Reconstruction of SBC axons innervating the MSO GCOE

1. Reconstruction of SBC axons innervating the MSO
Shotaro Karino

2. Introduction
Jeffress model
- A binaural cell is maximally active when the external acoustic delay (the ITD) is
compensated by an internal delay, so that the inputs from left and right ear to an MSO cell
are coincident.
Origin of the internal delays:
1)Axonal delays, i.e. differences in length or conduction velocity of the left and right inputs.
Anatomical studies
{Smith, 1993}
With physiological data: characteristic frequency (CF), spontaneous rate, spike patterns, etc.
Incomplete filling with HRP or neurobiotin
Only two examples of reconstructions of traced distal branches
{Beckius, 1999}
Direct gross injections to AVCN: adequate amount of fibers with complete filling
Single fibers with both ipsilateral and contralateral innervations.
Lack physiological property of spike patterns.

3. Physiological studies
{Yin & Chan, 1990}
Demonstrating the relationship
between physiologically measured
ITD and location of MSO cells.
A spatial map of ITDs across rostral-
caudal axis of the MSO. Low ITDs,
near 0 µs, were located near the
rostral pole, and more positive ITDs,
corresponding to delays of ipsilateral
stimulus, were mapped at more
caudal locations.
{Oliver, 2003}
Similar plot of best ITD and
rostrocaudal location in MSO,
however, they found no clear
relationship.

4. {McAlpine, 2001} observed that low-frequency
cells in the IC of guinea pigs generally show
large best delays, and high-frequency cells
show small best delays.
IC of the cat also showed a frequency-
dependent distribution of best delays {Joris,
2006}, and there was a range of best delays
at each frequency,
These observations are not explained, or only ad
hoc, by axonal delay lines.

5. 2) Internal delays based on inhibitory inputs, which dispenses entirely of axonal delay lines
{Brand, 2002}

6. 3) Consequence of mismatches in
the cochlear locations from which
MSO cells derive their inputs
{Schroeder, 1977}{Joris, 2006}.

7. Materials and Methods
Physiology
•A ventral surgical approach was used to expose the TB of cats
•Electrodes filled with either a 5% HRP or 2% neurobiotin
•Earphones inserted into each external auditory meatus.
•Characteristic frequency (CF), spontaneous activity, and threshold at CF were determined
•Poststimulus time(PST) histograms for short tones at CF
Histology
•Intraaxonal impalement was achieved either with brief current pulses or electrode
advancement, followed by confirmation of the physiological responses to assure identity of
the axon, and then iontophoretic injection of HRP or neurobiotin.
•Frozen or vibratomed 60 or 70 µm sections of the brainstem
•For preparation of HRP-filled axons: processed by using the DAB nickelicobalt intensification
method (Adams, ’81).
•For preparation of neurobiotin-filled cells: Sections were then left in phosphate buffer
containing avidin-biotin-HRP reagent (ABC kit, Vector Labs) overnight so the injected
neurobiotin molecules could bind to this ABC complex for subsequent visualization. HRP
visualized by using the standard DAB-nickel/cobalt intensification method

8. Tracing and Reconstruction
•3D reconstructions were made with a Neurolucida system (Microbrightfield, Inc., Colchester,
VT) : Olympus BX61 light microscope, a video camera (MBF-CX9000; Microbrightfield, Inc.,
Colchester, VT), a motorized stage controller (MAC5000; Ludl Electronic Products Ltd.,
Hawthorne, NY), and a personal computer.
•Axons were traced and digitized directly on the microscope using a UPlanFLN 40×objective
and the video camera.
•The XYZ data points accompanied with axon diameter were collected every 1.9 µm on average
(538 points/mm).
•Corrections were made for shrinkage only on Z-axis (rostrocaudal direction) according to
section thickness at cutting and thickness measured in observation by microscope.

9. Contralateral projection 1
Data points
EP: Endpoint
ML: Midline
FB: First branching point
RP: Rostral pole
CP: Caudal pole
→Normalized RC (0~1)
DP: Dorsal pole
VP: Ventral pole
→Normalized VD (0~1)
MB: Medial border
LB: Lateral border
→Normalized ML (0~1)

10. Contralateral projections 2-4
Innervate the MSO with a ladder-like
branching pattern (horizontal and
parasagittal planes).
The input from this fiber would reach the
anterior before the posterior MSO, i.e.,
there would be a neural delay from rostral
to caudal.

11. Contralateral projections 5-7
More complicated branching patterns: main
tract before FB is located near the center of
the rostrocaudal range of EPs. As a result
they demonstrate a mixture of ladder-like
structure from rostral to caudal and that
from caudal to rostral.

12. Ipsilateral projections 1
(Coronal, parasagittal, and horizontal views)
No apparent delay line.
Complex location of EPs from two kinds of
innervations, namely: forward and
backward innervations (coronal view)
Forward: the branch coursed along the
lateral aspect of the MSO. Four of the
seven ipsilateral fibers had such
components.
Backward: the other branches didn't leave
the axon until it crossed the MSO. These
branches looped back through the MSO to
innervate the same region. All of the seven
ipsilateral fibers had such components.

