Synaptic Transmission between End Bulbs of Held and Bushy Cells in the Cochlear Nucleus of Mice

22 Mice that carry a mutation in a calcium binding domain of Otoferlin, the putative calcium sensor at hair cell 23 synapses, have normal distortion product otoacoustic emissions (DPOAEs) but auditory brain stem responses 24 (ABRs) are absent. In mutant mice mechanotransduction is normal but transmission of acoustic information to 25 the auditory pathway is blocked even before the onset of hearing. The cross sectional area of the auditory nerve 26 of mutant mice is reduced by 54% and the volume of ventral cochlear nuclei is reduced by 46% relative to 27 hearing controls. While the tonotopic organization was not detectably changed in mutants, the axons to end 28 bulbs of Held and the end bulbs themselves were smaller. In mutant mice bushy cells in the anteroventral 29 cochlear nucleus (aVCN) have the electrophysiological hallmarks of control cells. Spontaneous miniature 30 EPSCs occur with similar frequencies and have similar shapes in deaf as in hearing animals but they are 24% 31 larger in deaf mice. Bushy cells in deaf mutants are contacted by about 2.6 auditory nerve fibers compared to 32 about 2.0 in hearing controls. Furthermore, each fiber delivers more synaptic current, on average 4.8 nA 33 compared to 3.4 nA, in deaf versus hearing controls. The quantal content of evoked EPSCs is not different 34 between mutants and controls; the increase in synaptic current delivered in mutants is accounted for by the 35 increased response to the size of the quanta. Although responses to shocks presented at long intervals are larger 36 in mutants, they depress more rapidly than in hearing controls. 37 38 Introduction 39 Depriving the cochlear nuclei of acoustic input after the onset of hearing has long been known to affect 40 neuronal circuits in the cochlear nuclei (Trune and Morgan, 1988; Trune and Kiessling, 1988; Pasic and Rubel, 41 1989; Ryugo et al., 1998; Redd et al., 2000; Stakhovskaya et al., 2008). However, electrical activity occurs even 42 before the onset of hearing in auditory circuits; that activity, especially in the second postnatal week, regulates 43 synaptic transmission from hair cells (Kros et al., 1998; Beutner and Moser, 2001; Johnson et al., 2011; Johnson 44 et al., 2013). Before hearing begins, supporting cells in the cochlea induce action potentials in small groups of 45 adjacent inner hair cells that in turn evoke periodic bursts of suprathreshold responses in the auditory nerve that 46 are propagated to the cortex (Tritsch et al., 2007; Tritsch et al., 2010; Tritsch and Bergles, 2010). Synaptotagmin 47 IV contributes to shaping synaptic responses in adult inner hair cells and immature outer hair cells (Johnson et 48 al., 2010) but after the third postnatal day (P3) synaptic transmission between inner hair cells and spiral ganglion 49 neurons, the cells whose axons are auditory nerve fibers (ANFs) depends largely on the detection of calcium by 50 Otoferlin (Roux et al., 2006; Beurg et al., 2010). The output from the cochlea is significantly reduced and 51 altered in mice that lack normal Otoferlin (Pangrsic et al., 2010; Beurg et al. 2010). Downstream synapses do 52 not depend on Otoferlin but likely depend on synaptotagmin I as a calcium sensor as do other central synapses 53 (Wang et al., 2011). Mice that lack Otoferlin are thus naive of most normal, patterned spontaneous activity that 54 originates in the cochlea and propagates throughout the auditory system before the onset of hearing in wild type 55 animals. Here we examine how the reduction of electrical activity from inner hair cells affects downstream 56 neuronal circuits. 57 ANFs innervate the ipsilateral ventral (VCN) and dorsal cochlear nuclei (DCN) tonotopically. Fibers 58 that encode low-frequencies innervate ventral regions and fibers that encode high-frequencies innervate dorsal 59 regions. ANFs terminate on bushy cells in the anterior VCN (aVCN) with large synapses, end bulbs of Held 60 (Held, 1893; Brawer and Morest, 1975; Sento and Ryugo, 1989; Ryugo and Sento, 1991; Lauer et al., 2013). 