A distinguishing characteristic of the dorsal cochlear nucleus (DCN) is that it is the first site in the ascending auditory pathway that integrates acoustic information relayed by the auditory nerve with multimodal inputs from several brain regions that convey somatosensory information important for sound source discrimination and localization. These two independent streams converge upon distinct dendritic regions of fusiform cells, principal neurons that provide the main output of the DCN. We are investigating the cellular and synaptic mechanisms underlying fusiform cell responses to sounds, and how these responses can be influenced by somatosensory stimuli. To address these questions we combine in-vivo recordings with brain-slice electrophysiology and cellular imaging techniques. Our progress in the past year is outlined below. 1. First, we have developed techniques to record in-vivo sound-evoked responses from individual neurons in the mouse DCN. Despite the importance of the mouse as a model for studying auditory function, the sound-evoked response properties of the different classes of neurons in the mouse DCN are not well-characterized. Therefore we developed surgical techniques for recording from the mouse dorsal cochlear nucleus in vivo using both decerebrate and anesthetized preparations. Determining how the different classes of neurons in the mouse DCN respond to sounds is a critical first step in developing hypotheses about the underlying synaptic mechanisms in this circuit. Our results show that fusiform cell responses in the mouse differ from those found in previous studies in the cat, primarily in exhibiting an overall lower amount of inhibition in their receptive fields. We have also identified a novel subset of broadly tuned responses that have not been observed previously. Our initial study has been completed and submitted for publication. Current experiments are underway to combine intracellular recordings and anatomical labeling techniques to establish a more direct link between specific neuron types and sound-evoked response properties. Intracellular recordings will also permit more detailed understanding of the underlying circuit mechanisms that generate the observed responses of fusiform cells, in particular the role of feedforward inhibition by tuberculoventral cells in shaping fusiform cell output. 2. We have also made progress in applying optogenetic techniques to study the influence of somatosensory inputs on sound-evoked responses in fusiform cells. We examined two transgenic mouse lines that express channelrhodopsin, a light-activated ion channel, in various cell types under a general promoter. However, after evaluating the channelrhodopsin expression patterns in their cochlear nuclei, we have determined that the lack of cell-type specificity in these lines renders them unsuitable for our in vivo and acute slice experiments. Currently, we are evaluating other transgenic mouse lines characterized by cell type-specific channelrhodopsin labeling patterns. We are also using stereotaxic injections of adenoviral constructs in the mouse brainstem to express channelrhodopsin in specific populations of neurons. These experiments will allow us to combine optical stimulation of non-auditory inputs to DCN with sound presentation in order to determine the mechanisms by which fusiform cells integrate auditory and non-auditory information. 3. In addition, we have investigated the role of neuromodulatory pathways that influence DCN function. Endocannabinoids act as retrograde messengers that are released from postsynaptic neurons and regulate synaptic transmission at synapses in many brain regions, including auditory synapses, but their role in auditory function is not known. We have demonstrated a novel mechanism of activity- and calcium-dependent endocannabinoid release from DCN cartwheel cells, glycinergic neurons that suppress fusiform cell activity. Our results show that parallel fiber excitation of cartwheel cells increases their firing rates and causes elevation of dendritic calcium that evokes release of endocannabinoids, suppressing parallel fiber synapses in a heterosynaptic manner. This study has been completed and submitted for publication. We also have recently identified a cartwheel cell-specific marker that will facilitate current experiments focused on characterizing the molecular components underlying relevant forms of synaptic plasticity that influence the output of DCN.

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National Institute on Deafness and Other Communication Disorders
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Sedlacek, Miloslav; Brenowitz, Stephan D (2014) Cell-type specific short-term plasticity at auditory nerve synapses controls feed-forward inhibition in the dorsal cochlear nucleus. Front Neural Circuits 8:78
He, Shan; Wang, Ya-Xian; Petralia, Ronald S et al. (2014) Cholinergic modulation of large-conductance calcium-activated potassium channels regulates synaptic strength and spine calcium in cartwheel cells of the dorsal cochlear nucleus. J Neurosci 34:5261-72
Brenowitz, Stephan D; Regehr, Wade G (2014) Presynaptic calcium measurements using bulk loading of acetoxymethyl indicators. Cold Spring Harb Protoc 2014:750-7
Brenowitz, Stephan D; Regehr, Wade G (2012) Presynaptic imaging of projection fibers by in vivo injection of dextran-conjugated calcium indicators. Cold Spring Harb Protoc 2012:465-71
Ma, Wei-Li Diana; Brenowitz, Stephan D (2012) Single-neuron recordings from unanesthetized mouse dorsal cochlear nucleus. J Neurophysiol 107:824-35
Wagner, Wolfgang; Brenowitz, Stephan D; Hammer 3rd, John A (2011) Myosin-Va transports the endoplasmic reticulum into the dendritic spines of Purkinje neurons. Nat Cell Biol 13:40-8
Sedlacek, Miloslav; Tipton, Philip W; Brenowitz, Stephan D (2011) Sustained firing of cartwheel cells in the dorsal cochlear nucleus evokes endocannabinoid release and retrograde suppression of parallel fiber synapses. J Neurosci 31:15807-17
Wang, Philip Y; Petralia, Ronald S; Wang, Ya-Xian et al. (2011) Functional NMDA receptors at axonal growth cones of young hippocampal neurons. J Neurosci 31:9289-97