The Auditory Periphery. 4 Afferent synaptic transmission by cochlear hair cells Structure and Function

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1 The Auditory Periphery 4 Afferent synaptic transmission by cochlear hair cells Structure and Function 2011 Dr. Elisabeth Glowatzki eglowat1@jhmi.edu Johns Hopkins School of Medicine 824 Ross Building 720 Rutland Ave Baltimore MD Websites: fiigures used are from: Promenade round the cochlea ( Auditory Animations, Univ. of Wisconsin ( Texts (at Welch or Eisenhower): From Sound to Synapse, C. D. Geisler, New York: Oxford Univ. Press, 1998 An Introduction to the Physiology of Hearing, J. O. Pickles, New York: Academic Press, 1982 Fundamentals of Hearing: An Introduction (3 rd ed.), W. A. Yost, San Diego: Academic Press, 1994

2 Hair cells and their innervation in the organ of Corti 1. Inner hair cells receive ~ 95% of afferent innervation (blue). 2. Outer hair cells receive ~ 5% of the afferent innervation (green). 3. Outer hair cells receive mainly efferent innervation from the auditory brain stem (red). 4. Inner hair cell afferent fibers (blue) receive efferent innervation (pink). Figures from: Stephan Blatrix, Promenade round the Cochlea In the cochlea, there are 2 types of sensory cells located in the organ of Corti, one row of inner and 3 rows of outer hair cells, as seen here in a cross section. Both cell types transform sound signals into electrical signals with their transduction apparatus in the apical part of the cells. However, both cell types have different functions, defined by their innervation. The main role of the inner hair cells is to transmit sound signals to the brain. 95 % of the auditory nerve fibers, that transmit information about sound to the brain (afferent fibers), contact only the IHCs, shown here by the blue synaptic contacts. Therefore the synapse between the IHC and afferent fibers represent the 1 st relay station in the auditory pathway as most of the information about sound has to pass through here. Outer hair cells receive about 5 % of the afferent innervation (here in green). Not much is known about the function of these fibers. Outer hair cells are also contacted by efferent fibers (in red), providing feedback from the brain and modulation the sensitivity of the hearing organ. The afferent fibers contacting the inner hair cells are innervated by efferent fibers (in pink) at their unmyelinated ending. In this lecture we will focus on properties of the afferent synapse at the IHC. 2

3 The inner hair cell ribbon synapse A striking feature of the IHC afferent synapse is the synaptic ribbon, a presynaptic structure that appears in EM micrographs. Here are serial sections through a synaptic ribbon in an inner hair cell of the cat. An electrodense structure, the synaptic body, is surrounded by clear core vesicles that are tethered to the synaptic body. Normally one row of vesicles surrounds the synaptic body and a few vesicles can be located between the synaptic body and the presynaptic membrane. We don t know exactly how this structure works, however, it is thought that it attracts vesicles to the presynaptic site of the synapse and thereby allows for constant and fast release. 3

4 Afferent innervation of the cochlea Each inner hair cell makes Type I afferent contacts. (This number varies along the tonotopic axis.) Each afferent is associated with a single ribbon synapse. Therefore, activity in an auditory afferent neuron represents the activity of a single synaptic ribbon! Stephan Blatrix, Promenade round the Cochlea Schematic of a cochlear turn with one row of IHCs and 3 rows of OHCs and afferent fiber innervation. Every Type I auditory nerve fiber contacts only one IHC with only one synaptic contact. We use the term afferent fiber (AF) or auditory nerve fiber or spiral ganglion neuron or Type I fiber for the same fibers! Type II fibers contact a number of OHCs. Both Type I and Type II fibers have their soma located in the spiral ganglion. Spiral ganglion neurons are bipolar cells. Their peripheral process contacts the hair cells, their soma is located in the spiral ganglion (in blue) within the cochlea, and their central process travels through the auditory nerve into the cochlear nucleus in the brain stem, to make synapses onto cochlear nucleus neurons. For the Type I fibers, only the ending that contacts the IHC is unmyelinated. Starting at the habenula perforata, the peripheral neurite is myelinated, as well as the soma. We therefore assume that the action potential is generated in the peripheral neurite, close to where the myelinization starts. Type II fibers are unmyelinated. 4

