Adeno-associated viral transfection and optogenetic manipulation of auditory brainstem regions

E. McCullagh1, S. Minkowicz2, A. Klug1

787.05

1Physiol.

And Biophysics, Univ. of Colorado Anschutz, Aurora, CO; 2Florida Gulf Coast Univ., Fort Myers, FL
Viral transfec2on of the MNTB with halorhodopsin

Introduc2on

Optogene2c control of MNTB

Optogene2c control of MSO

AAV2/1NpHR3= AAV1.hSyn.eNpHR3.0-eYFP.WPRE.hGH

How do humans localize low frequency sound?

Crea%on of interaural %me dierences. Depending on
the loca0on along the azimuth from which a sound
originates, the sound waves create systema0c
interaural 0me dierences (ITDs. For example, a sound
origina0ng from the midline will arrive at both ears at
the same 0me due to the equal path length and will
thus produce an ITD of zero (A). By contrast, a sound
origina0ng from the leF hemisphere will arrive earlier
at the leF ear than at the right ear due to the shorter
path length, resul0ng in a non-zero ITD (B).

13 days

Where does low frequency sound processing occur?

AVCN

AVCN

28 days

21 days

AAV2/9NpHR3= AAV1.hSyn.eNpHR3.0-eYFP.WPRE.hGH

How does the MSO process low frequency sound?

21 days

14 days

2160Hz, AAV2/1 ArchT (53d), 532nm, 40mW

Jeress Model
Jeress 1948

Fast Inhibi2on Model

Brand et al 2002

Goal: To test whether inhibi0on from the MNTB and LNTB ne tunes ITD
Predic0ons:
Jeress model predic2on: manipula2on of inhibitory projec2on from LNTB or MNTB
to MSO will have no eect on ITD tuning, and therefore sound localiza2on ability
Fast inhibi2on predic2on: Peaks of ITD curves will shic into the physiological range
when inhibi2on from the MNTB or LNTB is turned o and sound localiza2on will be
impaired
Joris lab model: If MSO neurons are impacted by previous membrane poten2al
changes, we will see a shic in ITDs that correspond with inhibi2on being altered
over 2me

Viral transfec2on of the MNTB with ArchT

AAV2/1Arch= AAV1.CAG.ArchT.GFP.WPRE.SV40
65dB, ITD 0.2ms, 2640Hz, AAV2/9 Halo(63d), 532nm, 60mW

Optogene2c control of LNTB

Future Direc2ons
Optogene2c Control of Behavior

Methods

28 days

24 days

The gerbil will ini0ate a trial by poking its head into the central port at which 0me one of the
speakers will present the gerbil with a sound, a second sound will then be played to the leF or
right of the original sound. The gerbil will make a correct choice by poking its head into the
corresponding food trough (right side for sound origina0ng from the right of the rst sound
and vice versa). Speakers will be separated by at least 20. For each trial there will also be a
masking light to hide any illumina0on that occurs from ac0va0on of the laser. Each trial will
also be recorded and viewed on a camera over the arena.

AAV2/9Arch= AAV9.CAG.ArchT.GFP.WPRE.SV40

Top: During in-vivo recordings an optrode, A/B was

used to illuminate the MNTB, LNTB or MSO with green
light. At certain 0me points during the recordings, the
laser (C, op0cal ber to connect to laser) was turned
on and halorhodopsin or ArchT channels in the brain
area ac0vated, which in turn shut down that area. (A)
includes a tungsten electrode to ensure the loca0on of
the MNTB when the virus is injected and the op0cal
ber placed. The same tungsten electrode is also used
to verify that MNTB ring ceases during light
ac0va0on. BoRom: We tested the light scaRering
proper0es of gerbil MNTB 0ssue with these same
op0cal bers. The graph shows rela0ve light intensity
as a func0on of 0ssue depth, and indicates that at a
depth of 500 micrometers, the light intensity is about
1-3% of the light emiRed at the glass ber (light
scaRering coecient of about 14/mm). Thus, our setup
will provide enough light to reach the threshold of light
ac0va0on for HR or ArchT at a depth of at least
500m.

References
Jeress L.A. 1948. A place theory of sound localiza2on. J. Comp. Physiol.
Psychol. 41:35-39.
Brand, A., Behrend, O., Marquardt, T., McAlpine, D., and B. Grothe. 2002.
Precise inhibi2on is essen2al for microsecond interaural 2me dierence
coding. Nature. 417:543-547.

56 days

23 days
50dB, 1391Hz, AAV2/9 ArchT (80d), 532nm, 27mW