Brace et al
Auditory responses in a rodent model of Attention Deficit Hyperactivity Disorder
Louise R. BraceA, Igor KraevA, Claire L. RostronA, Michael G. StewartA, Paul G. OvertonB and Eleanor J. DommettA,C*
ADepartment of Life, Health and Chemical Sciences, The Open University, Milton Keynes. MK7 6AA. U.K.
BDepartment of Psychology, University of Sheffield, Western Bank, Sheffield. S10 2TN. U.K.
CDepartment of Psychology, Institute of Psychiatry, Psychology and Neuroscience, King’s College London, London. SE1 3QD. U.K.
* Corresponding Author
Department of Psychology,
Institute of Psychiatry, Psychology and Neuroscience,
King’s College London,
9th Floor, Capital House,
Guy's Campus,
42 Weston Street,
London.
SE1 3QD.
U.K.
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Keywords:
Spontaneously Hypertensive Rat; Superior Colliculus; Orienting; Distractibility; Hearing loss; Validity
Abstract
A central component of Attention Deficit Hyperactivity Disorder (ADHD) is increased distractibility in response to visual and auditory stimuli, which is linked to the superior colliculus (SC). Furthermore, there is now mounting evidence of altered collicular functioning in ADHD and it is proposed that a hyper-responsive SC could mediate symptoms of ADHD, including distractibility. In the present study we conducted a systematic characterisation of the intermediate and deep layers of the SC in the most commonly used and well-validated model of ADHD, the spontaneously hypertensive rat (SHR), building on prior work showing increased distractible behaviour in this strain using visual distractors. We examined collicular-dependent orienting behaviour, local field potential (LFP) and multiunit activity (MUA) in response to auditory stimuli in the anaesthetised rat, and morphological measures, in the SHR in comparison to the Wistar Kyoto (WKY) and Wistar (WIS). We found no evidence of increased distractibility in the behavioural data but suggest that this may arise due to cochlear hearing loss in the SHR. Furthermore, the electrophysiology data indicate that the SC in the SHR may still be hyper-responsive, normalising the amplitude of auditory responses that would otherwise be reduced due to the hearing impairment. The morphological measures of collicular volume, cell density and ratios did not indicate this potential hyper-responsiveness had a basis at the structural level examined. These findings have implications for future use of the SHR in auditory processing studies and may represent a limitation to the validity of this animal model.
1. Introduction
Attention deficit hyperactivity disorder (ADHD) is the most common neurodevelopmental disorder, affecting 8–12% of children (Biederman and Faraone, 2005), with symptoms often persisting into adulthood (Spencer et al., 2002). A central feature of ADHD is increased distractibility (Douglas, 1983; Thorley, 1984), which has long been considered one of the most common symptoms of ADHD (Barkley and Ullman, 1975) and features in the inattentive and combined presentations of ADHD under DSM-5 (APA, 2013).
Distractibility can be measured by examining changes in task performance or ongoing behaviour as a result of the presentation of an extraneous or distracting stimulus (Bremer and Stern, 1976). Dykman et al. (Dykman et al., 1970) presented a loud unpredictable tone (90 dB SPL) during a reaction time task in which participants had to press a key in response to a visual target. They reported greater increases in reaction times from hyperactive children in comparison to non-hyperactive children, indicating increased distractibility to an auditory stimulus in this group. Similar results were found by Bremer and Stern (1976) who assessed distractibility during a reading task by repeated presentation of one of two types of distracting stimuli: a telephone ringing (65 dB SPL) and flashing or a sinusoidal oscilloscope display with an accompanying sound (75 dB SPL). They found that both types of multimodal stimuli elicited similar reactions with hyperactive children significantly more distracted by the stimulus. Hyperactive children showed orienting responses to a greater number of consecutive stimuli and prolonged duration of response in comparison to the non-hyperactive group. More recently, an increased distractibility in children with ADHD has been supported by further behavioural findings and electrophysiological measures. Gumenyuk et al (Gumenyuk et al., 2005) showed that a variety environmental ‘distractor’ sounds (e.g. rain, car horn; 200 dB SPL, 200 ms) enhanced the number of response omissions on a visual discrimination task significantly more in ADHD children than controls and altered evoked potential response components (P3a and LN) in such a way that the authors suggest there could be a deficit in control of involuntary attention in ADHD. Finally, Cassuto et al (Cassuto et al., 2013) showed that, on a visual continuous performance task (CPT), children with ADHD were significantly distracted by visual distractors, auditory distractors and multimodal (auditory and visual) distractors, whilst those without ADHD were only significantly distracted by the multimodal distractors. The authors suggest that this difference in distractibility on CPT could be a useful diagnostic tool for ADHD.