5 5 auditory nerve 4 spiral ganglion Stephan Blatrix This cross-section through the cochlea illustrates the location of the spiral ganglion (4, in yellow) within the cochlea. The spiral ganglion holds the somata of Type I and Type II auditory nerve fibers. Along the cochlear coil, like the hair cells, the spiral ganglion cells are also tonotopically organized in terms of the frequencies they code. 5

6 The inner hair cell receptor potential drives transmitter release Calcium channels are located close to the synaptic ribbon Stephan Blatrix AMPA receptors are located at the postsynaptic membrane How does the receptor potential drive transmitter release? The resting membrane potential of the inner hair cell is set at a level that allows for calcium channels to open and for inducing calcium dependent continuous transmitter release. This release is reflected in the spontaneous rate of action potentials in the auditory nerve fibers. When sound sets the basilar membrane in the inner ear in motion, the stereocilia bundles on top of the hair cells are deflected, the hair cell is depolarized by K influx and generates a receptor potential. Depolarization of the inner hair cell further opens voltage gated calcium channels and enhances transmitter release. Vesicles filled with glutamate fuse with the synaptic membrane (exocytosis) in a calcium dependent manner, activate glutamate receptors on the afferent fiber terminal and induce excitatory postsynaptic potentials. The excitatory postsynaptic potentials activate action potentials that travel down the auditory nerve. 6

7 Continuous release at rest resting membrane potential ~ -50 mv? Stephan Blatrix We don t know exactly what the inner hair cell membrane potential is exactly in vivo; however, we know that it is depolarized enough to induce transmitter release, as auditory nerve fibers fire spontaneously without sound. 7

8 The next slides review some features of auditory nerve fibers. Auditory nerve fibers are exhibit spon These figures show distributions of spontaneous rates for different species. Spontaneous rates occu 8

9 These example plots show another typical feature of auditory nerve fibers: Discharge rate is plotted versus the level of sound presented. The two fibers have different thresholds, one at 25 and one at 32 db. Additionally, the change in discharge rate compared to change in tone burst level is different. If these 2 fibers contact the same IHC, they code different signals. A will activate at low sound intensities, and highly sensitively code differences around 20 db. However A be we saturated already at 30 db. B will be insensitive to low sound levels, but cover a la Often fibers with a high spontaneous rate have a low threshold in responding to sound and fibers wi 9

10 Innervation of the IHC by low and high spontaneous rate fibers Merchan Perez and Liberman 1996 Liberman Merchan Perez (1996): Fibers with low spontaneous rates have usually high thresholds and vice versa. In the cat, low spontaneous rate fibers contact the IHC more often on the modiolar side and high spontaneous rate fibers contact the IHCs more often on the pillar side. 10

11 How are the properties of individual nerve fibers set?

12 Some important players in IHC synaptic transmission that may affect the properties of the auditory nerve fibers: 1) Calcium channels, location relative to vesicles, number 2) Calcium sensor, properties 3) Synaptic vesicle availability: properties of the synaptic ribbon and pools of vesicles 4) Transporters that fills glutamate into vesicles and remove glutamate after release 5) Postsynaptic glutamate receptors; types, numbers etc. 6) Postsynaptic ion channels that shape the EPSP 7) Action potential generation

13 Blocking potassium channels to isolate the hair cell s calcium current Fuchs et al., 1990 Hair cell calcium channels can only be studied after first blocking the much larger potassium currents. In this whole cell recording, after the cell membrane ruptured, potassium channel blockers entered the cell and over time blocked these currents. After 30 s the calcium inward current (downward deflection) can now be seen. Note that during the stimulus the calcium current does not inactivate much. This is a typical feature of hair cell calcium currents.

14 Calcium current in chicken hair cells: the peak current is at ~-20 mv calcium currents in hair cells do not inactivate much Mike Zidanic, Mike Evans As in other hair cells, calcium channels in chicken cochlear hair cells are rapidly activating at the beginning of the stimulation, and rapidly deactivating after the end of the stimulation. During the stimulation, they do not inactivate much. They are preferentially permeable to barium over calcium, activate at a relatively negative membrane potential, and their peak current is at about -20 mv.

