Auditory thalamic organization: Cellular slabs, dendritic arbors and tectothalamic axons underlying the frequency map

Neuroscience 136 (2005) 927–943

AUDITORY THALAMIC ORGANIZATION: CELLULAR SLABS, DENDRITIC ARBORS AND TECTOTHALAMIC AXONS UNDERLYING THE FREQUENCY MAP N. T. MCMULLEN,* D. S. VELENOVSKY AND M. G. HOLMES

hair cells with the dendrites of spiral ganglion cells, a frequency map is created. This frequency map is maintained at all levels of the ascending auditory system up to the auditory neocortex and is the fundamental template upon which other discontinuous maps are superimposed (Rouiller, 1997). While much is known concerning the neuronal circuits that underlie the frequency map in the auditory brainstem (Ryugo, 1992; Ryugo and Rouiller, 1988; Ryugo and May, 1993; Rouiller, 1997), little is known at the level of the auditory thalamus and above (de Ribaupierre, 1997). In contrast to the well-established laminar architecture of the lateral geniculate body (Jones, 1985; Sherman and Guillery, 2001) and the rod-matrix organization of the somatosensory thalamus (Rausell and Jones 1991a,b; Rausell et al., 1992), the cellular architecture of the auditory thalamus and its relationship to functional maps, remain obscure. The medial geniculate body (MGB) is the last obligatory synaptic site for relaying ascending auditory information to the cerebral cortex. Traditionally, this large complex of nuclei has been subdivided into three major anatomic divisions: the ventral (MGV), medial (MGM) and dorsal (MGD) subdivisions (Morest, 1964; Jones, 1985; Oliver and Hall, 1978; Winer et al., 1988; Clerici and Coleman, 1990; Ramón y Cajal, 1995). These subdivisions receive input from separate and parallel functional channels from lower centers and project to auditory neocortex (Calford, 1983; de Ribaupierre, 1997). The MGV receives its major input from the tonotopically-organized central nucleus of the inferior colliculus (ICC) and projects to primary auditory areas in the neocortex (Winer et al., 1977; Andersen et al., 1980; Winer, 1985). The MGV has been shown to have a well-defined tonotopic axis in a variety of species (Aitkin and Webster, 1972; Gross et al., 1974; Imig and Morel, 1985; Redies and Brandner, 1991). Functionally, the MGV is composed of layered, narrow-band sheets that form a complete cochleotopic representation. Golgi studies have revealed a complementary orientation of the dendrites of principal cells and axonal arbors believed to originate in the auditory midbrain (Morest, 1965; Jones and Rockel, 1971). These “fibrodendritic laminae” have been proposed as the anatomical substrate for the frequency map in the MGV (Morest, 1964; Aitkin and Webster, 1972). Correlative structural–functional studies of the cat and other species have been hampered by poorly defined boundaries between MGB subdivisions as well as the lack of visible laminae within the MGV itself (Morest, 1965; Calford and Webster, 1981; Calford, 1983; Jones, 1985; Rodrigues-Dagaeff et al., 1989). The MGV of the rabbit

Department of Cell Biology and Anatomy, University of Arizona College of Medicine, P.O. Box 245044, 1501 North Campbell Avenue, Tucson, AZ 85724, USA

Abstract—A model of auditory thalamic organization is presented incorporating cellular laminae, oriented dendritic arbors and tectothalamic axons as a basis for the tonotopic map at this level of the central auditory system. The heart of this model is the laminar organization of neuronal somata in the ventral division of the medial geniculate body (MGV) of the rabbit, visible in routine Nissl stains. Microelectrode studies have demonstrated a step-wise ascending progression of best frequencies perpendicular to the cell layers. The dendritic arbors of MGV neurons are aligned parallel to the cellular laminae and dendritic tree width along the frequency axis corresponds closely with the frequency steps seen in microelectrode studies. In the laminated subdivision, tectothalamic axons terminate in the form of bands closely aligned with the laminae and dendritic arbors of thalamic relay neurons. The bands of tectothalamic axons extend in the anteriorposterior (A-P) plane forming a dorsal–ventral series of stacked frequency slabs. In the pars ovoidea region, the homologous spiraling of somata, dendritic fields and tectothalamic axons appear to represent a low-frequency area in this species. At least two types of tectothalamic terminals were found within the bands: large boutons frequently arranged in a glomerular pattern and smaller boutons arising from fine caliber axons. We propose that the rabbit is an ideal model to investigate the structural–functional basis of functional maps in the mammalian auditory forebrain. © 2005 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: hearing, medial geniculate, cerebral cortex, inferior colliculus, laminar organization.

A fundamental process of hearing is the transduction of mechanical energy into electrical signals by hair cells of the basilar membrane. This membrane is “frequencytuned” and because of the strict topographic relationship of *Corresponding author. Tel: ⫹1-520-626-2248; fax: ⫹1-520-626-2097. E-mail address: [email protected] (N. T. McMullen). Abbreviations: AI, primary auditory cortex; A-P, anterior-posterior; BDA, biotinylated dextran amine; BF, best frequency; DAB, diaminobenzidine; ICC, central nucleus of the inferior colliculus; LV, pars lateralis of the ventral division of medial geniculate body; MGB, medial geniculate body; MGD, dorsal division of medial geniculate body; MGI, internal division of medial geniculate body; MGM, medial division of medial geniculate body; MGV, ventral division of medial geniculate body; NRS, normal rabbit serum; OV, pars ovoidea of the ventral division of medial geniculate body; PBS, phosphate-buffered saline; PV, parvalbumin; VL, ventral lateral division of ventral division of medial geniculate body.

0306-4522/05$30.00⫹0.00 © 2005 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2005.04.058

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offers several significant advantages for investigating structural–functional relationships in the auditory CNS: it has readily identifiable subdivisions (de Venecia et al., 1995) and a laminar cytoarchitecture visible in routine Nissl stains (Cetas et al., 2001). Recent electrophysiological and juxtacellular-labeling studies (Cetas et al., 2001, 2002) have revealed a direct correspondence between cellular laminae, dendritic orientation and the tonotopic map in the MGV. We have recently proposed a model of MGV organization that incorporates cellular laminae and oriented dendritic growth as a basis for the frequency map (Cetas et al., 2003). In the present paper, we review the studies which support this model and describe the results of recent experiments using the anterograde transport of biotinylated dextran amines (BDA) to label tectothalamic axons originating from physiologically-characterized sites in the inferior colliculus. Our updated model of auditory thalamic organization incorporates cellular laminae, the dendritic arbors of relay neurons and tectothalamic axons as a basis for the frequency map. Portions of this work have been presented in abstract form (McMullen et al., 2004; Velenovsky et al., 2004).

