In this study we measured axon length in the chicken brainstem sound localization circuit responsible for the processing of ITDs, as well as parameters responsible for conduction velocity, specifically axon diameter and internode distance. Surprisingly, axon length dimensions do not to comply with the proposed modified Jeffress model (Overholt et al., 1992
), as the contralateral axon is more than 1600 μm longer than the ipsilateral one, making coincidence detection based on travel distance impossible. However, the data indicate that variations in both axon diameter and internode distances may counterbalance the offset in axon length. This axon length disparity and anatomical evidence for temporal compensation provokes a reassessment of current thinking about the organization of the avian and mammalian sound localization circuits responsible for ITD coding.
In accord with the hitherto proposed model embodied by the NM-NL circuit (Overholt et al., 1992
), our results illustrate similar lengths of axons from NM onto the dorsal NL dendrites and confirm that each of the contralateral axons forms successive series of short collaterals stemming from a parent axon coursing immediately ventral to NL neurons orthogonal to the tonotopic axis. These collaterals form a “delay line” of axon terminals on NL neurons from the contralateral NM. The most interesting and unforeseen observation of this study is a large and reliable difference in the lengths of contralateral and ipsilateral pathways from NM to NL, which does not comply with the proposed modified Jeffress model. In their seminal paper about the barn owl sound localization circuit, Carr and Konishi (1990)
also noted a difference in axon lengths from NM to the ipsilateral and contralateral NL. The differences (~ 1 mm, and a much larger head) were not as great as those reported here, but that may be due to the fact that 3-dimensional reconstructions were not attempted. In this paper, Carr and Konishi also foreshadowed our findings of variations in axon caliber and internodal differences, noting that these two parameters varied along the pathways and may modulate conduction velocities in meaningful ways. Physiological data demonstrate the existence of neurons responding best to 0 ITD (Koppl and Carr, 2008
). Indeed, rough calculations of the conduction velocity for the ipsilateral and contralateral axon using response delay data from Figure 4 of Koppl and Carr (2008)
and from Koppl (1997)
yield values that average at about 3.8 and 8 m/s, respectively. Not only are the resulting values for the contralateral axon twice as large, the conduction velocity for the contralateral axon is also consistent with the values proposed for the XDCT in this paper (). Given the difference in length between ipsi- and contralateral axon collaterals, the encoding of ITDs in the physiological range for the chicken is impossible unless other morphological or physiological features regulate the timing in these circuits. In myelinated axons, their diameter is linearly proportional to conduction velocity (Gasser and Grundfest, 1939
; Rushton, 1951
; Hutchinson et al., 1970
). Our data show significantly smaller axon diameters in the terminal trees compared to the major branches of the axons as they traverse the XDCT. Additionally, the ipsilateral terminal tree has an average axon diameter that is only three quarters of the average diameter in the ventral terminal tree, further delaying the arrival of ipsilateral action potentials at NL relative to the contralateral signal. This effect is amplified by the fact that the average axon length in the ipsilateral terminal tree is more than double the length of the mean axon in the ventral terminal tree. We did not measure axon diameter in the ipsilateral loop, but variations in this segment might enable the system to further alter conduction velocities and to slow down the signal on the ipsilateral side.
Brill and colleagues showed that conduction velocity increases linearly with internode distance to up to 2000 μm (Brill et al., 1977
). Moreover, when the ratio of internode distance (L) and axon diameter (d) is small (< 150), conduction velocity is quite sensitive to variations in L/d (Brill et al., 1977
). Carr and Konishi (1990)
compared internode distance of axons as they traverse the cell body layers within the owl NL with axons outside NL, and also report shorter internode distances of the axons within NL. At that time, methods to label individual axons were not readily available; hence the origin of the axons they measured could not be confirmed. We were able to measure internode distances in different segments of labeled NM axons and found significant variations along the NM axon. In particular, internode distances along XDCT is significantly larger compared to other parts of the NM axon by at least 50%. It also appears that internode distances within the terminal arbors on both the ipsilateral/dorsal axons and the contralateral/ventral axons are smaller than the main branches leading up to these terminal branches. Again, this disparity of terminal tree length enhances the effect of short internode distances along the terminal endings and contributes to delaying the propagating signal ipsilateral relative to the contralateral side of the brain. Taking into account the axon diameter variations and the resulting sensitivity to L/d ratio, there seems to be ample possibility for variations in signal speed.
In summary, our results show that at least two parameters influencing conduction velocity, axon diameter and internode distances, are optimally regulated at different sites within individual axons of NM neurons to optimally adjust the conduction velocities of the ipsilateral and contralateral signals, presumably in order to optimize coincidence detection. Slowing down the propagating signal in the ipsilateral part of the axons allows the contralateral signal to “catch up” and enables encoding of ITDs in the physiological range in NL neurons. Interestingly, click delays measured in NL (from Koppl and Carr, 2008
) applied to our axon length data seem to indicate an ipsilateral transmission speed that is only half a fast as its contralateral counterpart. The regulation of these axonal parameters within individual axons seems quite remarkable from a cell biological point of view, but it is not unprecedented. Measurements in other neuronal systems have been shown to provide temporal compensation for conduction distance disparities. For example, in the electromotor system in fish internode distance is adjusted so that conduction velocity compensates for different axon lengths (Bennett, 1970
). In rat Purkinje cells, conduction velocity is varied to enable isochronicity of different length climbing fibers (Sugihara et al., 1993
). Other parameters, such as myelin sheet thickness (Rushton, 1951
), may also influence signal propagation in the NM-NL circuit. Moreover, one might inquire if there is a period of adjustment for one or several of these morphological features during development, wherein these circuits are temporally sharpened and compensated for changes in head size to optimize discrimination of binaural timing differences.
We show that axon length alone cannot be solely responsible to compensate for external ITDs in the chicken sound localization circuit. This unexpected result has implications for the mammalian brainstem binaural system as well. It is noteworthy that the mechanism responsible for low frequency sound localization in mammals has recently come under dispute. There is only marginal anatomical evidence for delay lines in mammals (Smith et al., 1993
; Beckius et al., 1999
) and a proposed alternative mechanism, incorporating the glycinergic inputs to MSO, requires a very fast inhibitory input (τdecay
= 0.1 ms) (Brand et al., 2002
) that has not been confirmed by physiological measurements (Magnusson et al., 2005
). Variations of parameters such as axon diameter, internode distance and others (e.g., see Pecka et al., 2008
) in the mammalian brainstem might be responsible for precise adjustments of physiological delays, thereby creating the framework and adjustments of the ITD detection circuit.