15 In hair cell, the gene underlying the dihydropyridinesensitive calcium current, is CaV1.3 (alpha 1D): CaV1.3 deficient mice are deaf and have highly reduced calcium currents Calcium currents are highly reduced in CaV1.3 deficient mouse IHCs compared to WT. Auditory Brainstem Responses Platzer et al., 2000 Figure 2. Effects of α1d Deficiency on Auditory Function. ABRs of 5-week-old wildtype (+/+) and α1d / ( / ) mice. Representative experiments are shown. The typical ABR waveform was present above 30 db SPL in wild type (+/+) but was absent in α1d / mice. The waves at 120 db SPL in α1d / mice represent background because they were also observed in control experiments without animals and did not show the typical ABR waveform. Figure 3.F and G shown here. (F) IBa in α1d+/+ (+/+) and α1d / mice ( / ) during 400 ms depolarizations to 2 mv. (G) Mean current densities (±SD) for α1d+/+ (+/+) and α1d / mice ( / ) obtained from the indicated number of cells.

16 Proteins involved in vesicular release; in CNS neurons, synaptotagmin is considered to be a calcium sensor From Neuron to Brain, 4 th edition, 2001, Sinauer Press synaptophysin, synapsin, synaptotagmin I and II are missing at the adult hair cell synapse. (Safieddine and Wenthold, 1990) A variety of proteins associated with the presynaptic plasma membrane, or with synaptic vesicle membrane, have been identified at chemical synapse throughout the brain. Ribbon synapses in hair cells, have some, but not all these release proteins including synaptophysin, synapsin, synaptotagmin I and II. Synaptotagmin has been proposed as the calcium sensor at CNS synapses, but has not been found in hair cells. 16

17 Otoferlin - a candidate for the calcium sensor for exocytosis in IHCs 1) Mutations in the human otoferlin result in nonsyndromic auditory neuropathy, (DFNB9) deafness resulting from deficits downstream of cochlear mechanotransduction. 2) Like synaptotagmin, otoferlin interacts with members of the SNARE complex, SNAP 25 and syntaxin I in a calcium dependent manner. 3) Otoferlin has been localized in IHCs in the cochlea. 4) Otoferlin deficient mice are deaf and their IHCs have greatly reduced transmitter release. Roux et al., 2006 A novel deafness gene product, otoferlin, has been proposed to be the calcium sensor in inner hair cells. 17

18 Otoferlin labeling in the mouse cochlea Roux et al., 2006 Figure 1. Otoferlin in Developing and Mature Mouse Cochlea (A F) Immunohistofluorescence analysis of otoferlin (green) in the cochlea. In (A), in the organ of Corti at E16, otoferlin is detected in the IHC but not in the OHCs. Actin filaments are labeled with rhodamine-conjugated phalloidin (red). In (B), longitudinal section of a P2 cochlea. Inset shows a close-up view of the organ of Corti, showing otoferlin labeling in both IHCs and OHCs. Note the baso(***)-apical(*) gradient of OHC labeling. At this stage, cochlear ganglion neurons (CGN) are faintly immunoreactive. (C) shows the peak of otoferlin labeling in the OHCs peaks at P6. In (D), at P60, otoferlin labeling is restricted to IHCs. (E) shows a confocal view of a set of P60 IHCs showing strong baso-lateral labeling in the area where these cells form synapses with the dendrites of afferent neurons (F). Scale bars: (A D) 25 μm, (E) 10 μm. (G H) Immunoelectron microscopy detection of otoferlin in the mature IHC synaptic region. The greatest density of 10 nm gold particles is associated with vesicles tethered to the ribbon (arrowheads), facing an afferent dendrite (a). Some gold particles are associated with the presynaptic plasma membrane. Inset shows a schematic illustration of the base of an IHC and its synaptic contacts with afferent dendrites. Scale bars: 100 nm. 18

