Sensory processing in the lower auditory pathway is generally considered to be rigid and thus less subject to modulation than central processing. However, in addition to the powerful bottom-up excitation by auditory nerve fibers, the ventral cochlear nucleus also receives efferent cholinergic innervation from both auditory and nonauditory top–down sources. We thus tested the influence of cholinergic modulation on highly precise time-coding neurons in the cochlear nucleus of the Mongolian gerbil. By combining electrophysiological recordings with pharmacological application in vitro and in vivo, we found 55–72% of spherical bushy cells (SBCs) to be depolarized by carbachol on two time scales, ranging from hundreds of milliseconds to minutes. These effects were mediated by nicotinic and muscarinic acetylcholine receptors, respectively. Pharmacological block of muscarinic receptors hyperpolarized the resting membrane potential, suggesting a novel mechanism of setting the resting membrane potential for SBC. The cholinergic depolarization led to an increase of spike probability in SBCs without compromising the temporal precision of the SBC output in vitro. In vivo, iontophoretic application of carbachol resulted in an increase in spontaneous SBC activity. The inclusion of cholinergic modulation in an SBC model predicted an expansion of the dynamic range of sound responses and increased temporal acuity. Our results thus suggest of a top–down modulatory system mediated by acetylcholine which influences temporally precise information processing in the lower auditory pathway.

Located in the midbrain, the inferior colliculus (IC) is the hub of the central auditory system. Although the IC plays important roles in speech processing, sound localization, and other auditory computations, the organization of the IC microcircuitry remains largely unknown. Using a multifaceted approach in mice, we have identified vasoactive intestinal peptide (VIP) neurons as a novel class of IC principal neurons. VIP neurons are glutamatergic stellate cells with sustained firing patterns. Their extensive axons project to long-range targets including the auditory thalamus, auditory brainstem, superior colliculus, and periaqueductal gray. Using optogenetic circuit mapping, we found that VIP neurons integrate input from the contralateral IC and the dorsal cochlear nucleus. The dorsal cochlear nucleus also drove feedforward inhibition to VIP neurons, indicating that inhibitory circuits within the IC shape the temporal integration of ascending inputs. Thus, VIP neurons are well-positioned to influence auditory computations in a number of brain regions.

10Located in the midbrain, the inferior colliculus (IC) is the hub of the central auditory system. Although 11 the IC plays important roles in speech processing, sound localization, and other auditory computations, 12 the organization of the IC microcircuitry remains largely unknown. Using a multifaceted approach in 13 mice, we have identified vasoactive intestinal peptide (VIP) neurons as a novel class of IC principal 14 neurons. VIP neurons are glutamatergic stellate cells with sustained firing patterns. Their extensive 15 axons project to long-range targets including the auditory thalamus, auditory brainstem, superior 16 colliculus, and periaqueductal gray. Using optogenetic circuit mapping, we found that VIP neurons 17 integrate input from the contralateral IC and the dorsal cochlear nucleus. The dorsal cochlear nucleus 18 also drove feedforward inhibition to VIP neurons, indicating that inhibitory circuits within the IC shape 19 the temporal integration of ascending inputs. Thus, VIP neurons are well-positioned to influence 20 auditory computations in a number of brain regions. 21 Results 67The VIP-IRES-Cre mouse line labels neurons in multiple subdivisions of the IC 68 By crossing VIP-IRES-Cre mice with Ai14 reporter mice, we obtained mice in which VIP + neurons 69 expressed the fluorescent protein tdTomato. VIP + neurons in these animals were visible in numerous 70 brain regions; the present report focuses on the IC. Figure 1 shows the distribution of VIP + neurons 71 (magenta) in transverse sections through the IC. Labeled neurons were present throughout most of the 72 rostro-caudal extent of the IC, but were most numerous in the caudal regions. Labeled neurons were 73 rare or absent in the IC rostral pole and intercollicular tegmentum. 74 75 VIP neurons are glutamatergic and represent 1.8% of IC neurons 76 Previous studies have shown that IC neurons are either glutamatergic or GABAergic (Merchán et al., 77 2005; Oliver et al. , 1994). To investigate the neurotransmitter content of VIP neurons, we performed 78 immunohistochemical staining against GAD67, a marker for GABAergic neurons, in brain slices from 79 three VIP-IRES-Cre x Ai14 animals, aged P58 (Figure 2A). We then counted tdTomato + neurons and 80 GAD67-labeled cell bodies in one caudal and one rostral IC slice per animal. Neurons located in the ICc, 81ICd, and IClc were combined for this analysis. Across 793 tdTomato + neurons, only 10 neurons co-82 VIP neurons exhibit sustained firing patterns and their intrinsic physiology varies along 101the tonotopic axis of the ICc 102Next, we investigated the firing pattern and intrinsic physiology of VIP neurons by targeting whole cell 103 patch clamp recordings to tdTomato + neurons in brain slices from VIP-IRES-Cre x Ai14 mice. Recordings 104 made from the ICc, ICd, and IClc were lumped together for this experiment because there were no clear 105 differences in VIP neuron physiology across these subdivisions of the IC. VIP neurons had a resting 106 membrane potential of -69.5 mV ± 4.4 mV (n = 216, correc...