Behavioural evidence suggests that distractibility is intimately linked with the superior colliculus (SC), a subcortical structure that is highly conserved across species (Ingle, 1973). The SC is involved in detecting and responding to novel, unexpected and salient stimuli across a range of modalities (Dean et al., 1989). In particular, it is responsible for orienting head and eye movements (Grantyn et al., 2004) and covert attention towards such stimuli (Rizzolatti et al., 1987). Work in a range of species has shown that collicular lesions cause a decrease in distractibility (Goodale et al., 1978; Milner et al., 1978; Sprague and Meikle, 1965) whilst removal of prefrontal cortex inhibitory control of the colliculus leads to an increase in distractibility in humans (Gaymard et al., 2003). This suggests that the SC remains important in the neural basis of distractibility in humans.
Furthermore, there is now mounting evidence indicative of a role for the SC in ADHD. Firstly, people with ADHD have difficulty inhibiting saccades (Klein et al., 2003; O'Driscoll et al., 2005) and shifts in covert attention (Swanson et al., 1991), consistent with collicular dysfunction (Ignashchenkova et al., 2004; Katyal et al., 2010; Robinson and Kertzman, 1995). Secondly, collicular dysfunction has been reported in rodent models of ADHD. For example, in the spontaneously hypertensive rat (SHR), the most commonly used rodent model of ADHD, altered height dependency of air righting reflexes has been found (Dommett and Rostron, 2011) which is linked to collicular dysfunction (Pellis et al., 1989; Pellis et al., 1991; Yan et al., 2010). More recently, orienting behaviour to a repeated visual stimulus has been shown to be increased in the SHR (Robinson and Bucci, 2014). In addition, in the New Zealand Genetically Hypertensive (GH) rat, a proposed model of ADHD, increased responsiveness to whole field light flashes has been found in the superficial layers of the colliculus (Clements et al., 2014). Thirdly, amphetamine which is used to treat ADHD, decreases the responsiveness of cells in the superficial layers of the colliculus to visual stimuli (Clements et al., 2014; Gowan et al., 2008) and is associated with increased Fos-like immunoreactivity in the intermediate/deeper layers (Hebb and Robertson, 1999a; Hebb and Robertson, 1999b). Amphetamine also reduces distractibility in healthy rats (Agmo et al., 1997) and humans both with (Brown and Cooke, 1994; Spencer et al., 2001) and without ADHD (Halliday et al., 1990). Finally, the colliculus is known to modulate ascending dopaminergic systems within the basal ganglia (Dommett et al., 2005) via a direct connection from the colliculus to midbrain dopaminergic neurons (Coizet et al., 2003; Comoli et al., 2003) and, therefore, alterations in collicular functioning could cause the dopaminergic abnormalities seen in ADHD (Sagvolden et al., 2005a; Solanto, 2002; Viggiano et al., 2003). Furthermore, connections to the basal ganglia, purported to act as a central selection device (Redgrave et al., 1999) would enable the SC, amongst other structures, to specify actions by putting ‘bids’ into the basal ganglia. Therefore, heightened activity within the SC may have the effect of strengthening the bid to the basal ganglia and increasing the likelihood of the bid ‘winning’ over competing action choices and generating an output, such as an orienting towards a stimulus (Grantyn et al., 2004).