19 Otoferlin-deficient mice are deaf and have greatly reduced transmitter release at the IHC afferent synapse Capacitance recordings reporting on exocytosis (Fig. 6) Calcium currents Auditory Brainstem Responses (Fig. 4A) Roux et al., 2006 Figure 4. Assessment of Hearing Impairment in Otof / Mice (A) Representative ABR recordings from Otof+/+ (black lines) and Otof / (gray lines) P30 mice at db SPL of broadband clicks. Roman numerals mark the peaks of the standard ABR waves. No ABR waveform is visible in the Otof / mouse recordings. Figure 6. Ca2+-Triggered Exocytosis Is Almost Completely Abolished in the Absence of Otoferlin (A) Ca2+ currents (top) and ΔCm responses (bottom) of representative Otof+/+ (black) and Otof / (gray) IHCs from P6 mice to 20 ms depolarization to peak Ca2+ current potential. The recordings in (A C) were performed in the perforated-patch configuration. (B) Ca2+ current I/V relationships of Otof+/+ (black) and Otof / (gray) IHCs from P6 (open circles) and P15 mice (closed squares): grand averages, including one I/V relationship of each cell (see C for n). 19

20 Synaptic transmission at the IHC afferent synapse is glutamatergic 1) vglut3 has been found to be the vesicular glutamate transporter that transports glutamate into the synaptic vesicles. 2) AMPA receptors have been localized at the IHC synapse by Immmunocytochemistry and Immuno-EM. 3) In in vivo recordings from auditory nerve fibers and in in vitro recordings from afferent terminals, activity can be blocked by AMPA receptor blockers. 4) In supporting cells the glutamate transporter GLAST has been located. These cells take up the glutamate released from the IHC. 5) A recent study proposes that under certain conditions NMDA receptors are activated at the afferent synapse. References: vglut3 has been found to be the vesicular glutamate transporter that transports glutamate into the synaptic vesicles. Seal et al., Neuron 2008, 57: Obholzer et al., J Neurosci 2008, 28: Ruel et al., Am J Hum Genet 2008, 83: ) AMPA receptors have been localized at the IHC synapse by Immmunocytochemistry and Immuno- EM. Parks, TM, The AMPA receptors of auditory neurons. Hear Res (1-2): review article. 3) In in vivo recordings from auditory nerve fibers and in in vitro recordings from afferent terminals, activity can be blocked by AMPA receptor blockers. Ruel et al., 2000, Neuropharmacology, 39(11): ) In supporting cells the glutamate transporter GLAST has been located. These cells take up the glutamate released from the IHC. Glowatzki et al., 2006, J Neurosci. 26(29): Furness, DN, Lehre, KP, 1997, Eur J Neurosci. 9(9): ) A recent study proposes that under certain conditions NMDA receptors are activated at the afferent synapse. Ruel et al., 2008, J Neurosci 28:

21 vglut3 is a vesicular glutamate transporter in hair cells vglut3 deficient animals are deaf Seal et al., 2008 vglut3 has been localized at the inner hair cells in the mammalian cochlea

22 vglut3 is a vesicular glutamate transporter in hair cells Seal et al., 2008 These experiments test whether there is glutamate release from the IHCs in vglut3 deficient mice. Recordings from the endings of afferents contacting the IHCs were performed in excised cochlea turns. The hair cell was depolarized with 40 mm potassium in the extracellular solution. This caused transmitter release, that could be blocked by a glutamate receptor blocker (NBQX). In the wildtype animal release was activated (C), whereas in the vglut3-deficient animal not release was activated (D)> E and F show, that in both, wildtype and knockout animal, glutamate receptors were present on the postsynaptic membrane., as kainate (KA) could activate a response in both cases. The conclusion was that in the vglut3 KO animals there was not glutamate released. 22

23 vglut3 is a vesicular glutamate transporter in hair cells Seal et al., 2008 vglut3 deficient animals are deaf vglut 3 loss is responsible for DFNA25, an autosomal-dominant form of progressive, high-frequency nonsyndromic deafness. Ruel et al., 2008