Principal neurons in the ventral cochlear nucleus (VCN) receive powerful ascending excitation and pass on the auditory information with exquisite temporal fidelity. Despite being dominated by ascending inputs, the VCN also receives descending cholinergic connections from olivocochlear neurons and from higher regions in the pontomesencephalic tegmentum. In Mongolian gerbils, acetylcholine acts as an excitatory and modulatory neurotransmitter on VCN neurons, but the anatomical structure of cholinergic innervation of gerbil VCN is not well described. We applied fluorescent immunohistochemical staining to elucidate the development and the cellular localization of presynaptic and postsynaptic components of the cholinergic system in the VCN of the Mongolian gerbil. We found that cholinergic fibers (stained with antibodies against the vesicular acetylcholine transporter) were present before hearing onset at P5, but innervation density increased in animals after P10. Early in development cholinergic fibers invaded the VCN from the medial side, spread along the perimeter and finally innervated all parts of the nucleus only after the onset of hearing. Cholinergic fibers ran in a rostro-caudal direction within the nucleus and formed en-passant swellings in the neuropil between principal neurons. Nicotinic and muscarinic receptors were expressed differentially in the VCN, with nicotinic receptors being mostly expressed in dendritic areas while muscarinic receptors were located predominantly in somatic membranes. These anatomical data support physiological indications that cholinergic innervation plays a role in modulating information processing in the cochlear nucleus.

In the avian nucleus magnocellularis (NM) endbulb of Held giant synapses develop from temporary bouton terminals. The molecular regulation of this process is not well understood. Furthermore, it is unknown how the postsynaptic specialization of the endbulb synapses develops. We therefore analysed expression of the postsynaptic scaffold protein PSD-95 during the transition from bouton-to-endbulb synapses. PSD-95 has been implicated in the regulation of the strength of glutamatergic synapses and could accordingly be of functional relevance for giant synapse formation. PSD-95 protein was expressed at synaptic sites in embryonic chicken auditory brainstem and upregulated between embryonic days (E)12 and E16. We applied immunofluorescence staining and confocal microscopy to quantify pre-and postsynaptic protein signals during bouton-to-endbulb transition. Giant terminal formation progressed along the tonotopic axis in NM, but was absent in low-frequency NM. We found a tonotopic gradient of postsynaptic PSD-95 signals in NM. Furthermore, PSD-95 immunosignals showed the greatest increase between E12 and E15, temporally preceding the bouton-to-endbulb transition. We then applied whole-cell electrophysiology to measure synaptic currents elicited by synaptic terminals during bouton-to-endbulb transition. With progressing endbulb formation postsynaptic currents rose more rapidly and synapses were less susceptible to short-term depression, but currents were not different in amplitude or decay-time constant. We conclude that development of presynaptic specializations follows postsynaptic development and speculate that the early PSD-95 increase could play a functional role in endbulb formation.

