Figures and data in Gradients in the biophysical properties of neonatal auditory neurons align with synaptic contact position and the intensity coding map of inner hair cells

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7 figures, 5 tables and 1 additional file

Figures

Figure 1 with 1 supplement

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Semi-intact preparation combines SGN biophysics with identification of pattern of connectivity with hair cells.

(A) Schematic of the innervation at auditory hair cell along with key anatomical structures provided with the semi-intact preparation presented in the XZ plane. (B) Confocal image of two type I spiral ganglion neurons (SGN) injected with biocytin in the acute semi-intact preparation of rat middle-turn cochlea. Hair-cells and biocytin-labeled fibers are visualized by immunolabeling for Myosin VI (red; hair cells) and streptavidin conjugated to Alexa Flour 488 (green, fiber). (C) Myelinating Schwann cells are mechanically removed by suction pipettes to make SGN somata accessible to recording electrodes. D.1-F.1. Confocal images in the XY plane show type I SGN afferent fibers making exclusive connections with inner hair cells. D.2-F.2 The synaptic position of the afferent fiber onto the inner hair cell is assessed by examining the cross-section of the hair cell in XZ plane. (G) Type II SGN projecting radially and turn within the outer hair cell region. (H) A schematic of an individual inner hair cell with guides showing how the normalized basal position (NBP) is measured for each type I SGN. NBP is measured by the synaptic position along the radial axis of the hair cell,c, divided by the maximum length of the inner hair cell, L. Positive NBP values indicate that the synaptic position is on the modiolar-side of the inner hair cell, while negative NBP values indicate that the synaptic position is on the pillar-side of the inner hair cell. (I) The distribution of type I SGN contacts as a function of NBP. NBP values averaged at 0.11 +/- 0.23 with a range from −0.27 to +0.52 (n = 38).

Figure 1—figure supplement 1

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Polarization vector shows that branching fibers have a preferred polarization to either modiolar or pillar face.

The net polarization of the fiber was computed by taking the sum of the individual polarizations divided by the total number of branches (Figure 1).

Figure 2

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SGN can be qualitatively grouped by firing patterns in response to step current injections.

(A) Rapidly-accommodating neurons fire a single action potential. (B) Intermediately-accommodating neurons fire more than one action potential and eventually reach a steady-state potential. (C) Slowly-accommodating neurons fire multiple actions and do not reach a steady-state potential. (D) Graded-response neurons do not produce an action potential, but produce graded-depolarization in response to incrementing current steps. These neurons are capable of firing action potentials after their membrane potential are held at −80 mV (inset). (E) Action potential features including current threshold (I_threshold), voltage threshold (V_threshold), and after hyperpolarization potential time constant (AHP tau) vary among spiking neurons. The p-values indicate when two means were assessed to be significantly different (see Materials and methods); asterisks indicate p<0.001. (F) Action potential features were significantly correlated with normalized basal position (NBP). As NBP values increased, current thresholds increased (r(20) = 0.68, p=0.0007), voltage thresholds hyperpolarized (r(20) = −0.45, p=0.045), and AHP time constants became faster (r(20) = 0.62, p<0.0001). Best fit lines with 95% confidence intervals are plotted to show the gradients along the normalized basal position axis. (G) The distribution of firing pattern subgroups as a function of NBP shows a significant relationship between the firing-pattern subgroup and NBP (p=0.0097, t-test), with rapidly-accommodating neurons found primarily on the modiolar-face and intermediately-accommodating neurons found on the pillar-face of the inner hair cell. Graded-firing neurons are found throughout the NBP scale on both the modiolar and pillar faces of the hair cell.

Figure 2—source data 1 Current-clamp features versus normalized basal position data set for Figure 2F.: https://cdn.elifesciences.org/articles/55378/elife-55378-fig2-data1-v1.xlsx
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Figure 3

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Response latencies are alternate predictors for normalized basal position.

There is a significant inverse relationship between first-spike latency and current thresholds. Black line shows a fit I_thresh = constant/latency. Color transition from red to blue indicates position on NBP scale. Type II SGN are colored black. Unlabeled and therefore unclassified SGN are in gray. (A) First spike latencies compared between three spiking SGN with different NBP values. Latencies are faster for more positive NBP (B) In non-spiking neurons, response latency is computed by averaging the time from the onset of the stimulus until peak depolarization potential over a series of current steps. Similar to the dependence of first-spike latency, average response latency is faster for modiolar-contacting SGN (NBP >0, red) than for pillar-contacting SGN (NBP <0, blue). (C) Response latencies for all spiking (black dot) and non-spiking (triangle) neurons plotted against NBP. In both spiking- and non-spiking SGN, response latencies become faster as NBP values increase (R² = 0.55, p<0.001). Latencies are highly variable across type II fibers (gray symbols).