24 Immunoreactivity for GluR2/3 and Glutamate at an inner hair cell (IHC) synapse IHC afferent synapse Parallel fiber to Purkinje cell synapse in the cerebellum Small particles: GluR2/3 Big particles: Glutamate Matsubara et al A key paper, Matsubara et al. 1996, presents immunogold labeling of glutamate receptor subunits at the IHC afferent synapse: An antibody for GluR2/3 labels postsynaptically to the synaptic ribbon. An antibody for Glutamate labels mostly presynapstically, in vesicles and mitochondria. Figure 2. Immunoreactivity for GluR2/3 at an inner hair cell (IHC) synapse in the organ of Corti (A, C) and a parallel fiber (Pf) to Purkinje cell synapse in the cerebellum (B). The 1.4 nm gold particles were made visible by silver enhancement. The section in A is not at the center of the synapse because the synaptic body (arrowhead) is cut near its periphery. C, Double immunolabeling. After demonstration of GluR2/3 by silver intensification (small particles, arrowheads), the sections were immunolabeled for glutamate (30 nm gold particles). Some of the large particles appear to be associated with vesicles (arrow) and with mitochondria (M). Inset shows a diagram of the organ of Corti. Frame indicates area represented in this and subsequent illustrations. Asterisk, Inner hair cell contacted by afferent dendrites; 1-3, the three rows of outer hair cells. s, Purkinje cell spine; A, afferent dendrite; TM, tectorial membrane. A, B, Fixative No. 1 (see Materials and Methods). C, Fixative No. 2. Freeze substitution. Scale bars: 0.5 µm in A, 0.2 µm in B and C. 24

25 Immunoreactivity for GluR2/3 and GluR4 at an inner hair cell (IHC) synapse GluR2/3 GluR4 Matsubara et al In this immage the ribbons are clearly visible. Again, GluR2/3 labels postsynaptically; GluR4 labels postsynaptically, but also presynaptically. Up until now no glutamate activated currents have been reported for the IHC; For the postsynaptic terminal there is evidence from electrophysiology that indeed, AMPA receptors mediate the postsynaptic response. Fig. 3. Immunoreactivity for GluR2/3 at an inner hair cell synapse (A, B) and at a hippocampal synapse (D) as demonstrated by 15 nm gold particles. Large arrowhead, Synaptic body surrounded by synaptic vesicles (small arrowheads). C, Electron micrograph of grid (square width mm) for accurate calibration (same magnification as A and B). T, Presynaptic terminal in stratum oriens of CA1; s, postsynaptic spine. Other abbreviations as in Figure 1. Fixative No. 2; freeze substitution. Scale bars: 0.3 µm in A-C, 0.2 µm in D. Fig. 4. Immunoreactivity for GluR4 at inner hair cell synapses as demonstrated by 15 nm immunogold particles using different fixatives and tissue preparation methods. A, C, D, Fixative No. 2, freeze substitution. B, Fixative No. 4, method of Phend et al. (1995). Note that some gold particles are associated with the presynaptic membrane. Large arrowhead, Synaptic body surrounded by synaptic vesicles (small arrowheads). D, Enlargement of C. Abbreviations as in Figure 1. Scale bars: 0.3 µm in A-C, 0.2 µm in D. 25

26 Afferent synapses on IHCs and OHCs are seen by immunostaining presynaptic ribbons with anti-ctbp2 (red) and postsynaptic densities with anti-glur2/3 (green). In the IHC area, most synapses include closely apposed red and green puncta (open arrows), and IHC nuclei are also weakly immunopositive for CtBP2. In the OHC area, only CtBP2-positive puncta are seen (filled arrows). A, An xy projection of a confocal z-stack through the synaptic regions of 12 IHCs and numerous OHCs from the 8 khz region. B, a yz projection of the same z-stack. Outlines (approximate) of individual hair cells are shown by dotted white lines in A and B.