When investigating neural circuits, a standard limitation of the in vitro patch clamp approach is that axons from multiple sources are often intermixed, making it difficult to isolate inputs from individual sources with electrical stimulation. However, by using channelrhodopsin assisted circuit mapping (CRACM) this limitation can now be overcome. Here, we report a method to use CRACM to map ascending inputs from lower auditory brainstem nuclei and commissural inputs to an identified class of neurons in the inferior colliculus (IC), the midbrain nucleus of the auditory system. In the IC, local, commissural, ascending, and descending axons are heavily intertwined and therefore indistinguishable with electrical stimulation. By injecting a viral construct to drive expression of a channelrhodopsin in a presynaptic nucleus, followed by patch clamp recording to characterize the presence and physiology of channelrhodopsin-expressing synaptic inputs, projections from a specific source to a specific population of IC neurons can be mapped with cell type-specific accuracy. We show that this approach works with both Chronos, a blue lightactivated channelrhodopsin, and ChrimsonR, a red-shifted channelrhodopsin. In contrast to previous reports from the forebrain, we find that ChrimsonR is robustly trafficked down the axons of dorsal cochlear nucleus principal neurons, indicating that ChrimsonR may be a useful tool for CRACM experiments in the brainstem. The protocol presented here includes detailed descriptions of the intracranial virus injection surgery, including stereotaxic coordinates for targeting injections to the dorsal cochlear nucleus and IC of mice, and how to combine whole cell patch clamp recording with channelrhodopsin activation to investigate long-range projections to IC neurons. Although this protocol is tailored to characterizing auditory inputs to the IC, it can be easily adapted to investigate other long-range projections in the auditory brainstem and beyond.

No abstract

When investigating neural circuits, a standard limitation of the in vitro patch clamp approach is that axons from multiple sources are often intermixed, making it difficult to isolate inputs from individual sources with electrical stimulation. However, by using channelrhodopsin assisted circuit mapping (CRACM) this limitation can now be overcome. Here, we report a method to use CRACM to map ascending inputs from lower auditory brainstem nuclei and commissural inputs to an identified class of neurons in the inferior colliculus (IC), the midbrain nucleus of the auditory system. In the IC, local, commissural, ascending, and descending axons are heavily intertwined and therefore indistinguishable with electrical stimulation. By injecting a viral construct to drive expression of a channelrhodopsin in a presynaptic nucleus, followed by patch clamp recording to characterize the presence and physiology of channelrhodopsin-expressing synaptic inputs, projections from a specific source to a specific population of IC neurons can be mapped with cell type-specific accuracy. We show that this approach works with both Chronos, a blue light-activated channelrhodopsin, and ChrimsonR, a red-shifted channelrhodopsin. In contrast to previous reports from the forebrain, we find that ChrimsonR is robustly trafficked down the axons of dorsal cochlear nucleus principal neurons, indicating that ChrimsonR may be a useful tool for CRACM experiments in the brainstem. The protocol presented here includes detailed descriptions of the intracranial virus injection surgery, including stereotaxic coordinates for targeting injections to the dorsal cochlear nucleus and IC of mice, and how to combine whole cell patch clamp recording with channelrhodopsin activation to investigate long-range projections to IC neurons. Although this protocol is tailored to characterizing auditory inputs to the IC, it can be easily adapted to investigate other long-range projections in the auditory brainstem and beyond.SUMMARYChannelrhodopsin-assisted circuit mapping (CRACM) is a precision technique for functional mapping of long-range neuronal projections between anatomically and/or genetically identified groups of neurons. Here, we describe how to utilize CRACM to map auditory brainstem connections, including the use of a red-shifted opsin, ChrimsonR.