Emu NL is a monolayer of bipolar neurons
In this paper we describe for the first time the anatomy and physiology of the auditory brainstem of the emu involved in encoding timing information of sound localization. The cochlear nuclei, magnocellularis and angularis, and the nucleus laminaris were clearly observed in Nissl material, as described for other avian species (). Auditory brainstem structures express calcium-binding proteins (Takahashi et al., 1987
; for review see (Braun, 1990
; Celio et al., 1996
). We used antibodies against the calcium-binding protein parvalbumin to investigate the cytoarchitecture of the auditory circuits in emu. Anti-parvalbumin immunoreactivity (PV-IR) was observed in NL () and the cochlear nuclei (not shown). In NL, PV-IR clearly labeled the cell bodies and dendrites ().
Figure 1 Auditory brainstem morphology and synaptic localization in emu nucleus laminaris. A) Schematic of transverse section of emu brainstem (left) and a micrograph of Nissl stained section (right). B, D) Anti-parvalbumin immunohistochemistry. C, E–G) (more ...)
Emu NL was a simple two-dimensional sheet of neurons stretching rostrocaudally and mediolaterally, with dendrites oriented dorsoventrally. Through most of NL, the cell bodies formed a compact monolayer. At the caudolateral extreme, NL was still laminar, but composed of a broader layer of cells, with 3 or 4 cell bodies in a slightly staggered formation. Throughout the nucleus, the most striking feature was the bitufted morphology of the NL neurons: typically a single thick primary dendrite extended from either end of an oblong cell body, which then branched into numerous short secondary and, in lateral areas, tertiary and higher-order dendrites with the appearance of “tufts” (). A tufted appearance was most apparent in the medial and rostral areas. A second feature apparent in the PV-IR sections was a gradual change in the lengths of the dendrites along the rostromedial to caudolateral axis. An analysis of this dendritic length gradient is reported below.
To investigate whether there might be a functional distinction between the primary dendrites and the tufts we used antisera against a presynaptic marker protein, synaptic vesicle protein 2 (SV2; ). Anti-SV2 immunoreactivity (SV2-IR) labeled axon terminals in both the cochlear nuclei and the nucleus laminaris (). In NL, SV2-IR was markedly absent from the afferent tracts containing the NM axons as they approached NL, but was dense within the NL dorsal and ventral dendritic neuropil (). SV2-IR was intense within the neuropil of the distal tufts, and inspection of sections double labeled for PV-IR and SV2-IR showed a close correspondence (not shown). Although weaker overall in the cell body layer, clear punctate staining could be seen along the proximal primary dendrites and cell bodies (). As with parvalbumin, SV2-IR highlighted the gradient in dendritic length ().
To determine the distribution of GABAergic inputs to NL, and to differentiate between the locations of excitatory and inhibitory synapses within NL, we used antibodies against GAD (glutamic acid decarboxylase), the enzyme that produces GABA and is found in the terminals of inhibitory axons. Anti-GAD immunoreactivity was observed in axon terminals throughout NL (), as well as in NM (); the principal neurons in these areas were GAD-negative. Unlike the anti-SV2 label, the GAD immunolabeling was observed as prominently in the cell body layer and around proximal dendrites as in the distal dendritic neuropil (). GAD-positive fibers were observed to penetrate the NL neuropil (). Although double labeling experiments for GAD and SV2-IR were not carried out, inspection of adjacent sections labeled with these antibodies suggests that most of the somatic and perisomatic SV2 label originated with GABAergic synapses.
Figure 2 Anti-GAD immunohistochemistry. A) GAD+ terminals surround unlabeled cell bodies in the nucleus magnocellularis. B) Transverse section through the NL shows the monolayer of cell bodies with the dorsal and ventral dendritic arbors outlined by GAD+ terminals. (more ...)
We created 3-dimensional reconstructions of NM and NL using Nissl stained transverse sections (). NL was a flat sheet of neurons, arcing dorsally toward the rostral and lateral edges (red nucleus, ). NM (blue nucleus, ) was positioned both dorsal and caudal relative to NL. A 2-dimensional horizontal projection of both nuclei is shown in . NM and NL have similar 2-dimensional profiles with their long axes running rostromedial to caudolateral. In birds, a tonotopic map of best frequency is oriented along this axis, with high best frequencies located rostromedially and low best frequencies located caudolaterally (Konishi, 1970
; Rubel and Parks, 1975
; Warchol and Dallos, 1990
). In emus, the basilar papilla has been shown to respond to acoustic frequencies from ~50 Hz to ~4.8 kHz, and is organized logarithmically (Köppl and Manley, 1997
). Therefore, we propose a hypothetical mapping of sound frequency as shown in . In this model, approximately two-thirds of NL is dedicated to best frequencies below 1 kHz, and nearly one-half to those below 500 Hz. This model map assumes no transformation of the tonotopic map between papilla and NL (i.e., expansion or compression of a subset of frequencies relative to the others).