In light of the mounting evidence supporting a role for the SC in ADHD and the previous work in the SHR and the GH rat focussing on visual processing, we conducted a detailed characterisation of the SC, examining the auditory-responsive intermediate and deep layers, in the SHR model of ADHD. Specifically, we hypothesized that the SHR would show increased distractibility as measured by an auditory orienting task and increased responsiveness to auditory stimuli at the neuronal level in the colliculus. Furthermore, we hypothesized that there would be changes in the underlying morphology (collicular volume, cell densities and neuron-glia ratio) of the colliculus.
2. Results
2.1 There were no strain differences in behavioural responses to auditory stimuli.
The majority of animals (89% SHR, 100% WKY and 88 % WIS) responded to the auditory stimulus on the first presentation, as expected for a novel stimulus. Responsiveness of the different strains remained similar across the consecutive presentations (Figure 1A) with the survival analysis showing no significant difference in median survival time (SHR=9.58; WKY=10.00; WIS=9.50) between strains (U(2)=25.0; p=0.882). In the 5 second periods either side of the stimulus tone being on, animals were not responsive to the stimulus object and this remained the case for all stimulus presentations. In terms of response duration, all three strains spent a similar amount of time responding to the first stimulus presentation (Figure 1B; SHR: 50.67±11.21% of total time or 2.53±0.56 s; WIS: 48.25±10.93% of total time or 2.41±0.58 s; WKY: 52.00±7.48% of total time or 2.60±0.37 s). Repeated measures ANOVA with STIMULUS PRESENTATION as the within-subjects factor and STRAIN as the between-subjects factor was conducted using the percentage of overall time responding to the stimulus as the dependent variable. There was a significant main effect of STIMULUS PRESENTATION (F (5.61, 129.13)=8.01; p<0.001), with all animals spending significantly less time responding to the stimulus at later stimulus presentations, with significant decreases in response duration compared to the first stimulus beginning at the sixth stimulus (F (1, 23)=10.51; p=0.004). By the final stimulus there was a highly significant time difference in response duration relative to the initial response (F (1, 23)=65.54; p<0.001). There was no main effect of STRAIN (F (2, 23)=1.05; p=0.365) or STIMULUS PRESENTATION x STRAIN interaction (F(11.23, 129.13)=0.583; p=0.843).
Repeated measures ANOVA with TIME as the within-subjects factor and STRAIN as the between-subjects factor was used to analyse locomotor activity for the four different measures (distance travelled, vertical activity, average velocity and stereotypic activity Figure 2) in order to be sure that locomotor activity did not confound measures of distractible behaviour. There was no main effect of STRAIN for average velocity (F(2, 23)=0.66; p=0.528) or stereotypic activity (F(2, 23)=0.44; p=0.650). However, there was a main effect of STRAIN for distance travelled (F(2, 23)=4.10; p=0.030), with post hoc (Tukey HSD) analysis revealing that there was a trend towards the WKY moving significantly less distance than the WIS (p=0.052) and SHR (p=0.056), and no significant difference between the WIS and SHR (p=0.994). There was also a main effect of STRAIN on vertical activity (F(2, 23)=4.12; p=0.029), with post hoc (Tukey HSD) analysis showing that the SHR were significantly more vertically active than WKY (p=0.023) but not the WIS (p=0.480). There were no significant differences between WIS and WKY (p=0.265). As may be expected for locomotor activity in a confined space, there was a main effect of TIME, with parameters decreasing with increasing time within the chamber as the environment became familiar through exploration for distance travelled (F (5, 115)=67.46; p<0.001), stereotypic activity (F(5, 115)=31.57; p<0.001) and vertical activity (F(3.07, 70.71)=11.02; p<0.001). There was no main effect of TIME on average velocity (F(3.63, 83.41)=2.38; p=0.064). There were no significant TIME x STRAIN interactions for average velocity (F(7.25, 83.41)=1.02; p=0.423), stereotypic activity (F(5, 115)=1.63; p=0.125) and vertical activity (F(6.15, 70.71)=2.08; p=0.070). There was a TIME x STRAIN interaction for the distance travelled (F(10, 115)=5.15; p<0.001). Restricted ANOVAs revealed this significant interaction to be due to differences between the WIS and the other two strains in the first ten minutes with the WIS showing a greater decrease during this period.