27 Excitatory postsynaptic currents (EPSCs) are blocked by glutamate (AMPA) receptor blockers Glowatzki and Fuchs, 2002

28 The glutamate aspartate transporter GLAST (EAAT1) is expressed in supporting cells surrounding the IHCs Glutamate transporter currents recorded in phalangeal cells (Fig. 1) Visualization of GLAST promoter activity in GLAST DsRed mice (Fig. 3) Glowatzki et al Figure 1. Glutamate transporter currents in cochlear IPCs. A, Infrared-DIC image from a 5-d-old apical cochlear turn. Black arrow, IPC soma; white arrow, triangular-shaped end of IPC phalynx contacting the cuticular plate. Scale bar, 10 µm. B, Responses of IPC to voltage steps ( 60 to 140 mv), in control and in 1 mm octanol. Vm = 90 mv. Calibration: top, 4 na, 50 ms; bottom, 100 pa, 50 ms. C, Inward currents elicited in an IPC in response to 300 µm D-aspartate. The top trace shows the duration of application. D, Current-to-voltage relationship of D-aspartate-evoked currents in IPCs (n = 10). Each current was normalized to the peak amplitude recorded at 80 mv. B D were recorded with a KNO3-based pipette solution. E, Average amplitude of D-aspartate-evoked currents recorded at Vm of 80 mv with different intracellular anions. F, D-Aspartate-evoked transporter currents recorded at room temperature (22 C) and at near-physiological temperature (36 C) (Vm = 80 mv, KMES-based pipette solution). Figure 3. Visualization of GLAST promoter activity in GLAST DsRed mice. A, DsRed fluorescence in a whole-mount apical turn; a narrow band of labeling was observed in the IHC area (red box). Scale bar, 500 µm. B, Region of the area outlined in A shown at higher magnification. Scale bar, 50 µm. C, D, Images taken at different focal planes of the IHC area. Arrow in C points to a labeled phalynx of an IPC. Asterisks in D mark unlabeled IHCs surrounded by DsRed+ supporting cells. Scale bars, 25 µm. E, DsRed fluorescence in a 14-µm-thick cochlear section. F, Merged fluorescence and DIC image of the section shown in E. In addition to intense labeling around the IHC, satellite cells in the spiral ganglion (SG) and cells in the spiral limbus (SLb) and spiral ligament (SLg) are labeled. SV, Stria vascularis. Scale bars, 50 µm. All images from apical turns of postnatal day 9 GLAST DsRed BAC mouse cochleas. 28

29 Disruption of glutamate homeostasis in GLAST KO mice Control After sound (Hakuba et al., 2000) After sound exposure, the glutamate concentration stays elevated in GLAST (-/-) animals.

30 ABRs stay elevated after noise exposure in GLAST KO animals (Hakuba et al., 2000) After sound exposure, ABR thresholds stay elevated in GLAST (-/-) mice.

31 The inner hair cell ribbon synapse: exocytosis A striking feature of the IHC afferent synapse is the synaptic ribbon, a presynaptic structure that appears in EM micrographs. Here are serial sections through a synaptic ribbon in an inner hair cell of the cat. An electrodense structure, the synaptic body, is surrounded by clear core vesicles that are tethered to the synaptic body. Normally one row of vesicles surrounds the synaptic body and a few vesicles can be located between the synaptic body and the presynaptic membrane. 31

32 Different ribbons for different hair cells: Chicken cochlear hair cells: A = low frequency, B = high frequency Martinez-Dunst, Micheals and Fuchs 1997 Ribbons vary in size and shape along the tonotopic axis of the chicken s cochlea. Low frequency ribbons are smaller than high frequency ribbons. Ribbons in mammalian cochlear hair cells also are rather small, mostly 100 but up to 200 nm in diameter, and with correspondingly fewer attendant vesicles. Hair cells in the mammalian cochlea would have ~60 vesicles on tethers, and an average of 5 vesicles docked at the plasma membrane. Martinez-Dunst C, Michaels RL, Fuchs PA. Release sites and calcium channels in hair cells of the chick's cochlea. J Neurosci. 1997, 17(23):

33 The ribbon synapse of frog saccular hair cells David Lenzi et al., 1999 The quantitative details of ribbon structure are best known from hair cells of the frog sacculus. David Lenzi, working with Bill Roberts at the U. of Oregon, reconstructed ribbons and surrounding vesicles and membranes from serial electron micrographs. The large dense bodies of frog, ~ 400 nm diameter, are surrounded by ~500 vesicles, and have room next to the plasma membrane for ~138 vesicles at closepacking. On average 32 vesicles were found in this docked position in the several ribbons that were entirely reconstructured. 33

34 Exocytosis of vesicles from the IHC measured with capacitance recordings The ready releasable pool; The fast and slow components of release Moser and Beutner, 2000 Hair cell exocytosis has been extensively studied with capacitance recordings. This method monitors the area (proportional to the capacitance) of the cell membrane. When vesicles fuse with the membrane, the area increases. Here the hair cells is depolarized for different times, and the capacitance changes are measured. This allows to measure the time course of vesicle release and estimate the different kinetic pools of release. A fast and a slower component of release has been found in hair cells by a number of laboratories and different preparations and species. For review see Nouvian R, Beutner D, Parsons TD, Moser T. Structure and function of the hair cell ribbon synapse. J Membr Biol. 2006;209(2-3):

