The localization and physiological effects of cannabinoid receptor 1 in the brain stem auditory system of the chick

Acoustic stimuli drive high rates of neural activity and these stimuli can be relatively long-lasting. In vivo spontaneous firing rates average 100Hz even lacking auditory stimuli (Warchol and Dallos, 1990). Neurons must be able to respond in a consistent manner throughout the entirety of the stimulus to code this information effectively. Additionally, the encoding of important information about acoustic stimuli is not simply a duration code as it requires that action potentials be precisely timed with respect to the acoustic stimulus. Neurons in the brain stem auditory system possess a number of features that optimize their ability to maintain precisely timed and sustained responses. Important adaptations to the rigorous demands of coding acoustic information have been identified in the cochlear nucleus of the chick, nucleus magnocellularis (NM). This area is key for processing time-locked signals from the auditory nerve (cnVIII) and relaying that information to nucleus laminaris (NL), where the analyses of interaural time differences assist in tasks such as the localization of a sound source (Sullivan and Konishi, 1984, Takahashi et al., 1984, Carr and Konishi, 1990, Warchol and Dallos, 1990, Overholt et al., 1992). One adaptation towards accomplishing NM's key role of accurately coding timed information can be seen in the nature of its innervation. NM neurons receive large excitatory calyceal input from only 2-3 cnVIII fibers (Carr and Boudreau, 1991). Each of these afferents is capable of producing suprathreshold currents through a large release of glutamate, resulting in a high neurotransmission safety factor (Parks and Rubel, 1978, Hackett et al., 1982, Zhang and Trussell, 1994b, a). NM neurons produce a single, strong excitatory post-synaptic potential (EPSP) mediated primarily via AMPA receptor activation. Little jitter or temporal summation is observed which is aided by low-threshold K+ channels to help ensure a minimal refractory period (Reyes et al., 1994, Zhang and Trussell, 1994b). While these synaptic adaptations allow NM to fire at very high rates, synaptic depression can become a serious impediment to the coding of ongoing auditory stimuli. This short-term effect is seen as a progressive decline in the size of postsynaptic responses to the same stimulus. AMPA receptor desensitization appears to be a major limitation of sustained responses (Trussell et al., 1993, Otis et al., 1996), but vesicle depletion presents a second limitation to sustained action potential (AP) firing (Brenowitz and Trussell, 2001). In addition, glutamatatergic autoreceptor activation may contribute to this phenomenon (Barnes-Davies and Forsythe, 1995, von Gersdorff et al., 1997). Failing to limit synaptic depression would be detrimental to temporal coding as it would prevent NM from coding throughout the duration of a stimulus. Strong synaptic depression is seen in both NM (Hackett et al., 1982, Zhang and Trussell, 1994a) and NL (Kuba et al., 2002, Cook et al., 2003) in brain stem slice preparations. It is possible that the procedure itself, i.e. extraction of a brain slice, removes endogenous signals necessary to attenuate depression. For example, descending GABAergic inhibition from the superior olivary nucleus (SON) may play a dual role, working both pre- and post-synaptically at the cnVIII-NM synapse to preserve temporal coding (Carr et al., 1989, Code et al., 1989, Lachica et al., 1994). GABAA receptors influence NM excitability, tightening the window for summation postsynaptically (Hyson et al., 1995, Monsivais et al., 2000) while GABAB receptors help conserve glutamate release during high rates of activity (Brenowitz et al., 1998). In doing so, GABAB activation reduces synaptic depression across a train of stimuli, limiting the progressive decrease in response. Another potential mechanism for regulating glutamatergic signaling is cannabinoid (CB) signaling, which has already been shown to have a modulatory role in other sensory systems (Straiker et al., 1999a, Straiker et al., 1999b, Czesnik et al., 2007, Deshmukh et al., 2007, Thornton-Jones et al., 2007, Nyilas et al., 2009). Our laboratory recently reported the distribution of the cannabinoid receptor 1 (CB1) mRNA throughout the brain of the chick (Stincic and Hyson, 2008). While little mRNA expression was found in NM or NL, the cnVIII ganglion (vestibular portion) displayed robust CB1 mRNA expression. This finding suggested that CB1 mRNA expression may be found in the cochlear ganglion (CG) as well. CB1 is expressed in the dorsal cochlear nucleus of rats (Mailleux and Vanderhaeghen, 1992) and CBs can modulate synaptic plasticity in auditory nuclei (Penzo and Pena, 2009, Zhao et al., 2009). CB mediated retroinhibition has also been reported in the medial nucleus of the trapezoid body (MNTB) (Kushmerick et al., 2004). This is significant because the MNTB is an auditory nucleus that also receives calyceal input. Taken together, these previous reports suggest that CB signaling in the chick auditory brain stem is likely as well. CB signaling acts primarily through metabotropic receptors that share some traits with classical neurotransmitter systems, but differs in key aspects. When activated, the receptors trigger 2nd messenger signaling cascades through Gi/o-proteins (Howlett and Fleming, 1984, Howlett, 1985, Howlett et al., 1986). Vesicles, however, are not required for transport since endogenous CBs (eCBs) are lipophillic and capable of crossing the cell membrane either unaided or by facilitated diffusion (Beltramo et al., 1997, Hillard et al., 1997). Once released into the synapse from postsynaptic cells, eCBs activate CB receptors located on the presynaptic cell. Functional CB1 has been localized presynaptically to axonal terminals with almost no exceptions in adult animals (Schlicker and Kathmann, 2001). A major effect of CB1 activation is inhibition of voltage-gated Ca2+ channels (N-type and P/Q-type) (Caulfield and Brown, 1992, Felder et al., 1993, Mackie et al., 1993), leading to a reduction in the number of vesicles released upon arrival of the action potential to the terminal. In this way, CB1 activation can reduce the amount of neurotransmitter released into the synaptic cleft, subsequently lessening the factors that contribute to synaptic depression. If CB signaling is an additional adaptation for optimizing coding at the cnVIII-NM synapse, it must be present and effective at modulating synaptic transmission. Our study first establishes the presence of CB1 at the cnVIII-NM synapse using in situ hybridization (ISH) and immunohistochemistry (IHC), and then documents the influence of CB1 activation on synaptic depression.