EXPERIMENTAL PROCEDURES The following methods were used to generate the results described in this manuscript: 1) microelectrode mapping of the auditory thalamus and midbrain to define the frequency organization (Cetas et al., 2001), 2) juxtacellular labeling of physiologically-characterized thalamic neurons with biocytin followed by immunocytochemistry to label dendritic arbors (Cetas et al., 2002), 3) three-dimensional reconstruction and quantitative spatial analyses of biocytin-labeled thalamic neurons (Cetas et al., 2003), and 4) anterograde labeling of tectothalamic axons via extracellular injection of BDA into physiologically defined sites in the ICC (Velenovsky et al., 2004; McMullen et al., 2004). Detailed methodologies can be found in the original reports. The anterograde labeling of tectothalamic axons is a new experiment and the methods are provided in more detail below.

Experimental subjects Normal adult New Zealand White rabbits (2–3 kg) were obtained from commercial suppliers. Animal protocols were approved by the University of Arizona Institutional Animal Care and Use Committee and conformed to NIH guidelines. All survival surgery was performed under sterile conditions. Care was taken to minimize the number of animals used and their suffering. Animals were anesthetized with ketamine (44 mg/kg i.m.) and xylazine (10 mg/kg i.m.) and placed in a Kopf stereotaxic device (David Kopf Instruments, Tujunga, CA, USA). After a small hole was drilled through the skull, a Carbostar-3 (Kation Scientific, Minneapolis, MN, USA) electrode with one barrel filled with 10% BDA (MW⫽10,000) in 0.1 M phosphate-buffered saline (PBS) was advanced in 100 ␮m steps into the ICC using a Kopf micromanipulator fitted with a remote-controlled microdrive (National Aperture, Inc., Salem, NH, USA). TDT System 3 (Tucker David Technologies, Alachua, FL, USA) hardware and software were used to deliver calibrated tone and noise bursts through custom transducers. When short latency, tightly tuned responses to tone bursts were seen, the site was physiologically characterized (best frequency (BF) and binaurality) and BDA was deposited using positive current (3 ␮A for 3–5 min) with a Midgard CS-3 (Stoelting Co., Wood Dale, IL, USA) current generator. The incision was then sutured and Chloromycetin administered (i.v.). When the

animal recovered from the effects of anesthesia, it was returned to its cage and monitored daily. Animals were allowed to recover for approximately 7–9 days and then deeply anesthetized and transcardially perfused with 4% paraformaldehyde. After cryoprotection in ascending sucrose solutions (to 30%), transverse sections through the MGB and IC were cut at a thickness of 50 ␮m on a freezing microtome. ICC injection sites and anterograde-labeled axons in the MGV were visualized immunocytochemically.

Localization of injection sites Sections through the auditory midbrain were incubated for 15 min in 1% H2O2 to suppress endogenous peroxidase activity. BDA was localized by avidin– biotin– horseradish peroxidase histochemistry (Vector Elite ABC Kit) with nickel– cobalt intensification of the diaminobenzidine (DAB) reaction product. Sections were mounted on gelatinized slides. In some cases, sections were counterstained with 1% aqueous Methylene Blue to help confirm the location of the injection sites.

Immunohistochemistry A sensitive immunoperoxidase method was used to visualize BDA-labeled axons in the MGV following ICC injections (McMullen and de Venecia, 1993). Sections were treated with 1% H2O2 for approximately 15 min to suppress endogenous peroxidase activity, followed by 3% normal rabbit serum (NRS) in 1% Triton X-100 to block nonspecific antibody labeling and to increase antibody penetration. Sections were incubated for 48 h at 4 °C in goat anti-biotin antibody (Vector, Burlingame, CA, USA) diluted 1:10,000 in PBS containing 3% NRS, followed by biotinylated rabbit antigoat IgG (Vector) diluted 1:200 in 3% NRS–PBS for 2 h at room temperature. After 90 min in Vector Standard ABC solution (90 ␮l each of reagents A and B per 10 ml of PBS), the sections were subjected to DAB with heavy metal intensification (Adams, 1981), mounted onto gelatinized slides and coverslipped with Permount. Alternate sections were lightly counterstained with 1% Methylene Blue for determining architecture of the MGV relative to the BDAlabeled axons.

Reconstruction of tectothalamic arbors and photomicroscopy An image-combining computer microscope equipped with a threeaxis motorized stage, Lucivid monitor and Neurolucida software (MicroBrightfield, Inc., Williston, VT, USA) was used to digitize brain section outlines, thalamic subdivisions, and BDA-labeled tectothalamic axonal bands in the MGV. The digitized outlines of the tectothalamic bands were used to measure the orientation and dimensions of the axon terminal fields. The orientation of tectothalamic bands was measured relative to the horizontal plane in each section (approximately nine sections/animal). The length and width (at three equally spaced locations in the transverse plane) of the axonal bands were measured in single transverse sections along their major axis of orientation. The mean orientation and dimensions of the tectothalamic axonal bands were calculated from four experiments whose ICC injection sites ranged from 1.2–9.6 kHz. Tectothalamic boutons in the MGV labeled by the BDA injections in the ICC were digitized from serial sections using a 40⫻ or 63⫻ oil-immersion objective. Brightfield images were acquired digitally with a Hamamatsu C5180 camera (Hamamatsu Photonics, K.K., Hamamatsu City, Japan) and Nikon E800 M microscope (Nikon Instruments Inc., Melville, NY, USA). Adobe Photoshop (Adobe Systems Inc., San Jose, CA, USA) software was used to optimize contrast and brightness only. Images were imported into CorelDraw (Corel Corp., Ottawa, Ontario, Canada) for final layout and labeling.

