Late postnatal shifts of parvalbumin and nitric oxide synthase expression within the GABAergic and glutamatergic phenotypes of inferior colliculus neurons
Extensive GABAergic inhibitory and glutamatergic excitatory networks have already been established at postnatal day 2 (P2) in the IC (Sturm et al., 2014). Following birth, sensitivity to auditory stimulus is rapidly acquired, and various functions of hearing achieve maturity during the early development. The first low‐frequency responses without tonotopy are recorded from neurons in the rostral and central parts of the ICC at P10, and refinement of the tonotopy is finished around P20 in house mice (Romand and Ehret, 1990). However, some aspects, such as sound source localization and temporal processing, do not achieve mature performance for a long time, even into adolescence in humans (Moore, 2002). Development of the auditory brainstem response characterized by rapid decrease in wave latency and increases in amplitude and sensitivity starts late in 2‐week‐old, and changes continue through the late postnatal period, in gerbils (Smith and Kraus, 1987).
GABAergic neurons in the auditory pathway play a wide variety of roles, such as the shaping of neuronal receptive fields and response profiles (Jones, 1993). To identify and classify neurons in the auditory brainstem, a number of studies have used various molecular markers, especially for GABAergic neurons in the cortex and hippocampus (Kawaguchi and Kubota, 1997; Jinno and Kosaka, 2006), e.g., parvalbumin (PV) (Fredrich et al., 2009), and nitric oxide synthase (NOS) that is also detected by nicotinamide adenine dinucleotide phosphate‐diaphorase (NADPH‐d) activity (Wu et al., 2008). The cellular distributions of PV and NOS in the IC have also been well analyzed: PV‐expressing (PV+) neurons are mainly distributed in the rat ICC (Lohmann and Friauf, 1996), while the majority of NOS+ neurons are located in the rat DC and LC (Herbert et al., 1991; Druga and Syka, 1993). Despite these findings, there have been few studies that have examined whether PV and NOS are actually expressed in GABAergic neurons in the auditory brainstem. Interestingly, however, recent studies using rodents and monkeys indicate that a certain population of IC neurons expressing these molecular markers may represent the non‐GABAergic phenotype (Fredrich et al., 2009; Gray et al., 2014). This drives us to the question whether PV and NOS are proper markers for GABAergic neurons in the IC.
In the present study we examined the phenotype of IC neurons expressing PV and/or NOS in C57BL/6J mice during the late postnatal period (2‐ and 8‐month‐old). Fluorescent triple immunostaining for PV, NOS, and glutamic acid decarboxylase 67 (GAD67) showed that IC neurons could be largely classified into four types. Fluorescent in situ hybridization showed that almost all GAD67‐lacking (GAD67−) IC neurons expressed vesicular glutamate transporter 2 mRNA, i.e., these neurons represented the glutamatergic phenotype.