We investigated the cytoarchitecture of the NL neurons in emu with PV-IR sections () and with fills of individual NL neurons that had been stained with the intracellular label biocytin during physiological recordings (; see below). During these physiological recordings we attempted to sample the full range of the nucleus, including the most rostromedial and caudolateral, and thus the filled cells should include examples across the full range of NL morphology. These materials showed two clear findings: first, the PV-IR sections suggested, and the biocytin fills confirmed, that the majority of NL neurons in emu were strictly bipolar, with two primary dendrites, one oriented dorsally, the other ventrally, while a smaller number of neurons had 3 or 4 primary dendrites. Among the filled neurons (n = 45), the majority had only two primary dendrites (; 30/45, or 66.7%), while a minority had three primary dendrites (13/45; 28.9%), and only a small number had four (2/45; 4.4%). No neurons were observed with more than 4 primary dendrites, although some dendrites branched very close to the cell body. Nearly all dendrites invaded exclusively the dorsal or ventral neuropil; in one case a dendrite was oriented horizontally.
Figure 4 Nucleus laminaris neuronal reconstructions. Sixteen examples of biocytin-labeled neurons as reconstructed with the Neuroludica computer assisted drawing system, arranged from the shortest dendritic length neurons (most rostromedial) to the longest dendritic (more ...)
The second major finding was a gradient of dendritic length, with the shortest lengths in the rostral and medial area, and the longest in the caudal and lateral area. The gradient was observed in both the PV-IR and the SV2-IR sections (e.g., ). Throughout most of NL, the cell bodies formed a compact monolayer, and thus the extent of the neuropil was defined by the dorsoventral neuronal dendritic length. When the dorsoventral depth of the neuropil was measured as an indicator of dendritic length, the lengths ranged from 30 µm to 430 µm, a ~14-fold range. Filled neurons that were reconstructed and measured as a straight line from dorsal to ventral dendritic tip had lengths that varied from 61 µm to 311 µm, a ~5-fold range. The discrepancy between these two measurements may be due to an underestimate of the extremes by an undersampling of NL during physiological experiments, an overestimate of the maximal length in the PV-IR sections because of the staggered organization of the cell body layer in the caudolateral area, or both.
To investigate the distribution of dendritic lengths throughout the nucleus, we sampled the dorsoventral dendritic neuropil extent at regular intervals from the serial sections of PV-IR material. These lengths were then plotted versus position expressed as percent rostrocaudal position on one axis and percent mediolateral position on the other (). Dendritic length varied smoothly from shortest in the rostral and medial region to longest in the caudal and lateral region. To quantify this pattern, we calculated the best isolength contour with a multivariable linear regression (red line, ). The dendritic length gradient was calculated as a line orthogonal to the isolength contour (black line, ) and was oriented in a rostromedial to caudolateral direction, similar to the gradient described in the chick (Smith and Rubel, 1979
; Smith, 1981
). We then used the gradient line to relate the average dendritic length with rostrocaudal-mediolateral position (% RC-ML; ). The dendritic lengths increased linearly (r = 0.97; p < 0.0001) with an average slope of ~2.4 µm/percentile. The exception to this pattern was the decrease in length measurements found at the extreme caudolateral pole; in this area, the neuropil depth measure may have underestimated the true dendritic length because caudolateral-most NL neurons were curved instead of being oriented strictly dorsoventrally. The cumulative distribution of dendritic lengths showed a steep slope at the middle lengths, with shallow tails above and below, suggesting that the median lengths were proportionately overrepresented (); 80% of the nucleus by area was devoted to lengths between 120 µm and 275 µm.
In order to describe the dendritic morphology of NL neurons, we used detailed reconstructions of individual neurons filled with biocytin during physiological recordings (physiological results are reported below). We were able to fully reconstruct 43 of 45 filled NL neurons with the Neurolucida system (). For these neurons we made two dendritic length measurements. 1) Linear dorsoventral length: a straight line length measure from most distal dorsal extent to most distal ventral extent, comparable to the neuropil depth measurements reported above. 2) Total dendritic length: the sum of all reconstructed dendritic segment lengths, and equivalent to the total dendritic length of Smith and Rubel (1979)
. Total dendritic length and linear dorsoventral length were well correlated (; linear fit, r = 0.73, p < 0.0001). The data was slightly better fit by a power law regression (y = 0.53 × 1.42
, r = 0.83, p < 0.0001), but with little improvement in the residual distribution. Total dendritic lengths varied widely from 165 µm to 2668 µm (a 16-fold range) and averaged 765 ± 677 µm.