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Fig. 1. Medial geniculate subdivisions in the rabbit. Photomicrographs of adjacent transverse sections through the rabbit MGB stained with Methylene Blue (A) and with antibodies against the calcium-binding protein parvalbumin (B). (A) The oval ventral nucleus (V) is readily identified in Nissl-stained sections and can easily be differentiated from the dorsal (D), internal (I) and medial (M) subdivisions. (B) Parvalbumin immunocytochemistry sharply delineates the ventral subdivision where neuronal somata, fibers and terminals are intensely labeled. Scale bar⫽1 mm. APT, anterior pretectal area. (Adapted from Cetas et al., 2002.)

RESULTS Laminar architecture of the MGV The MGB of the rabbit consists of four major subdivisions as revealed by Nissl stains, calcium-binding protein expression, and histochemical stains (Caballero-Bleda et al., 1991; de Venecia et al., 1995). The large MGV and MGD subdivisions are separated by the wedge-shaped internal division (MGI; Tarlov and Moore, 1966; Jones, 1985). A small medial division (MGM) lies dorsal and medial to the MGV (de Venecia et al., 1995). The MGV is readily distinguished from the other divisions of the MGB by its relatively high cellular density in routine Nissl stains and dense expression of the calcium binding protein parvalbumin (PV; Fig. 1). The MGV is surrounded on the ventral, lateral, and medial edges by the marginal zone (MZ), an area characterized by calbindin-immunoreactive somata and reduced PV expression (de Venecia et al., 1995). The ventral division can be further divided into a pars lateralis (LV), a pars ovoidea (OV), and a ventrolateral (VL) nucleus. Each of these three regions displays a unique cytoarchitecture, evident with Nissl and Golgi stains, and a distinct pattern of calcium binding protein expression (de Venecia et al., 1995; Cetas et al., 2002). The LV forms the major central component of the MGV and is marked by a laminar ar-

rangement of cell bodies oriented dorso-medially to ventrolaterally in the transverse plane (Fig. 2A). A similar laminar pattern in the LV is exhibited by PV-immunoreactive axonal fascicles and by the dendritic orientation of tufted principal neurons in Golgi sections (Cetas et al., 2002). The OV can be distinguished from the LV by the concentric organization of somata (Fig. 2), a spiraling pattern of dendrite systems and a higher density of PV immunoreactive somata and fibers (de Venecia et al., 1995). In comparison to the cat (Morest, 1965; Winer, 1985), the OV in the rabbit is rotated 180° and is situated at the dorsolateral edge of the MGV (Fig. 2). The VL lacks a laminar architecture and is characterized by weak PV immunoreactivity and a higher proportion of calbindin-immunoreactive neurons (de Venecia et al., 1995). Quantitative analysis of Nissl-stained sections of five animals used in MGV mapping studies (Cetas et al., 2001) revealed that the main axis of the cellular laminae in LV is inclined at an angle of 25° relative to the horizontal plane (Fig. 2; Cetas et al., 2001, 2003). Portions of the MGV surrounding this laminated core (the lateral, ventral and medial edges of the LV) do not appear to be laminated. The lateral boundary of the LV corresponds, in part, to the ventrolateral nucleus described in chemoarchitectonic studies (Caballero-Bleda et al., 1991;

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Fig. 2. (A) Photomicrograph of Nissl-stained transverse section through the middle portion of the MGV which is composed of the LV and the OV. Vertical lines indicate trajectory of two mapping penetrations. Lateral is to the left. Note the dorsomedial to ventrolateral orientation of cell somata in LV region of the MGV. Approximate boundary of the OV is indicated by dashed line. (B) BF as a function of depth for the two mapping penetrations shown in A. An ascending step-wise frequency progression is seen in the LV subdivision of the MGV (adapted from Cetas et al., 2001).

de Venecia et al., 1995). The cytoarchitecture of the LV is not homogeneous along the A-P axis: anterior and posterior regions also lack a laminar organization (Cetas et al., 2001). The anterior region may correspond with the dorsal cap described by Morest (1965), the most anterior part of the cat MGV, which also lacks a fibrodendritic architecture. The posterior region probably represents, in part, a transition between the laminated MGV and the non-laminated MGI that surrounds the MGV both dorsally and posteriorly in this species (Caballero-Bleda et al.1991; de Venecia et al., 1995). Vertical mapping penetrations through the LV revealed a close correspondence between the laminar organization and the tonotopic map. Results from two parallel electrode penetrations through the LV, shown in Fig. 2, demonstrate a low-high frequency gradient roughly perpendicular to the cell laminae. Within the central MGV, the range of best frequencies closely matched the most sensitive frequency region of the rabbits’ behavioral audiogram (approximately 1–16 kHz, Heffner and Masterton, 1980). Surprisingly, the frequency gradient within the LV was found to be discontinuous (Fig. 2B). This stepwise progression was quite consistent across animals and independent of the recording interval: similar best frequencies were observed for distances of roughly 300 ␮m at which point they would suddenly increase by almost one octave and remain fairly constant for another 300 ␮m. Along the dorsal–ventral axis, the shift in BF of clusters was at the rate of 2.7 octaves/mm (Cetas et al., 2001). In the OV, only clusters responding to low best frequencies, ranging from approximately 0.4 –2 kHz, were observed (Cetas et al., 2001). Penetrations through the anterior non-laminated MGV generally encountered large areas of neuronal clusters characterized by high best frequencies (⬎5 kHz) while those through the posterior region were tuned to low frequencies (ca. 0.4 – 6 kHz).

Dendritic architecture of MGV neurons In order to determine the response properties and dendritic morphology of single MGV neurons, physiologically-characterized units were labeled with the juxtacellular technique (Pinault, 1996). Single neurons were then reconstructed from serial sections with the aid of a computer microscope. The most common cell type labeled within the MGV was the bitufted variety, similar in morphology to those described in the auditory thalamus of many other species (Ramon-Moliner, 1962; Morest, 1964; Oliver, 1982; Winer, 1984; Winer et al., 1988, 1999; Clerici et al., 1990; Winer and Wenstrup, 1994; Bartlett and Smith, 1999). The bitufted profile arose from the repeated branching of four to five primary dendrites into numerous daughter branches a short distance from the cell body. The MGV neurons exhibited a small number of dendritic spines, with the majority located along the intermediate branches. MGV neurons are the bushy class of thalamic cells defined by Kolliker in 1896 (reviewed by Sherman and Guillery, 2001) which are common in the mammalian sensory thalamus (Ramón y Cajal, 1995). Unlike their counterparts in other sensory thalamic nuclei, the bitufted cells of the MGV have specialized, highly oriented dendritic arbors (Ramon-Moliner, 1962; Morest, 1964; Scheibel and Scheibel, 1966, 1970). A composite diagram of the location and dendritic morphology of 10 juxtacellularly-labeled MGV neurons is shown in Fig. 3. The predominant physiological response of MGV neurons (66%) was the onset type characterized by a transient burst of action potentials to the acoustic stimulus (Cetas et al., 2002). This response was often followed by a period of inhibition, the duration of which varied considerably from unit to unit. Spatial analyses of LV neurons The spatial properties of the dendrite systems were analyzed using three techniques: the dendritic prism, the den-

