Tinnitus, commonly referred to as ringing in the ears, is likely initiated by damage to the auditory periphery. The resulting partial deafferentation of the cochlear nerve initiates a series of complex maladaptive plastic changes at multiple levels of the central auditory pathway and in non-auditory, cortical, limbic and attentional structures (Kujawa and Liberman, 2009; Shore and Wu, 2019; Henton and Tzounopoulos, 2021; McGill et al., 2022; Kumar et al., 2023). Historically, compromised somatosensory and auditory inputs have been shown to cause plastic changes resulting in increased activity from deep-layer principal cells (Merzenich et al., 1983; Robertson and Irvine, 1989; Auerbach et al., 2014; Henton and Tzounopoulos, 2021; Kumar et al., 2023). These changes are posited to underpin neuropathic pain and tinnitus in their respective cortices (Kuner, 2010; Llano et al., 2012; Cichon et al., 2017; Henton and Tzounopoulos, 2021). Changes resulting from decreased auditory input canonically result in increased spontaneous activity, increased neural synchrony and increased bursting at multiple levels of the central auditory pathway (Brozoski et al., 2002; Kaltenbach et al., 2004; Brozoski and Bauer, 2005, 2005; Ma et al., 2006; Schaette and Kempter, 2006; Bauer et al., 2008; Roberts et al., 2010; Noreña, 2011; Auerbach et al., 2014; Geven et al., 2014; Kalappa et al., 2014; Ropp et al., 2014; Henton and Tzounopoulos, 2021). At the level of auditory cortex (A1), tinnitus-related maladaptive homeostatic changes in neurotransmission are often associated with decreased biomarkers for inhibitory GABAergic signaling and likely indirect increases in excitatory glutamatergic function (Scholl and Wehr, 2008; Roberts et al., 2010; Yang et al., 2011; Eggermont, 2013; Zhang, 2013; Caspary and Llano, 2017; Resnik and Polley, 2017; Ghimire et al., 2023a) (see review Table 2). Previous A1 studies describe tinnitus-related loss of GAD-65, an enzyme critical for GABA synthesis likely decreasing GABA synthesis and release (Miyakawa et al., 2019). The change in GABA availability can alter GABAAR number and/or GABAAR pharmacology via subunit switching (Wafford et al., 1993). Using a group of tinnitus rats from the same cohort as those used in the present study, Ghimire et al. (2023a) found that layer 5 principal neurons (PNs) were less sensitive to puff-applied GABA, producing smaller peak IPSCs in animals with behavioral evidence of tinnitus (Ghimire et al., 2023a). A recent mouse sound-exposure study found plastic decreases in selective A1 inhibitory interneurons after peripheral damage (Kumar et al., 2023). Collectively, studies suggest that neurons in deep A1 layers likely play an important role in tinnitus pathology through maladaptive changes in top-down excitability onto ascending subcortical auditory information as well as mis-integration of cortico-cortical information (Nelson et al., 2013; Williamson and Polley, 2019; Blackwell et al., 2020; Henton and Tzounopoulos, 2021). Excitatory PNs project glutamatergic information to subcortical nuclei, including the inferior colliculus and regions of the medial geniculate body (Barbour and Callaway, 2008; Stebbings et al., 2014; Sottile et al., 2017; Williamson and Polley, 2019).

Sound-exposures resulting in a partially compromised auditory periphery generally show significant increases in A1 gain/excitation which are seen from in vivo recordings as increases in sound-evoked potentials and single unit responses when compared to normal hearing controls (Rajan et al., 1993; Syka et al., 1994; Noreña, 2011; Eggermont, 2015). Few in vivo studies have used animal models of tinnitus to directly correlate an animal's behavioral tinnitus score to functional metrics (Kalappa et al., 2014; Wu et al., 2016; Ghimire et al., 2023a). In conjunction with a series of parallel in vitro studies (Ghimire et al., 2023a, 2023b), the present study compared single-unit responses from deep A1 layers in control and sound-exposed rats with behavioral evidence of tinnitus. There were significant positive correlations between behavioral measures of tinnitus and changes in sound-driven activity, spontaneous activity and burst metrics.