1. Introduction
The type 1 cannabinoid receptors (CB1Rs) are widely expressed throughout the central nervous system (CNS) (Domenici et al., 2006; Castillo et al., 2012; Kendall and Yudowski, 2016a; Barti et al., 2024; B et al., 2024a), particularly in areas such as the hippocampus, basal ganglia, and cerebellum. Activations of CB1Rs by endocannabinoids such as 2-arachidonoyl glycerol (2-AG) and anandamide (AEA) can modulate neurotransmitter release and impact a myriad of physiological functions, such as stress, learning, memory, cognitive and sensory processing (Marsicano et al., 2002; Stachowicz, 2023; Barti et al., 2024) (Zou & Kumar, 2018). CB1Rs are expressed in both excitatory and inhibitory neurons, and their specific functions can be different depending on the type of neuron in which they are expressed. (Kendall and Yudowski, 2016b; Busquets-Garcia et al., 2018b, 2018a; Barti et al., 2024). For instance, Busquets-Garcia et al. have found that CB1Rs are predominantly expressed on certain subpopulations of GABAergic interneurons in hippocampus and the deletion of CB1Rs in these neurons impairs incidental associative learning (Busquets-Garcia et al., 2018a, 2018b). Recently, Dudok et al. have employed advanced imaging techniques to observe endocannabinoid (eCB) signaling in the hippocampal CA1 region of mice during navigation tasks, and demonstrated that CB1Rs are selectively expressed at synapses of CCK-expressing interneurons and genetic deletion of CB1Rs in these interneurons results in altered place cell firing tuning, suggesting that CB1Rs-mediated modulation of inhibitory synapses is crucial for the proper encoding of spatial information during behavior (B et al., 2024a). These above researches highlight CB1Rs importance in modulating synaptic plasticity and neuronal activity during behavior and underscore the critical role of eCB signaling in fine-tuning neural circuits involved in spatial navigation and memory formation. While much has been uncovered regarding the specific functions of CB1Rs in distinct neuronal subtypes in the central nervous system, less attention has been paid to central auditory system specifically.
CB1Rs are highly expressed in the central auditory system across several species, including rodents, humans, and other mammals (Tzounopoulos et al., 2007; Zheng et al., 2007; Y et al., 2009; Zhao et al., 2011; X et al., 2020; S et al., 2022). For instance, the distribution of CB1Rs in the adult rat brain, including the cochlear nucleus, has been investigated using receptor binding radioautography and in situ hybridization histochemistry (Mailleux and Vanderhaeghen, 1992). In the cochlear nucleus, CB1Rs are predominantly found in regions associated with synaptic processing, particularly in the dorsal cochlear nucleus (DCN) (Mailleux and Vanderhaeghen, 1992). Further studies using immunohistochemistry have confirmed CB1Rs are present in the DCN of mice (Tzounopoulos et al., 2007; Y et al., 2009; Zhao et al., 2011). Moreover, Zheng et al. have demonstrated that CB1Rs are localised to many different cell types in the cochlear nucleus of rat (Zheng et al., 2007). More recently, Alejando et al. have found that CB1Rs are expressed in inferior colliculus (IC) and auditory cortex (AC) of hamster through RT-qPCR and immunostaining (Fuerte-Hortigón et al., 2021). Consistently, evidence has suggested that CB1Rs are expressed in AC of human and non-human primate (S et al., 2022). Liu et al. have investigated the transcriptional abundance of Cnr1 (CB1Rs encoding gene) mRNA in the brains from adult C57BL/6J mice of two sexes using RNAscope in situ hybridization (ISH) (X et al., 2020), and CB1Rs are found in the IC and AC. Although the expression of CB1Rs in the central auditory system has been extensively studied, the spatial distribution of CB1Rs across different auditory nuclei is still not fully understood.
Accumulating evidence indicate that CB1Rs in central auditory system modulate neurotransmitter release, synaptic plasticity, and may play roles in normal physiological function (18,19). CB1Rs have been implicated in the pathology of tinnitus (Zheng et al., 2007, 2015). Moreover, a previous study has suggested that deleting the CB1Rs gene does affect basic hearing abilities of mice (Anon, n.d.). However, the exact roles of CB1Rs in the central auditory system and in different types of neurons, such as inhibitory interneurons or excitatory projection neurons, remain largely unexplored.
To address these issues, we first systematically characterized the neuronal localization and distribution of CB1Rs in several central auditory nuclei of mice, including the cochlear nucleus (CN), superior olivary complex (SOC), inferior colliculus (IC), lateral lemniscus (LL), medial geniculate body (MGB), and auditory cortex (AC), using fluorescence immunohistochemical techniques. Our findings demonstrated that CB1R proteins are highly expressed in the CN, SOC and LL. We next generated conditional knockout mice with Cnr1 specifically deleted from PV interneurons, a major type of GABAergic interneurons in the central auditory system, and explored how CB1R deficiency in these neurons affects hearing function using auditory brainstem response (ABR) tests. Interestingly, the conditional deletion of Cnr1 in PV interneurons resulted in hearing loss at 32 KHz and abnormal auditory brainstem responses. This work may have broader implications for understanding how cannabinoid signaling influences sensory processing and provide further insights into CB1Rs as a potential pharmaceutical target for treating auditory dysfunctions.