Using Cortical Neuron Markers to Target Cells in the Dorsal Cochlear Nucleus

Schematics of sound and light triggers combined with digital time stamps using a two channel sound card. A, Schematic showing analog pulses and a small jitter of digital detected timestamps. Right, Histogram showing percentage of recorded pulses and delay to analog edge. B, Illustration of channel 1 producing a sound wave to the speaker; channel 2 producing the corresponding square wave with the positive portion with the same duration as the sound pulse (gray shading). Using a diode cuts the negative portion and can be detected as the sound timestamp. C, Channel 2 writes the light square wave and the positive portion is split to provide a laser trigger and the acquisition board analog input indicates the light timestamp. D, Channel 1 as in B, and channel 2 illustrates the sum of light and sound square waves. As in C, both timestamps can be extracted from the signal recorded from the analog input of the acquisition board.

CaMKIIα-ChR2-eYFP-positive neurons in the DCN and ABR in injected animals. A, Image of coronal brainstem sections with the DCN and VCN highlighted after DAPI nuclear staining (left), control CaMKIIα-eYFP (center), and CaMKIIα-ChR2-eYFP expression (right). Scale bar: 250 μm. B, High-magnification confocal images showing several elongated horizontal somas (white arrows, possibly fusiform cells) labeled with membrane expression of eYFP. Scale bar: 40 μm. C, Another high-magnification example of CaMKIIα-ChR2-eYFP labeling of the DCN. Two possible giant cells are in the deep layer (white arrow). Lateral is left and ventral is down for all images. Scale bar: 40 μm. D, E, ABR waveforms recorded using electrodes lowered into the DCN in response to sound (D) and sound+light (E) stimulation protocols. Left, Mean (black line) and SEM (red shadow) ABR traces (n = 13), with detected peaks marked with black asterisks. Center, Box plots show group amplitude of the first five ABR peaks, horizontal lines show the median, triangles show mean, circles show outliers, whiskers bounding 99% of the data points. Right, Group latency of the first five ABR peaks.

Activation of CaMKIIα-ChR2-eYFP expressing neurons in the DCN during sound stimulation can decrease unit firing. A, left, Photography of a mouse in the recording setup and schematic representation of recording electrode and optic fiber for blue light stimulation in a DAPI labeled section showing the DCN sub-regions (ML-molecular layer, FL-fusiform cell layer, DL-deep layer). Center, Confocal image showing an example of eYFP expression in the dorsal region of the DCN with the probe tract colored by DiI. Scale bar: 100 μm. Right, Example of a DCN neuron expressing eYFP along the somatic membrane and proximal dendrites. Scale bar: 20 μm. B, Example of a superficial unit (DV depth in mm) with its waveform shown, mean (black line), SEM (red shadow) at higher magnification (center), that responded to sound stimulation (red vertical bar) by increasing firing rate as seen by a PSTH. C, Example of a deeper unit that does not respond to sound stimulation (left), increases firing in response to blue light stimulation (blue vertical bar, center), but shows no increase on both light and sound stimulation (right). D, Example of a unit responding to sound (left), blue light (center) but with a slight decrease to concomitant sound and light stimulation (right). E, Group mean number of spikes for all units (n = 76) showing a significant increase after sound (S) comparing to baseline (B; left) or blue light (L; center) stimulation (p = 5.3e-5 and 0.016) and a significant decrease after concomitant sound and light stimulation (S+L; right; p = 8.8e-5). F, Group mean number of spikes for all units (n = 76) after sound is higher than after sound+light stimulation (p = 5.3e-4). *p < 0.05; **p < 0.005; ***p < 0.0005.

Inhibition of CaMKIIα-eArch3.0 expressing neurons can delay unit sound responses. A, Example of a unit (mean, black line; SEM, red shadow) that responds to sound, while during inhibition of CaMKIIα-eArch3.0-positive DCN cells using green light (5- to 7-mW/mm² laser intensity, green background) this unit shows delayed excitation in response to sound. B, Another unit showing time-locked excitation in response to sound stimulation. This excitation is delayed by ∼20 ms when CaMKIIα-eArch3.0-positive DCN cells are inhibited by green light. C, Example of a DCN unit with a negative response following sound stimulation. This pause in firing is delayed by CaMKIIα-eArch3.0-positive DCN cells inhibition using green light. D, Group latency in response to sound is significantly increased for all units responding to both stimulation (n = 7, p = 6.7e-6). E, Quantification of numbers of units. Left, Out of 86 units (black), 17 (gray; 20%) responded to the provided stimulation. Right, Out of 12 units responding to sound, 10 (orange; 83%) increased and two (purple; 17%) decreased firing in response to sound. Out of 17 responding units, five (red, 29%) responded only to sound (S), five (green, 29%) responded only to sound and light combined (S+L), and seven (olive, 41%) responded to both stimulations. F, Responding (R) units showed lower firing rate than non-responding (NR) units (p = 5.8e-4). **p < 0.005; ****p < 0.00005.

