant increase in the gap-detection threshold (Figure 35-C) (p=0.0035), whereas the comparison of gap-gap-detection thresholds between control animals and MBP^+/- or MBP^emx mice yielded no significant difference (p=0.66, p=0.78 respectively). Additionally, a significant difference in gap-detection threshold was seen between MBP^neo and MBP^+/- (p=0.017), and between MBP^neo and MBP^emx (p=0.0052). This means that a strong loss of myelin (~80%) elicits temporal acuity deficits in mice, consistent with the electrophysiological measurements of responses to gaps in the ACx. Interestingly, I observed also a strong reduction in the amplitude of the only-startle response of MBP^neo mice (p=0.0012) (Figure 35-D). No differences were seen between control animals and MBP^+/- or MBP^emx mice (p=0.43, p=0.83 respectively). This means that the MBP^neo mice tend to startle less compared to control animals, when a loud sound is presented without a pre-pulse. This effect was striking, and I hypothesize is caused by an abnormality in the motor-related circuit that elicits the reflex.