I set out to characterize the organization of the posterior lateral line by first quantifying the number of afferent neurons (herein referred to as afferents) in the ganglion for 5-day post fertilization (dpf) HUC GFP transgenic larval zebrafish (D. rerio
; Park et al. 2000
). At this stage, it has been established that larvae have a functional lateral line system that is integrated with the motor system (McHenry et al. 2009
). Fish were anesthetized with 0.02 per cent solution of MS-222 (Sigma) and embedded in 1.4 per cent low melting point agar (Fisher Scientific). Images of posterior lateral line ganglia (one for each left and right side) were collected with a Zeiss LSM 510 inverted confocal microscope (b–d
) and individual afferents were identified with image recognition software (Neurolucida, MicroBrightField Inc.).
To see if there was a relationship between neuromast body location and afferent position in a ganglion, I electroporated (Axoporator 800A, Molecular Devices) tetramethylrhodamine dye (3000 MW, Molecular Probes Inc.) into individual afferents in 5 dpf larvae. To confirm that electroporation thoroughly labelled afferents and their long processes, I also genetically labelled afferents by injecting a HUC-eGFP construct (25 mg µl−1) into wild-type embryos (pico-injector PLI-100, Harvard Apparatus). Fish were then screened with a Leica MZ 16FA fluorescent microscope to confirm single-cell expression (e–h). Backfilling neuromasts with rhodamine-loaded glass pipettes provided yet another way to label afferents, since the dyes readily travel from hair cells to afferent soma. This provided the advantage of marking larger numbers of afferents specific to a neuromast and supplemented the other labelling methods that were more time-intensive.
Afferent projections to neuromasts coalesce along the horizontal midline, so it was important to backfill only those neuromasts located at the terminal ends (D2 and P9 neuromast, a
). This avoided accidental labelling of other projections and therefore afferents that were not connected to the neuromast of interest. For example, backfilling the P3 neuromast would risk labelling all afferents that innervate neuromasts located caudal to P3 (e.g. P4–P9). In addition, labelling widely spaced neuromasts would most quickly reveal any general pattern of afferent organization in the ganglion. Note that afferents can innervate more than one neuromast (Faucherre et al. 2009
), such that backfilling P9 does not indicate that the corresponding afferents project to P9 exclusively. To standardize the X
position of neurons in the three-dimensional ganglia across individuals, the distal tip of the cleithrum was chosen as the reference focal depth for each image. Because ganglia varied in shape, I digitized each ganglion outline and bisected it with two lines into equal areas of left/right and top/bottom halves (Matlab
v.2007a, Mathworks). I took the centre as the location where these two lines intersected and measured afferent positions relative to this reference point.
Whole cell patch recordings of afferents were conducted in paralysed larvae (1 mg/1 ml α-bungarotoxin, Sigma) to determine changes in their firing rate in response to jets of water directed at specific neuromasts along the body. At the same time, extracellular motor root recordings were performed to be able to evaluate if motor activity, whether spontaneous or elicited by the water jet, was affecting the firing response of the afferents. Both patch and motor root electrodes were pulled from borosilicate glass (model G150-F-3, Warner Instruments) on a Model P97 Flaming/Browning puller (Model P-97, Sutter Instruments). Patch electrodes were pulled to 5–10 MΩ resistances and filled with 125 mM K gluconate, 2.5 mM MgCl2, 10 mM EGTA, 10 mM HEPES buffer, 4 mM Na2ATP, 0.1 per cent sulphorhodamine B, and adjusted to a pH of 7.3 with KOH. Recordings were amplified with a Multiclamp 700A amplifier at a gain of 20 with a low-pass filter set at 30 kHz, with a sampling rate of 63 kHz and converted to digital signals with Digidata 1322A (Axon Instruments). Motor root electrodes were pulled to approximately 30 µm diameter tips, beveled and flame polished with a microforge (MF-830 Narishige USA) and placed on myotomal clefts. Recordings were amplified at a gain of 1000 with a low pass filter set at 5 kHz and a high-pass filter set at 50 Hz. To reveal the sensitivity of afferent neurons to hydrodynamic stimuli, individual neuromasts were deflected with a water micro-jet triggered by a computer-controlled pico-spritzer (Harvard Apparatus). I used a motorized micromanipulator (Siskiyou Co.) to carefully position the pipette to direct the jet orthogonal to the neuromast kinocilia and parallel to the rostrocaudal axis of the body. Water velocity was calibrated by tracking suspended particles (Potters Industries Inc.) ejected from the stimulus pipette (aperture approx. 30 µm, length 3.5 cm) over a range of velocities. At the highest velocities, particles in the jet traveled approximately four neuromast diameters (about 200 µm). All values reported are mean ± standard error.