61 End bulbs release glutamate which binds to postsynaptic AMPA receptors in the bushy cell membrane (Wang et 62 al., 1998; Gardner et al., 1999; Gardner et al., 2001). To understand how the lack of input from inner hair cells 63 even before the onset of hearing affects the function of the auditory pathway, we examine mice with a mutation 64 that prevents synaptic transmission between inner hair cells and spiral ganglion cells and measure synaptic 65 events in the bushy cell targets of spiral ganglion cells that receive input through end bulbs of Held. 66 The mice studied in these experiments have a point mutation resulting in a non-conserved substitution 67 of I319N in the second of six calcium domains of Otoferlin that results in the absence of its expression in 68 cochlear hair cells (Longo-Guess et al., 2007). Otoferlin is thought to sense calcium and regulate synaptic 69 transmission by inner hair cells (Roux et al., 2006; Johnson and Chapman, 2010). Otoferlin is a member of the 70 Ferlin family of proteins, all of which have been implicated in calcium regulated membrane fusion events and 71 are membrane-anchored, cytosolic proteins that are involved in vesicle fusion and/or membrane trafficking 72 (Yasunaga et al., 2000; Lek et al., 2012). Mutations in Otoferlin were first described from human pathologies in 73 several unrelated, consanguineous Lebanese families (Yasunaga et al., 1999). These deaf individuals have an 74 autosomal recessive, nonsyndromic prelingual form of deafness, DFNB9 (Yasunaga et al., 1999). A mutation 75 associated with a splice site in Otoferlin has been associated with auditory neuropathy spectrum disorder in 76 humans (Runge et al., 2013). In humans, Otoferlin has multiple long and short, alternatively spliced isoforms 77 but mice have only the long isoform (Yasunaga et al., 1999; Yasunaga et al., 2000). In mice Otoferlin appears in 78 inner hair cells at P4, after which it is responsible for the calcium induced exocytosis in inner hair cells; 79 synaptotagmin isoforms 1, 2 and 7 are also present in inner hair cells but none appear responsible for calcium 80 induced exocytosis before P3 (Beurg et al., 2010). Otoferlin also mediates synaptic transmission from outer hair 81 cells (Beurg et al., 2008) and is required for the replenishment of synaptic vesicles in hair cells (Pangrsic et al., 82 2010). 83 84 Materials and Methods 85 Mice 86 Otoferlin mutant animals Otof deaf5Jcs/Kjn, were purchased from Jackson Labs, stock #006128. An ENU 87 induced point mutation from Thymine to Adenine in exon 10 of the otoferlin gene causes a non-conserved 88 amino acid change from isoleucine to asparagine in the second calcium binding domain of the protein. Breeding 89 colonies of mutant mice, on a mixed background of C57BL/6J and C3HeB/FeJ, were maintained by crossing 90 deaf Otoferlin mutant males, with hearing, heterozygous females. Their offspring were either homozygous deaf 91 mutants, or hearing heterozygotes and could be distinguished before experiments by the presence or absence of 92 a Preyer reflex. Wild type mice were created by mating heterozygous animals; resulting wild type mice were 93 bred and maintained separately. Genotypes of all mice were confirmed post hoc (Longo-Guess et al., 2007). 94 Homozygous, Otoferlin mutant animals will be referred to as “deaf Otoferlin mutants”; heterozygotes and wild 95 type animals will in some cases be lumped and referred to as “hearing controls” or “Otoferlin controls.” 96 Animals of both sexes, aged from P11-60, with the majority of animals aged P17-23, were used for anatomical 97 experiments and P16-22 for the electrophysiological experiments. Otoferlin knockout mice were kindly given to 98 us by Dr. Isabel Roux with permission from Dr. Christine Petit (Roux et al., 2006). Genotyping of these mice 99 were also confirmed post hoc. All procedures were approved by the Institutional Animal Care and Use 100 Committee at the University of Wisconsin, Madison. 101 102 Solutions 103 The dissection of the cochlear nuclei from the brainstem was done in a high sucrose extracellular saline 104 solution that contained reduced Na and Ca (345 mOsm/kg). The composition of the solution (in mM) is as 105 follows: 99 NaCl, 3 KCl, 1.2 KH2PO4, 1 CaCl2, 1.3 MgSO4, 20 NaHCO3, 6 HEPES, 10 glucose, 72 sucrose, pH 106 7.3. The cutting solution was kept around 28°C. 107 The extracellular physiological saline (osmolarity 308 mOsm/kg) used to perfuse the tissue (1.5-2 108 hours) after biocytin injections and also for whole-cell recordings, contained (in mM): 130 NaCl, 3 KCl, 1.2 109 KH2PO4, 2.4 CaCl2, 1.3 MgSO4, 20 NaHCO3, 6 HEPES, 10 glucose, and 0.4 ascorbic acid, pH 7.3. Whole-cell 110 recordings were made in the presence of 10 μM strychnine to block glycinergic inhibition. All salines were 111 saturated with 95% O2-5% CO2, and maintained at 32-33°C. Chemicals were from Sigma-Aldrich, except that 112 sucrose was purchased from Fisher. 