35 Preparation from a P19-21 rat cochlea * * * 10 μm Grant et al., 2010 Lisa Grant, postdoc in the laboratory worked out how to record from afferent fibers in 3 week old rats. You see well preserved IHCs and afferent terminals contacting the IHCs 35

36 Performing whole cell recordings from individual auditory nerve fibers directly where they contact the IHC, reveals excitatory postsynaptic currents. The procedure for recording is demonstrated in an online video: Postsynaptic recordings at afferent dendrites contacting cochlear inner hair cells: monitoring multivesicular release at a ribbon synapse. Grant L, Yi E, Goutman JD, Glowatzki E. J Vis Exp Feb 10;(48). pii: doi: /2442.

37 Simultaneous recordings from IHCs and afferent fiber terminals Goutman & Glowatzki, 2007 To now directly look at the transfer function between the IHC and afferent fiber, we performed simultaneous whole cell recordings from IHCs and afferent fibers directly where they contact the IHC. 37

38 Afferent fiber response adapts during constant stimulation calcium current: activation ~ 500 s, inactivation < 16 % AF response: ~ 4 ms delay, adaptation to 6 % of peak value Average AF response: 1 = 10 ms 2 = 190 ms Goutman & Glowatzki, 2007 This shows how the experiments are typically done. The hair cell is depolarized by a voltage step to -30 mv, to activate calcium currents and thereby induce transmitter release. The calcium currents are pharmacologically isolated, and as you see don t inactivate much. The afferent fiber is held at a negative membrane potential, and every 30s the response to an IHC stimulation is recorded, so here you see the average response of many stimulations. You see that the afferent response adapts. More remarkably, during a long stimulation, a steady state activity found, showing the incredible power of the IHC to constantly release transmitter. From these experiments we estimate the time course of different kinetic components (pools) of vesicle release. 38

39 In CTZ, the afferent response still adapts Goutman & Glowatzki, 2007 Is this synaptic depression due to a pre- or postsynaptic mechanism? : First we tested the impact of AMPA receptor desensitization, which can be removed by CTZ. You see, that the peak of the afferent response becomes wider in CTZ, as single EPSCs slow down. On the right you see the comparison of average single EPSCs from these records. However, the amount of synaptic depression is still prominent in CTZ, and therefore we think this is due to a presynaptic mechanism, most likely exhaustion of presynaptic vesicles ready for release. To look directly at the time course of exhaustion of vesicles, we used deconvolution, a mathematical procedure that tells is the rate of EPSCs in the afferent signal. 39

40 Rate of release during afferent fiber stimulation calculated by deconvolution Goutman & Glowatzki, 2007 Adaptation in the auditory nerve (mouse) Taberner and Liberman, 2005 You see at a the beginning of the pulse (and in the extrended trace) an EPSC is activated in the first ms and then the rate drops to about 0.2 EPSCs per ms. Here you see the deconvolved afferent response. In comparison, this plot shows the rate of APs in response to a tone burst, a typical auditory nerve fiber response showing adaptation in the auditory nerve. As has been suggested before, we conclude that synaptic depression and the exhaustion of vesicles could account for adaptation in the auditory nerve. 40

41 Ribbon: 55(w) x 278(l) x 230(h) nm Postsyn. Density: 740 nm (l) Vesicles/ribbon: ~ Docked vesicles: ~16 (Khimich et al. 2005) Goutman & Glowatzki, 2007: linear transient release total release/s Tau (ms) A (EPSCs) /s /s A (Vesicles) /s /s The table shows you the time constants for the different kinetic components of release as measured for the data shown in the last slide. We estimate that about 11 vesicles are released with a time constant of 2.1 ms, 27 vesicles with a time constant of 27 ms, and 163 vesicles with a time constant of 175 ms. The remaining constant release occurs at a rate of 280 vesicles/s. And remember that these numbers only represent a ballpark!!! These numbers can now be compared with numbers from EM (Khimich et al. 2005). An average of 16 vesicles were found to be docked at the membrane, vesicles were found to be tethered to the ribbon. Interestingly, our total calculated number of vesicles for the transient component of release is about as big as the number of vesicles tethered to the whole ribbon. And the total number of vesicles release seems to be bigger that the number of vesicles tethered to the membrane. 41