Figure 3—source data 1 Current threshold versus latency dataset for Figure 3B.: https://cdn.elifesciences.org/articles/55378/elife-55378-fig3-data1-v1.xlsx
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Figure 3—source data 2 Latency versus normalized basal position dataset for Figure 3D.: https://cdn.elifesciences.org/articles/55378/elife-55378-fig3-data2-v1.xlsx
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Figure 4 with 1 supplement

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Net whole-cell conductances increase as SGN synaptic position moves from pillar to modiolar-face of the inner hair cell.

(**A–C**) Voltage-clamp responses of a modiolar-contacting (A), pillar-contacting type I (B), and a type II SGN (C). For each cell, we held the potential at −60 mV, followed by a series of voltage 400 ms steps from −120 mV to 70 mV, and then finally back to the holding potential. For each cell, we measured current-voltage relationships at approximate steady-state (400 ms indicated by the arrow). We measured the time course for outward current inactivation by fitting a single exponential curve to the current evoked by a +70 mV voltage step (inset). (D) Steady-state conductance in microSiemens as a function of command voltage of all SGN recorded with three measurements: the magnitude of conductance at −30 mV (g_-30), the maximum conductance (g_max), and the half-activation potential (V_1/2) via Boltzmann function. (E) Steady-state conductances of identified type I SGN as a function of normalized basal position. (**F–I**) Voltage clamp properties as a function of normalized basal position (NBP) (Spiking, black dot; non-spiking, triangle). Linear regression models were fitted to each parameter with statistical significance test of p<0.05. All parameters were significant except for V_1/2.

Figure 4—source data 1 Voltage-clamp features versus normalized basal position for Figure 4F through 4I. Conductance values are shown in units of micro Siemens.: https://cdn.elifesciences.org/articles/55378/elife-55378-fig4-data1-v1.xlsx
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Figure 4—figure supplement 1

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Considering the influence of space-clamp and series resistance errors.

(A) Circuit diagram of an acute SGN in the semi-intact preparation clamped in whole-cell configuration. This diagram shows the series resistance (Rpip), membrane resistance (Rm), axial resistance (Ra), and a secondary compartment resistance from the neurite (Rcomp) with corresponding capacitors (C). (B) Voltage-clamp response of the transient capacitance current of an SGN in the semi-intact preparation fitted with a bi-exponential function (green trace). The fast (pink trace) and slow (blue trace) components of the bi-exponential function are plotted to show the contribution of the current coming for the somata (fast/pink) and neurite (slow/blue). (C) The relative size of the magnitude of the fast (A2) and slow (A1) components of the trace, indicating that the fast component dominates the fit and currents are measured primarily from the soma. (D) The fast component of the bi-exponential (A2) relative proportion to the sum of the bi-exponential function (A1+A2). A2 relative proportion dominates in both modiolar(red) and pillar (blue) contacting SGN. (E) Cell capacitance, measured as the integral of the capacitance current, is not correlated with basal position (r = 0.05, p=0.85). (F) Series resistance as a function of normalized basal with the mean trend line is shown in red. (G) After performing series resistance error corrections per cell, we see that the position correlation of maximum outward current (Imax) and NBP does not collapse, and remains correlated (R² = 0.23, p=0.0003). If we simulate increasing levels of systematic series resistance errors by three (blue trace) and six (black trace) times the amount we measured, trends along NBP do not collapse.

Figure 5

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Combining voltage-clamp and current-clamp features in multiple-variable regression models to predict normalized basal position (NBP) based on biophysics.

(A) Prediction model for spiking neurons (P3–P10) relies on I_thresh, g_max and t_inact. (B) Prediction model that pools spiking and non-spiking neurons (P3–P10) is successful by using latency in place of current threshold (**C,D**), respectively. Biophysical gradients are still successful at predicting spatial position even after filtering data into narrow age bins. P3-P5 in C and P6-P8 in D. Predictor variables used in each model are presented at the bottom right of the predicted vs. actual plot. (E) Current threshold increase with increases in g_-30. (F) Response latency as a function of g_-30. Fit is a function of the form latency = constant/_g-30.

Figure 6

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Biophysical properties of SGN change with age but remain diverse throughout 3 weeks of post-natal development.