13. Ipsilateral projections 2
(Coronal, parasagittal, and horizontal views)
This fiber has both forward and backward
innervations.
However, they innervate different position in
rostrocaudal direction (parasagittal and
horizontal views).
In this example, backward branches
innervated more rostral part of the MSO but
showed a little longer length of axon from
FB than forward branches.

14. Ipsilateral projections 3
(Coronal, parasagittal, and horizontal views)
Different location of EPs between forward
and backward branches.
In this example, backward branches
innervated more caudal part of the MSO
and showed longer length of axon from FB
than forward branches.

15. “Normalized “ parasagittal
distribution of endpoints
Normalized VD of endpoints
of each fiber are compactly
distributed, and the width of
the range is 0.1 – 0.2.
→ Isofrequency organization
of MSO.
Contralateral fibers:
seven of the nine fibers
showed broad distriburion in
rostrocaudal axis. The width
of the range is about 0.4 –
0.6. This broad range
reflects ladder-like structure
including 2-4 clusters.
Ipsilateral fibers:
each fiber has more
scattered distribution of
endpoints.
Our data are deviated to low-
CF area compared with
Guinan’s data {Guinan,
1972}.
CF = 1498
CF = 1345
CF = 2397
CF = 2470
CF = 7184
CF = 2018
CF = 1345
CF = 10508
CF = 841
CF = 1498
CF = 2470
CF = 2694
CF = 2300
CF = 1903
CF = 200
CF = 5388

16. Tonotopic distribution in
normalized dorsoventral axis
Black lines: relative position of
four MSO cells whose CF is 1000,
4000, 10000, and 20000 Hz,
respectively from Guinan’s paper
{Guinan, 1972},
Most of our endpoints are located
more dorsally than the Guinan’s
data points.
→Low-CF MSO neuron occupy
broader area in dorsal part of
MSO than low-CF SBC neuron do.
Green line: frequency converted
into position on basal membrane
according to the formula by
Greenwood (Greenwood and Joris
1996).
Most of our endpoints whose CFs
are less than 3000 Hz are located
more dorsally than the green line.
→Disproportionate representation
of low frequencies in MSO.

17. Axon length as the delay line in rostrocaudal axis (Contralateral fibers)
The ordinate indicates:
Normalized axonal length = (axonal length from FB)/(distance between RP to CP).
This normalization enables comparison across brainstems.
Six fibers showed significantly linear distribution (p < 0.05), and five of the six showed
correlation coefficient higher than 0.80.
→ rostrocaudal gradient: the more caudal endpoints had relatively longer axonal length.
Dashed line: line of equalty
CF = 1498
CF = 1345
CF = 2397
CF = 2470
CF = 7184
CF = 2018
CF = 1345
CF = 10508
CF = 841

18. Axon length as the delay line in rostrocaudal axis (Ipsilateral fibers)
All the seven fibers had significantly linear regression line (p < 0.01).
Three of the seven showed positive slope
→ more caudal endpoints had relatively longer axonal length.
Four fibers showed positive slope.
→ more rostral endpoints had relatively longer axonal length.
Dashed line: line of equalty
CF = 1498
CF = 2470
CF = 2694
CF = 2300
CF = 1903
CF = 200
CF = 5388

19. Location of endpoints in mediolateral axis (Contralateral fibers)
Four of the nine fibers showed endpoints whose normalized ML are larger than 0.7.
→ These endpoints are located adequately lateral side of MSO.
Such lateral location of inputs from contralateral side is not consistent with a classical observation {Stotler,
1953}.
CF = 1498
CF = 1345
CF = 2397
CF = 2470
CF = 7184
CF = 2018
CF = 1345
CF = 10508
CF = 841

20. Location of endpoints in
mediolateral axis (Ipsilateral
fibers)
Almost all endpoints from ipsilateral
side were located medial part in the
MSO.
Especially, endpoints of forward
components occupied restricted
lateral position.
CF = 1498
CF = 2470
CF = 2694
CF = 2300
CF = 1903
CF = 200
CF = 5388

21. Do low-CF fibers have longer axons than high-CF fibers?
In nine contralateral fibers, there found no clear relationship between CF and axonal length from ML to
endpoints.
Red symbols in right panel: two fibers which innervate the same MSO.

22. Axon diameter
We measured mean diameter of
“segment”, which is a part of
axon between branching points
or terminal.
White bars show all segments
from ML/FB to endpoints, and
the histogram showed a peak
around 1.9 µm.
Black bars show distribution of
most distal.
Because precedent paper
{Beckius, 1999} reported
diameter less than 1 µm at
terminals of SBC axons, our
sections might lack complete
filling of axons.

23. Discussion
Cascade pattern on contralateral fibers, sometimes on ipsilateral fibers but less systematic.
Pattern of delay lines is not consistent with distribution of BDs as assessed in the IC. Delay lines as nice at
high CFs as at low CFs.
Current findings of BD distribution show that there should be little delay at high CFs, which is not observed.
Moreover, it has since become clear that the range of BDs at low CFs is larger than previously appreciated.
So if differences in axonal delay are the source of internal delays, there should be a clear difference between
low and high CFs in the anatomy, which is not observed. Possibility that this would be “lost” on the way to the
IC is remote.
Ipsilateral fibers can also show delays running in the same direction as contra, which would counteract
delays on the contra side.
Low CFs under- rather than overrepresented.