113 The internal pipette solution for voltage and current clamp recordings was (in mM): 108 potassium 114 gluconate, 9 HEPES, 9 EGTA, 4.5 MgCl2, 14 phosphocreatinine (Tris salt), 4 ATP (Na salt), and 0.3 GTP (tris 115 salt) that had a final osmolarity ~303 mOsm/kg. The pH was adjusted to 7.4 with KOH. The final holding 116 potentials were corrected for a −12 mV junction potential. 117 118 Brain Slices 119 For electrophysiological studies, parasagittal slices of the left cochlear nucleus were cut from the 120 brainstem with a vibrating microtome (Leica VT 1000S) in sections of 200 μm. Cochlear nuclei were superfused 121 continually at ~ 3-6 mL/min. The temperature was measured with a Thermalert thermometer (IT-23, Physitemp) 122 and was controlled with a custom-made, feedback-controlled heater to remain at 32 or 33°C. 123 124 Biocytin injections 125 For the anatomy, cochlear nuclei were removed bilaterally from the brainstem by hand with scissors so 126 that the circuitry of the cochlear nuclei could be assessed in its entirety. In a few cases, parasagittal slices up to 127 420 μm thick of cochlear nuclei were cut with a vibrating microtome (Leica VT 1000S). Tissue was transferred 128 to a holding chamber ~0.6 mL and superfused continually at ~6-8 mL/min for 1.5-2 hours. Temperature was 129 kept at 33C with a custom-made, feedback-controlled heater that was connected to a Thermalert thermometer 130 (IT-23, Physitemp). Injections were made under the control of a Wild (M5) dissecting microscope. 131 1% Biocytin was dissolved in extracellular saline and was applied as the fibers were disrupted with a 132 glass pipette. Extracellular injections were made with a Picospritzer, through a glass pipette with a tip diameter 133 ~ 5 μm, along the dorsal/ventral axis of the pVCN. Some injections were made only at the nerve root, while 134 others were made in groups of three with one made ventrally in the nerve root, one medially in the pVCN and 135 one dorsally in the pVCN. Single injections into the aVCN were made to visualize the tuberculoventral cell 136 projections, which also labeled end bulbs. Tissue was fixed in 4% paraformaldahyde, stored at 4°C, cryo137 protected in 30% sucrose, embedded in a gelatin-albumin mixture, and resectioned at 60 μm in frozen sections. 138 Biocytin was visualized with Vectastain ABC Peroxidase Kits (Standard) purchased from Fisher (Golding et al., 139 1995). Tissues were mounted on subbed slides, dehydrated with alcohol and stained with cresyl violet to 140 visualize cellular nuclei. Photomicrographs were taken through a Zeiss Axioskop with a Zeiss Axiocam. 141 142 Volume and Area measurements 143 The volumes of the magnocellular regions in the ventral and dorsal cochlear nuclei were measured from 144 camera lucida reconstructions in serial sections from each nucleus. Images were scanned into a computer, 145 outlined and analyzed with Image J software, wherefrom the final volume measurements were imported into 146 Excel and compared by Student’s t-tests for statistical analysis. The area of the auditory portion of the eighth 147 nerve was measured in Image J from photomicrographs of cross sections through the nerve. Measurements are 148 presented as means ± standard deviation. 149 150 Auditory Brainstem Responses (ABRs) and Distortion Product Otoacoustic Emissions (DPOAEs) 151 Animals aged between P20 and P60 were anesthetized with Ketaject, 150 mg/kg and Xylazine, 5 mg/kg. 152 Once animals were unresponsive to a paw pinch, they were placed on a heating pad to maintain body 153 temperature. ABRs and DPOAEs were recorded with Tucker Davis Technologies, Sig Gen Software. 154 To measure ABRs, animals were placed next to a calibrated freefield speaker (ES1) positioned 10 cm 155 away from their left ear, grounded by a subcutaneous electrode behind the contralateral pinna and a 156 subcutaneous reference electrode at the apex of the skull and a subcutaneous recording electrode directly behind 157 the left ear, as near as possible to the ear. Animals were presented with clicks, 0.1 msec in duration and of 158 alternating polarity between 90 and 10 dB; or with tones, 5 msec duration, with a 3 msec gating time, at 4 kHz, 8 159 kHz, 16 kHz and 32 kHz ranging between 90 to 10 dB SPL in 10 dB steps. Final traces were averages of 50016