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Fig. 3. Transverse composite diagram of the MGV showing the location of 10 fully reconstructed neurons labeled with juxtacellular injections of biocytin. Gray-scale coded bands represent the frequency gradient (in octave steps in kHz) of the MGV determined from the electrophysiological study of Cetas et al. (2001). Thin lines within the MGV indicate orientation of cellular laminae revealed by Nissl stains. The tufted dendritic fields of many cells exhibit a dorsomedial to ventrolateral orientation, an axis that parallels the cellular laminae. Some cells (arrow) exhibit pronounced asymmetry of their dendritic fields. Inset shows the rabbit MGB in transverse section and the location of the LV and OV relative to other MGB subdivisions including the dorsal (D), internal (I), medial (M) and suprageniculate nucleus (SG). Diagonal lines in LV of the inset represent orientation of cell layers in Nissl-stained preparations. Scale bar⫽250 ␮m. ot, optic tract. (Adapted from Cetas et al., 2003.)

dritic stick analysis and the fan-in projection system. For the dendritic prism analysis, a rectangular prism was constructed that completely enclosed the dendritic system of the neuron. This technique is useful for examining the overall form and dimensions of a dendrite system in a context-independent manner (Colonnier, 1964; Valverde, 1978; McMullen et al., 1984). In order to compare neurons with different axes of orientation, each neuron was first rotated in the transverse plane to align the maximal extent of the dendritic field with the horizontal plane (Blackstad et al., 1993; Malmierca et al., 1993). Results of the dendritic prism analysis, shown in Table 1, revealed that MGV neurons were quite large. The average dendritic field of an MGV neuron was encompassed by a rectangular prism whose dimensions were approximately 430 (L) by 310 (W) by 440 (D) ␮m. For LV neurons, the longest dimension of the dendritic prisms was length, the axis which corre-

sponds to the transverse plane in our coordinate system. In contrast, OV neurons and neurons from non-laminated (NL) regions had their longest axes (corresponding to dendritic field depth) parallel to the A-P axis of the brain (Table 1). Table 1. Dendritic prism analysis of MGV neurons* Subdivision

LV (n⫽13) OV (n⫽10) NL (n⫽7) Mean (n⫽30) a

Dimensions of dendritic field (␮m) Length

Width

Depth

436.4⫾15.2a 418.6⫾17.6 432.4⫾30.1 429.5⫾10.9

272.5⫾10.0b 326.8⫾18.4 353.2⫾21.9 309.4⫾9.8

406.6⫾21.5 470.0⫾44.0 449.2⫾21.7 437.7⫾18.2

Standard error of the mean. Significantly different from the OV (P⫽0.005) and NL (P⫽0.004). * From Cetas et al., 2003.

b

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These important differences in dendritic orientation were seen in single neurons as well as in population spatial analyses. The mean dendritic field width of MGV neurons (measured on an axis perpendicular to the cell laminae) was 309 ␮m (Table 1). This value was remarkably close to the frequency step size found in our physiological mapping studies (ca. 300 ␮m; Cetas et al., 2001). Results of the dendritic stick analysis for both a single cell as well as a population of MGV neurons are shown in Fig. 4. The dendritic stick analysis (Colonnier, 1964; Glaser et al., 1979; McMullen et al., 1984) disassembles each dendrite into somatofugally directed chords or sticks. Data derived from the stick analyses were displayed in the form of polar histograms that show the total length of dendritic sticks in 3.6° bin widths covering 360° in a specified plane. Because data from a single cell or a population of neurons can be displayed, polar histograms represent a powerful method for analyzing dendritic orientation. The polar histograms were tested for orientation by means of a combined Fourier and statistical analysis (Glaser et al., 1979; McMullen et al., 1984). A computer microscopic reconstruction of a representative LV neuron is shown in Fig. 4. Physiologically, the neuron had an onset response to acoustic stimulation with a BF of 9.1 kHz (Cetas et al., 2002). The neuron exhibited the classic profile of MGV neurons in the central laminated core: a bitufted, highly oriented dendritic field closely aligned with the cell laminae. When viewed in the sagittal plane (parallel to the axis of the cell laminae), the dendritic field was also strongly oriented in the A-P direction (Fig. 4, top right). Polar histograms confirmed the strong orientation of the dendritic field in both the transverse and sagittal planes (Fig. 4, middle). Population polar histograms for LV neurons (n⫽13) in both the transverse and sagittal planes are shown in the bottom of Fig. 4. The ellipsoidal envelope of the histogram in each plane indicates a high degree of orientation by LV neurons. A Fourier analysis of dendritic sticks in the transverse plane revealed a significant orientation (␹2⫽91.0, df⫽2, P⬍0.001) whose major axis was at 29o (relative to the horizontal plane). This orientation is within 4o of the cellular laminae measured in Nissl preparations (25o). These data confirmed highly directed dendritic growth by LV neurons parallel to the cellular laminae. For the polar histograms of LV neurons in the sagittal plane, neurons were rotated to examine orientation from the vantage point of the laminar axis. In this modified sagittal plane, LV neurons also exhibited a strong preferred orientation. Fourier analysis revealed a significant orientation at 9o relative to the A-P axis (␹2⫽85.1, df⫽2, P⬍0.001). There are two problems inherent in the use of polar histograms for determining orientation of neuronal dendrite systems. First, complex dendritic trees are disassembled into sticks whose spatial angles and lengths are preserved but not their positional relationship with the whole dendritic tree. The second problem is the distorting effects of depth foreshortening when three-dimensional data are projected onto a two-dimensional plane (Glaser et al., 1979; Glaser and McMullen, 1984). The fan-in projection method for