2.2 The SHR is less likely to respond to low intensity auditory stimulation and has lower amplitude LFP responses and greater onset latency in multiunit responses.
Eighty-three auditory responses were recorded from the intermediate and deep layers of the SC (Figure 3); 46 were positioned in the Intermediate Grey (InG; 14 SHR; 15 WIS; 17 WKY), 3 were recorded from the Intermediate White (InW; 1 SHR; 1 WIS; 1 WKY), with the remaining 34 responses were recorded in the deep layers (Deep Grey, DpG; 10 SHR; 12 WIS; 10 WKY; Deep White DpWh; 2 SHR). Chi-square analysis showed there was no significant association between strain and the layer from which recordings were made (χ2(6)=4.58; p=0.60).
The percentage of animals showing responses at each of the five stimulus intensities is shown by strain in Table 1. Chi-square analysis revealed a significant association between strain and responsiveness for the lowest stimulus intensity in both local field potential responses (χ2(2)=9.902; p=0.0.007) and multiunit activity (χ2(2)=6.696; p=0.035). Examination of the effect size of these analyses showed that the association between strain and responsiveness had a larger effect size for the LFP responses (φ=0.345) than the MUA (φ=0.284). Restricted Fisher’s Exact tests showed that for the LFP response the WKY was more likely to respond than the SHR (p=0.003) but not the WIS (p=0.282) and there was no significant strain associations when only the SHR and WIS (p=0.089) were compared. The same pattern was observed for the multiunit responses with the WKY more likely to respond than the SHR (p=0.026) but not the WIS (p=0.102) and with no differences between the WIS and SHR (p=0.758) in terms of responsiveness.
Local field potential responses: In order to analyse the impact of stimulus intensity on local field potential responses (example shown in Figure 4A) onset latency, peak-to-peak amplitude and duration, data from the animals that responded to the highest three stimulus intensities were analysed (SHR n=22; WKY n=24; WIS n=22) using repeated measures ANOVA with STIMULUS INTENSITY as the within-subjects factor and STRAIN as the between-subjects factor. These analyses revealed a significant main effect of STIMULUS INTENSITY (F(2, 130)=5.44; p=0.005) for onset latency, with the only significant difference being a decrease in latency between the 65 and 70 dB SPL (F(1, 65)=10.24; p=0.002). There was no significant main effect of STRAIN (F(2, 65)=2.79; p=0.069) or STIMULUS INTENSITY x STRAIN interaction (F (4, 130)=0.94; p=0.442) (Figure 4B). Analysis of peak-to-peak amplitude revealed no significant main effect of STIMULUS INTENSITY (F(1.78, 115.48)=2.67; p=0.080), but a significant main effect of STRAIN (F(2, 65)=8.46; p=0.001). Post hoc Tukey (HSD) analyses revealed that the SHR had significantly smaller responses than both the WKY (p=0.001) and WIS (p=0.012). The WKY and WIS did not differ from each other (p=0.513). There was no significant STIMULUS INTENSITY x STRAIN interaction (F (3.55, 115.48)= 1.99; p=0.108) (Figure 4C). The duration of the responses is shown in Figure 4D. As with amplitude, there was no significant main effect of STIMULUS INTENSITY (F(1.73, 112.59)=0.067; p=0.913), but there was also no significant main effect of STRAIN (F(2, 65)=0.598; p=0.553) or STIMULUS INTENSITY x STRAIN interaction (F(3.46, 112.59)=2.35; p=0.067).