Individual hair cells of the bullfrog's amphibian papilla release neurotransmitter most effectively during voltage stimulation at frequencies in the range of acoustic stimuli to which the organ best responds. The distribution of best frequencies along the organ accords with the tonotopic map based on afferent-fiber activity (Lewis et al., 1982
) and with the pattern of electrical resonance (Smotherman and Narins, 1999
). Hair cells of the amphibian papilla therefore display a tonotopic gradient in the efficacy of transmitter release.
What accounts for the difference between the present findings and an earlier investigation that showed no frequency-selective exocytosis in the amphibian papilla of the grass frog (Quiñones et al., 2012
)? First, the previous study pooled data from numerous cells in two large segments of the papilla, potentially obscuring sharp tuning curves. Second, the frequency resolution employed here exceeded that used in the earlier study and was therefore more likely to have revealed sharp tuning. Finally, the earlier recordings were conducted in the conventional whole-cell recording configuration as opposed to the perforated-patch mode. This difference suggests that soluble proteins, perhaps including Ca2+
buffers, are of critical importance for the mechanism of frequency selectivity. Supporting this notion is the papilla's conspicuous gradient of the Ca2+
-buffering proteins parvalbumin 3 and calbindin-D28k. Our observations accord with those of buffer gradients in other hearing organs (Hackney et al., 2003
; Hiel et al., 2002
; Schnee et al., 2005
), raising the possibility that synaptic tuning occurs elsewhere as well, particularly in non-mammalian systems lacking sharply tuned travelling waves (Gummer et al., 1987
; Manley et al., 1988
; O'Neill and Bearden, 1995
The release-site model shows how buffer concentration might influence synaptic tuning. Although tonotopic gradients of additional proteins are likely, the frequency range of the model could be matched to that of the amphibian papilla by systematic variation of only the buffer concentration and the rate constant for vesicle fusion. In this model a rise in the buffer concentration would not increase the resonant frequency but rather ensure that the system operates near a Hopf bifurcation, thereby keeping the system sensitive and sharply tuned. It is a noteworthy prediction of this model that lowering the buffer concentration, for example by whole-cell recording, might bring a hair cell into a regime in which spontaneous release of transmitter by individual synapses becomes rhythmic. This effect might be apparent in recordings of postsynaptic activity.
Because we have assumed no concurrent endocytosis during the 1 s voltage stimuli, the measured capacitance changes are likely underestimates of the actual exocytotic responses. Our results nevertheless imply that tuning can result from interactions involving primarily the readily releasable pool of synaptic vesicles. If each vesicle contributes a capacitance of 34 aF, about 1000 vesicles are released by 50 synaptic ribbons during 1 s of stimulation (Graydon et al., 2011
). This increment of 20 additional vesicles released per synapse is well within a ribbon synapse's exocytotic capacity of several hundred per second (Parsons et al., 1994
). Our experiments are unlikely to have wholly depleted the readily releasable pool of 15 vesicles per active zone (Graydon et al., 2011
), which can refill in 15–200 ms (Spassova et al., 2004
; Cho et al., 2011
). Because natural tones are seldom present for longer than a few tens to hundreds of milliseconds, this pool likely mediates tuned synaptic responses in vivo
The frequency dependence of synaptic release constitutes a bandpass filter. From recordings at closely spaced frequencies in more depolarized cells, we observed low-pass responses that decreased by half as the frequency doubled and high-pass responses that sometimes declined even more rapidly. These results may therefore account for at least two orders of the overall sharpening of auditory-nerve responses. It is possible that synaptic filtering in vivo may be still sharper, for the accumulation of Ca2+ in the presynaptic cytoplasm during prolonged depolarization might saturate synaptic release and thus broaden the tuning. However, given the small synaptic responses to short stimuli near the resting potential, the resolution of capacitance recording did not allow us to precisely delineate tuning at the resting potential.
The afferent fibers of the amphibian papilla display frequency selectivity comparable to that of the auditory organs in other vertebrates including mammals (Schoffelen et al., 2008
; Yu et al., 1991
). Although electrical resonance contributes to frequency selectivity in the low-frequency region of the papilla (Smotherman and Narins, 1999
), it is insufficient to account for the documented sharpness of tuning. The amphibian papilla lacks a flexible basilar membrane that could support traveling waves (Wever, 1973
). Although the overlying tectorium might bear traveling waves (Hillery and Narins, 1984
), its function in tuning remains hypothetical. Other tuning mechanisms must therefore account for the sharp tuning found in auditory afferents. We propose that the tonotopic tuning of hair-cell synapses plays an important role in sharpening the frequency selectivity of the amphibian papilla.