Inhibition of CaMKIIα-eArch3.0-positive cells in the DCN can both increase and decrease excitation of DCN units not responding directly to sound. A, DCN unit (−3.8 mm DV) showing high spontaneous firing but not responding directly to sound (center) showing decreased firing rate on green light stimulation (inhibiting CaMKIIα-eArch3.0-positive DCN cells, right). B, Unit at similar depth as A showing increased firing rate on inhibition of CaMKIIα-eArch3.0-positive DCN cells, but no response directly to sound. C, Group firing rate of all units not responding directly to sound (n = 69) in the presence of sound (red) or concomitant sound and green light (olive). Units were divided into high and low firing rate, and a significant increase in firing was found for low firing units (p = 0.03). Units were also divided into units that decrease or increase firing rate comparing both stimulations, and a significant decrease and increase was found (p = 4.7e-4 and 0.01, respectively). *p < 0.05; ***p < 0.0005.

Confocal images showing tdTomato expression and Chrna2-cre/DIO-ChR2-eYFP expression in transversal brainstem sections from Chrna2^cre/tdTomato^lox mice. A, Mosaic of images showing a coronal overview of Chrna2-tdTomato expression in the cochlear nucleus (left) and MNTB (right) AP coordinate. B, Zoom-in image showing tdTomato expression in the VCN and DCN. Red cell bodies can be clearly seen in the VCN area while red expression is more diffuse in the DCN, suggesting this area is showing dense axonal terminations from VCN T-stellate cells. C, Another example showing Chrna2-tdTomato expression overlayed with DAPI staining. D, Image showing unilateral expression of eYFP following local injections with cre-dependent ChR2 (Chrna2-cre/DIO-ChR2-eYFP) constructs in the VCN. The VCN contains strongly labeled cell bodies and the DCN shows diffuse green labeling. The strong green edge of the DCN is an artifact of the mounting medium. The ipsilateral S-shaped LSO is also strongly labeled by eYFP. E, Image of a coronal slice from the CLARITY dataset of a Chrna2cre^lox mice showing the cell bodies in the VCN and projections going up to the DCN. Highlighted bundles project to LSO (1) and MNTB (2). F, top, Zoom-in showing strong labeling of the MNTB and the lateral superior olive (LSO; arrow) suggests that also anteroventral VCN bushy cells are expressing the Chrna2 promoter. Bottom, Subsequent brainstem section (from the same injected animal from D showing strong labeling of the contralateral MNTB (arrow). Scale bars: 1 mm (A) and 500 μm (B–F).

Chrna2-cre-positive neurons of the VCN can be targeted to drive activity of DCN neurons. A, left, Confocal image showing tdTomato (red) expression in VCN cell bodies and strong axonal arborizations in the DCN fusiform and deep layers. Right, Expression of ChR2-eYFP (green) in Chrna2-cre-positive neurons of the VCN, with diffuse green innervation of the DCN. B, Example of a unit from the DCN that responds to sound but not light stimulation. Line PSTH bin size is 1 ms for sound and 2 ms for light responses. C, Example of a unit that does not respond to sound stimulation, but responds to light stimulation (center). In 200 trials of combined light and sound stimulation the unit responds to both stimuli, with what appears as an anticipation of sound. D, Example of a unit not responding to sound pulses but responds with high fidelity to light stimulation of the VCN. When sound and light stimuli were combined, the unit failed to respond. E, Quantification of number of units according to response. Left, Out of 76 units, 15 (gray) responded to stimulation. Right, Out of 15 responding units, eight (red; 53%) responded to sound, four (blue; 27%) responded only to light, five (yellow, 33%) responded to both sound and light, and only one (magenta, 7%) responded to light or concomitant sound and light stimulation. F, left, Group firing rate of responding (R) units show lower firing than non-responding (NR) units (p = 2.6e-7). Right, Group mean number of spikes for all DCN units, showing a significant increase after light stimulation of the VCN circuit (black, baseline; blue, light stimulation; n = 76 units, p = 0.026). *p < 0.05; ****p < 0.00005.