42 Transfer function at the hair cell afferent synapse in the frog papilla Keen and Hudspeth,, 2006 Hair cells operate with a linear calcium dependence of release. These data are based on simultaneous recordings from hair cells and afferent fibers in the frog papilla. The hair cell was clamped to different membrane potentials, and afferent activity was recorded. Figure C shows that the average postsynaptic current relates to the amplitude of the presynaptic current in a linear fashion. 42

43 Transfer function at the IHC afferent synapse: Voltage- and calcium- dependence of release Goutman & Glowatzki, 2007 Here the same experiment as in the last slide was performed in the rat cochlea: The IHC is depolarized to different potentials and the calcium current is recorded. Simultaneously, afferent activity is monitored. Again: Both, calcium current and afferent response grow linearly, resulting in a calcium dependence of release with a power close to 1. This is unexpected, as the calcium sensor is thought to have 4 calcium binding sites, and in other systems for similar experiments a power of 4 is found. One interpretation of this experiment would be that the calcium sensor is saturated and the linear rise in AF response is due to the linear rise of calcium channels. This would be expected if one channel releases one vesicle, in a nanodomain. 43

44 Multivesicular release: EPSC amplitudes distributions are highly skewed with a peak at ~ 40 pa and ranging up to 20 times higher pa Glowatzki and Fuchs, 2002 Multivesicular release at the IHC afferent synapse: Here you see an overlay of EPSCs with varying amplitudes in a single recording and the amplitude distribution of that recording. We found that EPSC amplitudes at single IHC afferent synapses showed wide variation in amplitudes. and had quite impressive sizes up to 800 pa. Please be reminded that this is recorded at a single ribbon. We concluded that the IHC afferent synapse releases multiple vesicles in a coordinate manner and that different sizes of EPSCs were due to a different number of vesicles being released. We interpret the first peak of the distribution pa as the quantal size. The biggest events would be due to release of about 20 vesicles at once. 44

45 Basically, we saw EPSC, with sharp rise times and monoexponential decays but also we saw a large number of events with multiple bumps, as if vesicles were release in coordinated groups, but not completely synchronous, causing multiple distinct inflections during events. 45

46 The charge transfer is similar for mono- and multiphasic EPSCs Grant et al The number of vesicles released per monophasic and per multiphasic event (here represented as charge transfer) are on average the same, suggesting that multiphasic events are a group of vesicles that are just less synchronized in their release compared to the synchronized group of vesicles that cause a monophasic event.

47 Multivesicular release: compound fusion is discussed as a mechanism Parsons and Sterling, 2003 One hypothesis on how multivesicular release occurs is that (like in secretory cells) vesicles fuse (compound fusion) with each other and when fusing with the membrane induce events based on multiple quanta. For review see Parsons and sterling

48 Afferent innervation of the cochlea Each inner hair cell makes Type I afferent contacts. (This number varies along the tonotopic axis.) Each afferent is associated with a single ribbon synapse. Therefore, activity in an auditory afferent neuron represents the activity of a single synaptic ribbon! Stephan Blatrix, Promenade round the Cochlea Remember, type II afferent fibers innervate outer hair cells. 48

49 Type II projections receive excitatory post-synaptic currents (EPSCs) Weisz et al Type II afferent fibers also show AMPA mediated EPSCs.

50 Comparison of Type I and Type II inputs Glowatzki and Fuchs 2002 Weisz et al Type II afferent fibers do not show multivesicular release as the type I afferent fibers do.

51 Summary Some important players in IHC synaptic transmission: 1) Transporters that fills glutamate into vesicles, vglut3 2) L-type calcium channels, low level of inactivation 3) Calcium sensor, Otoferlin as a candidate 4) Linear calcium dependence of release 5) Synaptic vesicle availability: the synaptic ribbon and pools of vesicles, 6) multivesicular release; not found at all ribbon synapses 7) Postsynaptic glutamate receptors, GluR2/3, 4; NMDA?