analyzing dendrite systems eliminates both of these problems (Glaser and McMullen, 1984). In the present study, the tonotopic axis, a vector perpendicular to the orientation of the MGV cellular laminae as revealed by Nissl stains, was chosen as the polar or fan-in axis. This permitted a graphical analysis of dendrite orientation relative to the tonotopic axis and cellular laminae (Cetas et al., 2001). Results of the fan-in analysis for a single LV neuron as well as a sample of LV neurons (n⫽13) are shown in Fig. 5. A reconstructed LV neuron in the transverse plane is shown in Fig. 5A. The cell was an onset type physiologically, had a BF of 1.0 kHz and responded maximally to binaural stimulation (EE). A fan-in diagram of this same neuron (Fig. 5B) revealed preferential growth of dendrites relative to the cell laminae. A population fan-in histogram of LV neurons (n⫽13) illustrating mean dendritic length in 3.6° increments is shown in Fig. 5C. This analysis revealed the highly directed growth of LV dendrites parallel to the cellular laminae (Fig. 5) with the majority of dendritic growth (ca. 60%) within 30° of the laminar axis (Cetas et al., 2003). Although a bitufted dendritic field was the norm, it was not uncommon for LV neurons to have asymmetric dendritic domains relative to the cell laminae. The high proportion of such neurons was readily apparent in Golgi preparations of the MGV. Two examples of LV neurons exhibiting pronounced asymmetry in their dendritic fields are shown in Fig. 6. Both neurons exhibited onset responses to acoustic stimuli and responded best to stimulation of the contralateral ear (EI binaural class). Neuron A (BF⫽8.0 kHz) had a preferred growth of dendrites dorsally with limited growth in the ventral direction. This dendritic field bias was confirmed by the polar histogram of dendritic sticks (Fig. 6). Neuron B (BF⫽7.1 kHz) in Fig. 6 showed the opposite pattern: although the major axis of the dendritic tree was parallel to the cellular laminae, the majority of dendritic growth was directed ventrally. Because approximately 30% of labeled MGV neurons in the LV and OV exhibited asymmetric dendritic fields, such neurons are incorporated into our current model of MGV organization. Laminar architecture of the rabbit ICC A laminar architecture was visible in both Nissl and Golgistained preparations of the ICC when the inferior colliculus was sectioned along its major axis in the transverse plane (Fig. 7). In Nissl preparations, laminar orientation was nearly vertical in the ventral ICC. This laminar organization was complemented by the dendritic domains of Golgistained principal neurons (Fig. 7B). Similar to the results of Aitkin et al. (1972), microelectrode mapping studies revealed a low-high frequency gradient orthogonal to the laminae. In most cases, BDA injections at physiologicallylocalized sites resulted in a slab-like pattern of BDA-labeled axons in the ICC (cf. Malmierca et al., 1995). An example of a BDA injection made at a 2.9 kHz BF site in the ICC is shown in Fig. 7C. The axonal labeling was closely aligned with the Nissl and dendritic architecture

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and extended throughout the dorsal–ventral extent of the ICC (Fig. 7). Tectothalamic bands in the MGV BDA injections at physiologically-characterized sites in the ICC resulted in robust labeling of tectothalamic arbors in the MGV that terminated in the form of highly-oriented axonal domains. The orientation of these terminal arbors corresponded quite closely to that of the Nissl laminae and the dendritic arbors of thalamic neurons (Fig. 8). Furthermore, the projection of tectothalamic axons to the MGV was tonotopic: higher frequency sites in the ICC projected to successively more ventral regions of the LV (Figs. 8, 9); low frequency sites (⬍1 kHz) in the dorsal ICC projected to the OV (Fig. 10). Tectothalamic arbors to OV conformed to the circular cyto- and dendritic architecture of this MGV subdivision (Fig. 10). Serial section reconstructions revealed that tectothalamic axonal bands formed slabs in the A-P axis. In order to quantify the relationship between the tectothalamic bands and the laminar architecture of the

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MGV, the orientation and dimensions of the axonal bands were measured in four animals in which BDA injections were placed at ICC sites ranging in BF from 1.2–9.6 kHz. An example of this angular measurement is illustrated in Fig. 11 where a band of tectothalamic axons resulting from a BDA injection at an 8.5 kHz site in the ICC is shown. Results of this quantitative analysis are shown in Table 2. The orientation of tectothalamic arbors in the four animals ranged from 24o to 32o relative to the horizontal plane. Mean overall orientation was 28.6o, a value quite close to the orientation of dendritic arbors of MGV neurons (29o, Fig. 4). The mean length and width of tectothalamic arbors within the MGV were 970 and 286 ␮m, respectively (Table 2). The elongated arbors of tectothalamic axons were more than twice the dendritic field length of MGV neurons (ca. 430 ␮m, Table 1). In contrast, the width of the tectothalamic bands (286 ␮m, Table 2) closely matched the dendritic field width of MGV neurons (309 ␮m, Table 1) and the distance between frequency steps observed in multiunit mapping studies (300 ␮m, Cetas et al., 2001).

Fig. 5. Fan-in analysis of LV neurons in the MGV. (A) Fully reconstructed LV neuron in the transverse plane. Dotted lines represent cellular laminae and frequency axes. The cell exhibited an onset-type response to acoustic stimulation, had a BF of 1.0 kHz and responded maximally to binaural stimulation (EE). (B) Fan-in diagram of neuron shown in A around frequency axis. Note preferential growth of dendrites relative to the cell laminae. Scale bar⫽50 ␮m for A and B. (C) Population fan-in histogram of LV neurons (n⫽13). Each histogram bar represents mean dendritic length in 3.6° increments. The majority of dendritic growth (ca. 60%) is within 30° of the laminar axis. (Adapted from Cetas et al., 2003.)

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Tectothalamic terminals and synaptic nests in the MGV Two types of tectothalamic axons were labeled by the BDA injections (Fig. 12). The majority of axons terminated in the form of large boutons approximately 2 ␮m in diameter (Fig. 12). The boutons were frequently clustered and formed ring-like structures similar to the synaptic nests described by Morest (1975, 1997) and Majorossy and Kiss (1976). The synaptic nests were distributed along the long axis of the tectothalamic slabs. A second, finer type of tectothalamic axon with smaller terminal boutons (ca. ⬍1 ␮m dia.) was also present within the MGV (Fig. 12).