(A) 1-A.3 Steady-state current/voltage curves show that net outward currents grow in three age bins (P1-P5; P6-9; and P10-P16). Small and large currents are evident in all age ranges. (**B,D and F**) Log-normal plots showing the distribution of maximum conductance (g_max), current threshold (I_threshold) and latency in three age bins, respectively. Points are color coded to indicate normalized basal position (red to blue gradient), unlabeled cells (gray) and type II neurons (black). Within each age bin, biophysical properties of type I SGN change as a gradient along the NBP scale. The relative dispersion in each distribution is quantified by the CV (coefficient of variation indicated above each distribution) and is not systematically dependent on age. **C,E and F** Plots of same three features, g_max, current threshold, and latency plotted with age as a continuous variable but with spatial position binned as ‘modiolar-contacting’ (NBP >0; red symbols) and ‘pillar-contacting’ (NBP <= 0, blue symbols). Solid lines in **C and E** show the best regression fits through modiolar-contacting and pillar-contacting groups $(g_{m a x}^{m o d} = 4.93 * A g e + 10; g_{m a x}^{p i l l a r} = 2.29 * A g e + 10)$ . Solid lines in F plot curves defined by the inverting the regression plots in C; $L_{(m o d, p i l)} = 420 / g_{m a x}^{(m o d, p i l)}$ .

Figure 6—source data 1 Maximum conductance versus age for Figure 6C. Conductance values are in microSiemens.: https://cdn.elifesciences.org/articles/55378/elife-55378-fig6-data1-v1.xlsx
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Figure 6—source data 2 Current threshold versus age dataset for Figure 6E.: https://cdn.elifesciences.org/articles/55378/elife-55378-fig6-data2-v1.xlsx
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Figure 6—source data 3 Latency versus age dataset for Figure 6G.: https://cdn.elifesciences.org/articles/55378/elife-55378-fig6-data3-v1.xlsx
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Figure 7

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Morphological development of spiral ganglion terminal branches.

(A) Three-dimensional reconstructions of SGN terminals are made to quantify the number of branches at each terminal and the approximate diameter of the fibers. (B) The number of branches exponentially decreases over the course of development. One-to-one connections with the inner hair cell can be seen at P5. In some cases, terminals have multiple branches at P10. (C) Pillar-contacting SGN are not observed to have the mature one-connection morphology. (D) Fiber diameters do not differ between type I SGN along the NBP scale. Type II SGN have significantly smaller fiber diameters than type I SGN.

Tables

Table 1

Analysis of covariance.

Figure	Source	d.f.	Sum of squares	F-score	p-value
3D Latency	Spike (Spike/Non-Spike)	1	0.158	0.0052	0.94
	NBP	1	967.79	32.04	<0.0001
	Spike * NBP	1	4.14	0.13	0.71

4F g_max	Spike	1	150.99	1.37	0.25
	NBP	1	1578.89	14.29	0.0006
	Spike * NBP	1	0.038	0.0004	0.98

4 G _g-30	Spike	1	41.27	1.97	0.17
	NBP	1	568.15	27.16	<0.0001
	Spike * NBP	1	12.14	0.58	0.45

4 H_V1/2	Spike	1	632.64	3.53	0.068
	NBP	1	743.44	4.15	0.0495
	Spike * NBP	1	107.86	0.60	0.44

4I $τ_{i n a c t}$	Spike	1	18715.3	3.64	0.068
	NBP	1	42318.4	8.24	0.0084
	Spike * NBP	1	9.85	0.0019	0.96

6C. g_max	Class (Modiolar/Pillar)	1	1508.38	7.32	0.0107
	Age	1	1963.45	9.53	0.0041
	Class* Age	1	1232.78	5.98	0.0199

6E I_threshold	Class (M/P)	1	3827.39	8.87	0.0087
	Age	1	2333.04	5.35	0.0335
	Class * Age	1	280.75	0.64	0.4334

6G Latency	Class (M/P)	1	577.732	36.97	<0.0001
	Age	1	483.887	30.96	<0.0001
	Class * Age	1	131.247	8.4	0.0066

Table 2

Correlations with normalized basal position.

		Spiking neurons P3-P10		Non-spiking P3-P10		Spiking plus non-spiking P3-P5		Spiking plus non-spiking P6-P8
		R	p-value	R	p-value	R	p-value	R	p-value
Current-clamp features	*I_Threshold	0.68	0.0007			0.81	0.0080	0.44	0.0772
	*First-spike Latency	−0.65	0.0013			−0.76	0.0168	−0.45	0.22
	*Voltage Threshold	−0.39	0.0169			−0.61	0.0837	0.15	0.7390
	*AHP Tau	−0.61	0.0031			−0.65	0.0217	−0.21	0.5616
	*AP Height	0.31	0.17			0.03	0.5634	0.63	0.0069
	Average Response Latency			−0.89	<0.0001	−0.76	0.0010	−0.77	0.0003
	Resting Potential	−0.54	0.0112	−0.31	0.229	−0.07	0.8048	−0.48	0.0487
Voltage-clamp features	g_max	0.52	0.0166	0.58	0.0177	0.78	0.8799	0.62	0.0083
	V_1/2	−0.27	0.233	−0.39	0.137	−0.10	0.71	−0.09	0.74
	g_-30	0.59	0.0045	0.74	0.0010	0.44	0.10	0.59	0.0122
	$τ_{i n a c t}$	0.5	0.0066	0.67	0.0179	0.48	0.10	0.45	0.1459

Table 3

Multiple variable regression models.