Figure 7 Correlation of total dendritic length with linear dorsoventral dendritic length. Total dendritic length and linear length measurements from biocytin-filled NL neurons (n = 43). Total dendritic length is the sum of all dendritic branch lengths drawn with (more ...)
To determine whether other morphological features varied across the nucleus, we plotted several measures versus total dendritic length (n = 43). Cell bodies were generally ovoid or ellipsoid throughout, with an average area of 244.0 ± 76.2 µm2 and an average length and width of 24.4 ± 6.6 µm and 14.2 ± 2.5 µm, respectively (). The average aspect ratio was 1.7 ± 0.4. Cell body area did not change significantly across NL (, r = 0.036). There was no correlation between cell body length, width, or aspect ratio with total dendritic length (data not shown).
Emu NL Morphological Properties1
Figure 8 Morphological characteristics of biocytin-filled NL neurons and their variation with total dendritic length. A) Cell body area. B) Length of primary dendrites, from cell body to first branch point. C) Average primary dendritic thickness. D) Total number (more ...)
Emu NL neurons had notable thick primary dendrites that extended some distance before branching into a dense “tuft”. We measured the length of each primary dendrite from the cell body to its first branch point. Individual primary dendritic lengths ranged from 1.0 µm to 92.5 µm. Primary dendrites in our sample had an average length of 22.7 ± 18.6 µm and thickness of 4.2 ± 1.4 µm (n = 102 primary dendrites). Primary dendritic length was not correlated with total length (, r = 0.014); this was because many of the primary dendrites of longer NL neurons branched close to the cell body. Average primary dendritic thickness also did not vary with total dendritic length (, r = 0.045).
The number of branch points, or nodes, was correlated with total dendritic length, however, demonstrating an increase in complexity across the nucleus (, r = 0.82, p < 0.0001). Total surface area likewise increased with total dendritic length (, r = 0.82, p < 0.0001). The number of primary dendrites was not strongly related to position, as the majority of neurons that had only two primary dendrites were distributed broadly across NL; however, neurons with 3 or 4 primary dendrites were proportionately more likely to have longer dendritic lengths ().
Ventral total dendritic lengths were longer on average than dorsal total dendritic lengths (452 ± 507 µm, ventral, versus 334 ± 272 µm, dorsal; paired Student’s t-test, p < 0.05; ). This reveals a slight overall bias toward longer total lengths in ventral trees, although dorsal and ventral lengths were correlated (, r = 0.64, p < 0.0001). While the ventral dendritic trees had slightly more branch points than dorsal, indicating greater complexity, this difference was not significant on average (ventral, 19.0 ± 19.2, dorsal, 15.8 ± 13.0; p = 0.086). The numbers of ventral and dorsal nodes were highly correlated (, r = 0.80, p < 0.0001).
Comparison of ventral and dorsal dendritic trees. A) Ventral and dorsal dendritic lengths were correlated, although ventral lengths were slightly longer. B) Ventral and dorsal dendritic trees had nearly equal numbers of nodes.
Emu nucleus laminaris contains contralateral delay lines
The avian nucleus laminaris is characterized by physiological specializations that contribute to the computation of interaural time difference. One such specialization is the formation of delay lines composed of the axons of nucleus magnocellularis neurons as they approach NL (Young and Rubel, 1983a
; Young and Rubel, 1986
). We performed in vitro
physiological experiments to determine whether delay lines were present in emus. In brainstem slices cut in the presumed isofrequency plane, we recorded local field potentials (LFP) in NL in response to electrical stimulation of the afferent inputs from NM (). These experiments were conducted at room temperature (22–24°C). We made serial recordings of field potential responses across the (oblique) mediolateral extent of the nucleus, while maintaining the position and stimulation strength of the stimulation electrodes. In some slices, we used two stimulation electrodes and recorded at the same NL site with both contralateral and ipsilateral stimulation. When we stimulated fibers from the contralateral NM, a pronounced, consistent shift occurred in the latency of the evoked postsynaptic field potentials across the medial-to-lateral extent of NL (). In the example in , the most medially recorded field potential evoked by contralateral stimulation (closed circles) had a latency of 470 µs, while the most laterally recorded field potential, at a distance of 800 µm from the first recording, had a latency of 1.5 ms, a shift of 1,030 µs. This shift can be expressed as the slope of the linear fit to the data, which in this example was 1.3 ms/mm (r = 0.91, p < 0.001). In contrast, ipsilateral stimulation produced a much smaller shift in latency (0.31 ms/mm; r = 0.84, p < 0.01).
Figure 10 Emu NM afferents form delay lines to contralateral NL. A) Right: Schematic of the brainstem slice preparation showing electrical stimulation of contralateral tract and/or ipsilateral tract during sequential recordings of the local field potential (LFP) (more ...)