ICC. In the transverse plane, each slab would span multiple cellular laminae and its width in the transverse plane (ca. 300 ␮m) corresponds to the dendritic field of thalamic relay neurons and tectothalamic axons from the ICC. The dorsal–ventral arrangement of these frequency slabs forms the basis of the tonotopic map in the central laminated MGV. In contrast, tectothalamic axons arising from low frequency sites in the ICC project to the OV and conform to its spiral organization. This model is consistent with the dendritic and afferent architecture of the MGV (Andersen et al., 1980; Malmierca et al., 1997; Cetas et al., 2003) and with the frequency steps reported in physiological studies (Cetas et al., 2001).

Model of frequency organization of the MGV Our model of auditory thalamic organization incorporating cellular laminae, the dendritic arbors of relay neurons and tectothalamic axons is shown in Fig. 13. In this model, a functional unit in the MGV corresponds to a frequency slab formed by the complementary organization of principal cell dendritic trees and tectothalamic axons originating in the

DISCUSSION History of MGV laminar organization Our model of auditory thalamic organization is an extension of the one proposed by Morest (1965). In his early description of a laminar organization in the MGV, Morest

Fig. 6. Examples of MGV neurons with asymmetric dendritic fields. Computer microscope reconstructions (left) and corresponding dendritic stick polar histograms (middle) of two neurons from the LV region of the MGV. The BF, binaural class and response category of each neuron are shown on the right. Scale bar⫽50 ␮m. (Adapted from Cetas et al., 2003.)

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Fig. 4. Dendritic orientation of LV neurons in the MGV. Center inset: Location of neuron in the MGV. Arrow indicates perspective of sagittal plane shown on top right. Computer microscope reconstruction (top) and corresponding dendritic stick polar histogram (middle) of an LV neuron in the transverse (left) and sagittal (right) plane. The cell exhibited an onset-type response to acoustic stimulation, had a BF of 9.1 kHz and responded maximally to monaural stimulation of the contralateral ear (EI). Note the highly oriented dendritic field in both the transverse and sagittal planes. Scale bar⫽50 ␮m. Bottom. Population polar histograms of dendritic orientation in the transverse and sagittal plane for LV neurons (n⫽13). Each histogram bar represents the mean dendritic length in 3.6° increments for a total of 100 bins in each plane. Dotted line at 25° in the transverse plane represents orientation of cellular laminae revealed by Nissl stains. In the transverse plane, LV neurons have highly oriented dendritic fields whose major axis (ca. 29°) closely corresponds to the cell laminae. In the sagittal plane, when viewed parallel to the laminar axis, the population polar histogram reveals significant orientation in the A-P axis. (Adapted from Cetas et al., 2003.)

relied on Golgi impregnations in the cat to define an anteroventral pars lateralis and a coiled OV located ventro-

medially. The complementary organization of afferent axons and bitufted dendritic trees of thalamic neurons was

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Fig. 7. Laminar labeling resulting from BDA injection in the ICC (IC-18). Triptych illustrating Nissl (A), Golgi (B) and BDA-labeling at physiologicallycharacterized injection site (BF⫽2.9 kHz) in the ICC (C). Arrow in A indicates marker lesion placed during mapping experiment. BDA injection in the ICC (arrow in C) labels local axons whose arborizations parallel the fibrodendritic architecture. Scale bar⫽500 ␮m.

the basis of the fibrodendritic architecture in both structures (Morest, 1965). Morest correctly predicted that this spatial relationship and laminar architecture was related to the acoustic frequency map. Although few mammalian species have visible laminae in the MGV, some of the earliest studies of the auditory thalamus described a laminar organization in this structure. In his study of the MGB (which included the rabbit), Ramón y Cajal (1995) included a description of the ventral division where “cells . . . form dense linear groups recognizable in Nissl preparations.” In addition, Poljak (1926) reported that “the medial geniculate body . . . shows well-marked cell stratification.” It is worth noting that cell laminae are distinctly lacking in the cat (Morest, 1965), a species heavily used in physiological and anatomical studies of the auditory thalamus (Morest, 1964, 1965; Aitkin and Prain, 1974; Calford, 1983; Calford and Webster, 1981; Winer, 1985; Morel et al., 1987; Imig and Morel, 1985). The rabbit’s contribution to this model derives from its visible cellular laminae which are clearly

related to the frequency map, the dendritic arborization of thalamic relay neurons and the termination of afferent axons arising from the ICC. Frequency slabs in the LV Our model proposes narrow-band, slab-like domains composed of multiple laminae within the LV (for frequencies above 1.0 kHz). There is both anatomical and physiological support for this organization. Quantitative estimates of dendritic field width (and hence slab width) for LV neurons (ca. 300 ␮m) correspond closely with frequency steps (0.8 octaves/300 ␮m) reported in physiological mapping studies (Cetas et al., 2001, 2003) and with the width of tectothalamic axonal bands arising from the ICC. The number of slabs encountered in vertical electrode penetrations (ca. three to five) cannot account for the behavioral hearing range in this species and it is likely that a frequency gradient exists parallel to the laminae as well as in the A-P

Fig. 8. Correspondence between tectothalamic axons and laminar architecture of the MGV. Triptych illustrating dendritic organization (A), Nissl architecture (B) and tectothalamic axons in the MGV (C) originating from BDA injection in ICC (BF⫽2.9 kHz) shown in Fig. 7. Tectothalamic axons parallel the cellular and dendritic architecture of MGV. Scale bar⫽200 ␮m.

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Fig. 9. Band of tectothalamic axons in MGV resulting from BDA injection at 8.5 kHz site in the far posterior ICC (IC-11). Tectothalamic axons parallel Nissl laminae as well as dendritic fields of thalamocortical relay neurons in the MGV. Dotted line defines ventral MGV border. Inset, lower left. Nissl-stained transverse section through the center of BDA injection site in posterior ICC (arrow). Note that BDA uptake is confined to narrow band within the ICC. Scale bar⫽100 ␮m.

plane. A similar slab-like organization has been described in the ICC where the laminae have been proposed as the anatomical substrate of critical bands (Schreiner and Langner, 1997; Braun 1999, 2000). Further, Braun (1999, 2000) has hypothesized that the slab-like structure of the ICC is optimal for fundamental frequency extraction, an important component for higher mammalian communication. While the functional architecture of the MGV is mir-

rored by the laminar organization of cell bodies, there is no simple relationship between cellular laminae and isofrequency contours: a single slab comprises multiple laminae. Indeed, there is no a priori reason for cellular laminae in our model: frequency slabs can arise from the spatiallyrestricted and complementary growth of thalamic neuron dendritic fields and tectothalamic arbors (much as originally proposed by Morest, 1965). The existence of cellular

Fig. 10. Low frequency sites in the ICC project to the OV region of the MGV. Serial section reconstruction of BDA-labeled boutons in MGV following BDA injection at low frequency site in ICC. The distribution of tectothalamic boutons conforms to the spiral organization of the OV. Inset: Photomicrograph of 0.85 kHz injection site in dorsal ICC. Scale bar⫽250 ␮m.