Figure	Variable	ß	SE	Df	/t/	VIF	RMSE	p-value
5A	I_threshold	0.0099	0.0016	11	6.102	2.420	0.090	0.0004
	g_max	−0.0182	0.0047		3.858	3.858
	$τ_{i n a c t}$	0.0012	0.0005		2.625	2.165
5B	Latency	−0.0231	0.0045	24	5.158	1.241	0.136	<0.0001
5B	$τ_{i n a c t}$	0.0005	0.0004	24	1.404	1.241	0.136	<0.0001
5C	Latency	−0.0192	0.00454	14	4.23	1.00	0.123	0.0010
5D	Latency	−0.0310	0.0067	16	4.62	1.00	0.140	0.0003

Table 4

Table of regression slopes.

Figure	Term	Estimate	Standard error	T	p-value
3D Latency	Slope	−22.8	4.02	−5.66	<0.0001
	Spike	−2.98	8.05	−0.37	0.71
	Non-Spike	2.98	8.05	0.37	0.71

4F g_max	Slope	27.87	2.55	10.89	<0.0001
	Spike	−0.29	15.4	−0.02	0.98
	Non-Spike	0.29	15.4	0.02	0.98

4 G_g-30	Slope	17.46	0.80	10.6	<0.0001
	Spike	5.1	6.7	0.76	0.45
	Non-Spike	−5.1	6.7	−0.76	0.45

4 H_V1/2	Slope	−19.98	9.8	−2.04	0.0495
	Spike	−15.22	19.62	−0.78	0.44
	Non-Spike	15.22	19.62	0.78	0.44

4I $τ_{i n a c t}$	Slope	178.9	62.3	2.87	0.0084
	Spike	−5.46	124.63	−0.04	0.97
	Non-Spike	5.46	124.63	0.04	0.97

6C g_max	Slope	2.80	1.06	2.63	0.0127
	Pillar	−2.60	1.06	−2.45	0.0199
	Modiolar	2.60	1.06	2.45	0.0199

6E I_threshold	Slope	4.68	2.26	2.07	0.054
	Pillar	−1.81	2.26	−0.8	0.4334
	Modiolar	1.81	2.26	0.8	0.4334

6G Latency	Slope	−1.752	0.29	−5.97	<0.0001
	Pillar	−0.85	0.29	−2.9	0.0066
	Modiolar	0.85	0.29	2.9	0.0066

Key resources table

Reagent type (species) or resource	Designation	Source or reference	Identifiers	Additional information
Strain, strain background (species)	Long-Evans (rat)	USC Vivarium and Charles River	RRID:RGD_2308852	Freshly isolated cochlear middle turns
Antibody	anti-myosin-VI (rabbit polyclonal)	Proteus Biosciences	Cat# 25–6791, RRID:AB_10013626	IF(1:1000)
Antibody	anti-peripherin (rabbit polyclonal)	Millipore	Cat# 183 AB1530, RRID:AB_90725	IF(1:500)
Antibody	streptavidin Alexa Fluor 488 conjugate	Molecular Probes	Cat# S32354, 182 RRID:AB_2315383	IF(1:200)
Antibody	Alexa Fluor 594 (goat anti-rabbit, polyclonal)	Thermo Fisher Scientific	Cat# A-11080, 185 RRID:AB_2534124	IF(1:200)
Other	Vecta-shield	Vector Laboratories	Cat# H-191 1000, RRID:AB_2336789
Software, algorithm	JMP	SAS Institute	RRID:SCR_002865
Software, algorithm	Matlab	Mathworks	RRID:SCR_001622
Software, algorithm	pClamp	Molecular Devices	RRID:SCR_011323
Software, algorithm	OriginPro	Origin Labs	RRID:SCR_015636
Software, algorithm	Imaris	Oxford Instruments	RRID:SCR_007370
Software, algorithm	Prism 8	Graph Pad	RRID:SCR_002798
Software, algorithm	FilamentTracer in Imaris	Oxford Instruments	RRID:SCR_007366

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Alexander L Markowitz
Radha Kalluri

(2020)

Gradients in the biophysical properties of neonatal auditory neurons align with synaptic contact position and the intensity coding map of inner hair cells

eLife 9:e55378.

https://doi.org/10.7554/eLife.55378