Contralateral stimulation evoked LFP responses whose latencies showed clear, positive shifts (n = 7; ), with an average slope at 22–24°C of 0.91 ± 0.55 ms/mm (range: 0 to 1.67 ms/mm), which corresponds to an average conduction velocity of 1.09 mm/ms. Ipsilateral stimulation produced LFP responses whose latencies were more difficult to quantify, or which could only be recorded over a portion of the mediolateral length. Ipsilateral stimulation could produce positive, negative, or negligible latency shifts, whose average slope was close to zero (0.09 ± 0.37 ms/mm, n = 8; ). Individual slopes ranged from −0.47 to +0.53 ms/mm; most had low correlation constants and thus slopes that were not statistically reliable.
To compare ipsilateral and contralateral latency shifts, we plotted the slope of the linear fit in the range of positions where both ipsilateral and contralateral responses were recorded in the same slice (n = 5; closed symbols; ). The average slope shift (gray closed symbols, ) for contralateral stimulation (1.11 ± 0.88 µs/mm) was significantly larger than for ipsilateral stimulation (0.26 ± 0.37 µs/mm; paired Student’s t, p < 0.05; n = 5).
In one experiment, we also made latency measurements while varying the temperature (). The change in latency with temperature was well fit by a log fit with a slope of −0.027, and the Q10
(factor change for every 10°C change in temperature) was 1.84. We extrapolated that at physiological temperatures (41°C), the conduction velocity would be ~3.6 m/sec, and thus the maximal delay across the extent of NL in our slice preparation (1 mm) would be ~280 µs. These results suggest the contralateral conduction velocities found in the emu laminaris could implement delays within the physiological range. Measurements from 4 emu skulls yielded interaural distances of 48.62 ± 1.85 mm, similar to those measured in the barn owl (Haresign and Moiseff, 1988
Emu nucleus laminaris and magnocellularis display timing circuit physiological specializations
In chick, it has been well established that the neurons of NM and NL express voltage-gated ionic conductances that contribute to their ability to phase lock and encode the timing cues present in sound (Reyes et al., 1994
; Reyes et al., 1996
; Trussell, 1997
; Rathouz and Trussell, 1998
; Kuba et al., 2002a
; Kuba et al., 2005
). To determine whether these conductances were present in the emu, we performed whole cell intracellular recordings to measure the voltage responses to steps of current input (at room temperature, 22–24°C). We recorded from 45 NL neurons sufficiently to fill with the intracellular label biocytin (see morphology section, above). In 11 of these, we recorded the full range voltage response to current injections; in NM, we made similar recordings from 3 neurons. Both NL and NM neurons showed physiological characteristics similar to those observed in chick NL and NM recordings (Reyes et al., 1994
; Reyes et al., 1996
; Kuba et al., 2002a
; Kuba et al., 2005
In emu NL, we found a single-spiking profile in response to depolarizing current injection, and a prominent ‘sag’ following hyperpolarizing current injection, and thus NL demonstrated both inward and outward rectification (). These results suggest the presence of a low-threshold potassium current activated by depolarization, and an Ih
-like current activated by hyperpolarization (Reyes et al., 1996
; Trussell, 1997
; Carr and Soares, 2002
; Kuba et al., 2002a
; Kuba et al., 2002b
; Kuba et al., 2005
). In some recordings, a rebound voltage overshoot was seen with repolarization of the membrane potential, which could result in an action potential (, inset). There was a significant difference in input resistance above and below resting potential (n = 11, p < 0.01). For depolarizing steps, emu NL neurons had an average input resistance of 30.2 ± 19.9 MΩ, while for hyperpolarizing steps, average input resistance was 177.2 ± 77.6 MΩ (). Membrane time constants were also different above and below resting potential (p < 0.05). For small (25–75 pA) current steps around resting potential, the average membrane time constant was 12.8 ± 3.8 ms above resting potential, and 30.2 ± 19.9 ms below resting potential.
Figure 11 Membrane properties of emu NL and NM neurons. A) Left: Voltage response (top traces) of a single NL neuron to current injection steps (bottom traces). Half of NL neurons showed a voltage overshoot at the offset of a hyperpolarizing step (asterisk) that (more ...)
Emu Neuronal Membrane Properties1
Voltage responses from NM neurons showed a similar profile (n = 3; ). For steps depolarizing from the resting potential (−59.8 ± 3.1 mV), emu NM neurons had an average input resistance of 49.0 ± 25.6 MΩ, and a average membrane time constant of 3.9 ± 0.2 ms (). For steps hyperpolarizing from rest, the input resistance was 260.0 ± 139.8 MΩ, and membrane time constant was 14.1 ± 3.9 ms.