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Fig. 11. Transverse section of the MGV showing band of tectothalamic axons resulting from a BDA injection at an 8.5 kHz site in the ICC. The axonal band is oriented 35o relative to the horizontal plane. Scale bar⫽500 ␮m.

laminae suggests a finer level of organization, as yet unidentified, in the auditory thalamus (cf. Oliver et al., 1997). The spiral organization of the OV While the structure and organization of the LV is generally agreed upon, the OV is less understood. Golgi preparations of the OV in the cat reveal coiled, spiraling laminae formed from the curved dendritic trees of tufted neurons and afferent axons (Morest, 1965, 1975). Similarly in the rabbit, tufted neurons in the OV have dendritic fields that follow the general spiraling pattern visible in Nissl and Golgi stains (Cetas et al., 2002). Quantitative studies have documented significant differences in OV dendritic fields relative to those in the LV: OV neurons are oriented primarily in the A-P plane, a direction orthogonal to that of LV neurons (Cetas et al., 2003). In his study of the cat MGV, Morest (1965) concluded that the coiled laminae of the OV were continuous with those of the LV, an organization

supported by the anterograde studies of Andersen et al. (1980) and with the anterograde results in the present report. The OV of the rabbit appears to be a low-frequency region of the auditory thalamus. It is located in the dorsal part of the MGV where mapping studies have only found best frequencies of 1.0 kHz and below (Cetas et al., 2001). In addition, injections of anterograde tracers at low frequency sites in the ICC labeled axonal projections to the OV whose terminal arbors complemented the spiral organization of this structure. The OV may represent an expanded low frequency domain in this species. This organization differs from that of the cat where the OV is reported to have a concentric high-low frequency gradient (Calford and Webster, 1981; Imig and Morel, 1983, 1985; Morel et al., 1987). Interestingly, Imig and Morel (1988) describe the core of the OV as being a low frequency area, which is similar to that of the rabbit. Although a possible counterpart to the OV may exist in the dorsolateral ICC

Table 2. Quantitative analysis of tectothalamic axons Exp #

IC injection frequency (kHz)

Axonal band angle (degrees)

Axonal band length (␮m)

Axonal band width (␮m)

IC-9 (n⫽6)a IC-18 (n⫽7) IC-11 (n⫽19) IC-14 (n⫽10) Mean (n⫽42)

1.2 2.8 8.5 9.6

24.4⫾1.4b 30.4⫾2.1 31.9⫾0.7 23.8⫾1.1 28.6⫾0.8

933.0⫾59.4 660.1⫾19.8 919.6⫾37.1 1304.4⫾33.2 969.9⫾38.4

295.5⫾16.9 228.7⫾28.8 248.0⫾17.2 392.0⫾19.1 285.9⫾14.1

a b

Number of sections. Standard error of the mean.

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Fig. 12. Photomicrographs of two types of ICC axons that terminate in the MGV. (A) Large boutons terminate in clusters forming synaptic nests (arrows) within the tectothalamic bands. (B) Small boutons arising from fine axons also terminate within the bands. Scale bar⫽20 ␮m.

(Rockel and Jones, 1973; see also Faye-Lund and Osen, 1985), more recent studies do not support such an organization (Brown et al., 1997). Clearly, more work is needed to define the functional organization of this MGV subdivision. ICC projections to the MGV Morest’s (1965) description of the fibrodendritic architecture of the MGV derived from the complementary pattern of principal cell dendritic fields and afferent axons believed to originate in the ICC. Earlier anterograde degeneration studies, while establishing the topographic projection from the ICC to the MGV, lacked the resolution to address this relationship (Moore and Goldberg, 1963; Tarlov and Moore, 1966). The first anterograde studies of ICC projections with modern techniques were carried out by Andersen et al. (1980) and Kudo and Niimi (1980) using tritiated leucine in the cat. ICC projections to the MGV were tonotopically organized and terminated in the form of bands. The terminal bands extended dorsoventrally and rostrocaudally and their orientation paralleled the fibrodendritic laminae described by Morest (1965). Interestingly, there were discontinuities in the terminal bands creating patches 300 –500 ␮m in diameter (Andersen et al., 1980), a feature shared by thalamocortical axons to primary auditory cortex (AI; McMullen and de Venecia, 1993; Romanski and LeDoux, 1993; de Venecia et al., 1998; Cetas et al., 1999). More recent studies of tectothalamic axons using horseradish peroxidase bulk-filling (Pallas and Sur, 1994; Pallas et al., 1994; Wenstrup et al., 1994) or iontophoretic injections of biocytin (Malmierca et al., 1997) have also reported oriented terminal arbors that paralleled the fibrodendritic laminae. Interestingly, axonal terminal arbors in these different species were consistent and quite narrow with a width of only 75–100 ␮m (Pallas and Sur, 1994; Wenstrup et al., 1994; Malmierca et al., 1997). In the present study, tectothalamic bands were closely aligned with the dendritic fields of MGV neurons and measured approximately 300 ␮m in width, a value which is quite close to MGV dendritic field width and frequency steps derived in physiological studies. It is not clear whether

methodological or species factors underlie these differences. Reconstruction of single tectothalamic axons may shed light on this question. Corticothalamic projections also form slab-like arrays in the MGV (Hazama et al., 2004) suggesting the possibility of synaptic domains similar to that of the ICC (Oliver and Schneiderman, 1991; Oliver and Huerta, 1992). Evidence for such domains includes the diverse response properties of identified tufted neurons in vivo (Cetas et al., 2002) and in vitro (Bartlett and Smith, 1999), and the excitatory and inhibitory inputs to MGV originating in the ICC (Winer et al., 1996; Peruzzi et al., 1997; Bartlett and Smith, 1999; Bartlett et al., 2000). One of the earliest anterograde studies of ICC projections reported two types of tectothalamic fibers in the MGV (Andersen et al., 1980). More recently, the auditory tectothalamic system has been shown to consist of excitatory (glutamatergic) and inhibitory (GABA-ergic) projections (Winer et al., 1996; Peruzzi et al., 1997). This unusual dual system is in contrast to the strictly excitatory projections to thalamic nuclei of other sensory modalities (Bartlett et al., 2000; for review, Sherman and Guillery, 2001). In the present study, two types of tectothalamic axons were labeled by the BDA injections. The majority of axons terminated in the form of large boutons (ca. 2 ␮m dia.) that were oriented parallel to the tectothalamic bands. The large boutons were frequently clustered and formed ring-like structures similar to the synaptic nests described by others (Majorossy and Réthelyi, 1968; Morest, 1975, 1997). A second, finer type of tectothalamic axon with smaller terminal boutons (ca. ⬍1 ␮m dia.) was also present within the MGV. In terms of size, these two types of boutons correspond to GABA-negative and GABA-positive ICC terminals in the MGV of the rat (Bartlett et al., 2000). The convergence of excitatory and inhibitory ICC axons onto MGV neurons has been demonstrated electrophysiologically (Peruzzi et al., 1997) and at the ultrastructural level (Bartlett et al., 2000). These ascending circuits most likely mediate frequency tuning in MGV neurons (Suga et al., 1997) as well as the significant transformation

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MGV

OV LV

Te ct ot ha la m ic A xo

ns

Lo

Hi

D

Lo Mid Hi (OV)

M

L

~300 um ~0.8 octaves

V Fig. 13. Current model of MGV organization based on cellular lamination, frequency organization and tectothalamic axons. Inset (lower right) illustrates how cellular laminae (open circles) and MGV neurons with symmetric and asymmetric dendritic fields (closed circles and lines) form a slab-like region approximately 300 ␮m in width (spanning approximately 0.8 octaves). Narrow-band frequency slabs in the LV are stacked to form a step-wise, frequency map in the MGV. Tectothalamic axons form afferent sheets that parallel the laminar organization. In contrast, tectothalamic axons projecting to the OV conform to its spiral organization. Alternating dendritic and axonal bands are illustrated. Synaptic nests formed by large boutons indicate high degree of convergence onto MGV dendrites. For simplicity, small boutons are not shown.

in response properties of thalamic neurons relative to those of the ICC (Wenstrup, 2005). Comparative architecture of the ICC and MGV The ICC is the major source of ascending afferent input to the MGV (Oliver and Huerta, 1992). It, too, has a highly visible laminar architecture closely related to the tonotopic

map (Aitkin et al., 1972). Similar to the MGV, the major cell type is the disc-shaped cell whose dendritic orientation corresponds to the laminar, as well as tonotopic architecture (Oliver and Morest, 1984; Malmierca et al., 1993). Ascending projections to the ICC from a variety of brainstem nuclei terminate in the form of bands that are directly related to the tonotopic and binaural maps (Merzenich and

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Reid, 1974; Oliver, 1987; Shneiderman and Henkel, 1987; Oliver and Huerta, 1992; Loftus et al., 2004). Oliver and Huerta (1992) have hypothesized that these banded inputs may create synaptic domains that could account for different functional classes in the ICC. Despite their close synaptic relationship and similar tonotopic representation, the MGV and ICC possess quite different functional architectures. The frequency progression in the ICC of the cat is also discontinuous and step-wise (Schreiner and Langner, 1997), but the step size between frequency plateaus (ca. 0.3 octaves) is much smaller relative to that of the rabbit MGV (0.8 octaves). The distance over which a given BF dominates in the ICC ranges from 100 to 180 ␮m (Merzenich and Reid, 1974; Schreiner and Langner, 1997). While this may reflect species differences, a comparison of ICC and MGV neurons suggests that step size is closely correlated with dendritic field width of principal neurons (70 ␮m vs. 300 ␮m, respectively) in the two nuclei (Oliver and Shneiderman, 1991; Oliver and Huerta, 1992; Malmierca et al., 1993; Cetas et al., 2001) and with the dimensions of afferent terminal fields (Loftus et al., 2004). These data suggest a major transformation in computational circuits from the ICC to the MGV which may account for the significant differences in response properties between these auditory nuclei (Rees et al., 1997; Wenstrup, 2005). MGV projections to AI Our model can be useful for understanding the derivation of frequency and binaural maps in AI. Recently, we demonstrated that physiologically characterized sites in the MGV diverge to multiple axonal patches in AI coding for similar frequency and binaurality (Velenovsky et al., 2003). In this model’s extension to thalamocortical pathways, MGV axons originating from binaural-specific sites within frequency slabs terminate in patches along a frequency strip in AI. Adjacent MGV neurons, within the same frequency slab but coding for a different binaural class, project to separate fields within the same frequency strip. Our model of auditory thalamic organization may explain how continuous (frequency) and discontinuous (e.g. binaurality) maps in AI derive from patchy thalamocortical circuits. For most thalamic neurons, the relationship between the shape of the dendritic arbor and its function remains unresolved (Sherman and Guillery, 2001). In the auditory thalamus, however, the dendritic fields of relay neurons in the LV are closely related to the frequency map and to a laminar architecture visible with routine Nissl stains. To our knowledge, this is the highest level in the ascending auditory system in mammals where a structural–functional relationship has been defined. As such, we believe the rabbit auditory thalamus may be a useful model for defining the structural basis of functional maps, and for addressing neuroplasticity mechanisms during development (McMullen and Glaser, 1988; McMullen et al., 1988a,b) and aging of the central auditory system. Acknowledgments—The authors wish to thank Dr. Naomi Rance and Sally Krajewski for useful comments on this manuscript. This

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work was supported by grants from the NIH/NIDCD: DC02410 (N.T.M.), DC05108 (D.S.V.) and DC006825 (D.S.V.) as well as awards from the Deafness Research Foundation and the American Academy of Audiology (D.S.V.). M. Holmes was supported by the Undergraduate Biology Research Program at the University of Arizona and the Howard Hughes Medical Institute.

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(Accepted 17 April 2005)