Quantitative comparisons of corticothalamic topography within the ventrobasal complex and the posterior nucleus of the rodent thalamus

Brain Research 968 (2003) 54–68 www.elsevier.com / locate / brainres

Research report

Quantitative comparisons of corticothalamic topography within the ventrobasal complex and the posterior nucleus of the rodent thalamus Kevin D. Alloway a , *, Zachary S. Hoffer a , John E. Hoover b a

Department of Neuroscience and Anatomy, H109, Hershey Medical Center, 500 University Drive, Hershey, PA 17033 -2255, USA b Department of Biology, Millersville University, Millersville, PA 17551 -0302, USA Accepted 18 December 2002

Abstract To compare the topographic precision of corticothalamic projections to the ventrobasal (VB) complex and the medial part of the posterior (POm) complex, different anterograde tracers were placed in neighboring parts of the primary (SI) and secondary (SII) somatosensory cortical areas. The location of labeled corticothalamic terminals and their beaded varicosities were plotted, and the digital reconstructions were analyzed quantitatively to determine the extent of overlapping projections from the cortical injection sites. Among animals that received all tracer injections in SI cortex, tracer overlap in the thalamus varied according to the proximity of the cortical injection sites. Regardless of which combination of somatic representations were injected in SI, within each animal the amount of tracer overlap in POm was similar to that observed in VB, and a matched-sample statistical analysis failed to reveal significant differences in the proportion of the labeled regions that contained overlapping projections from the injected cortical sites. Among those animals in which the tracers were injected into the whisker representations of SI and SII, the amount of tracer overlap in the thalamus was not affected by the proximity of the cortical injection sites. Instead, tracer overlap appeared to be related to the degree of somatotopic correspondence. Furthermore, within each of these animals, the amount of tracer overlap in POm was similar to that found in the VB complex. These results indicate that POm has a well-defined topographic organization that is comparable to the degree of topography observed in the VB complex.  2002 Elsevier Science B.V. All rights reserved. Theme: Sensory systems Topic: Somatosensory cortex / thalamocortical relationships Keywords: Anterograde tracing; Convergence; Corticothalamic projection; Somatotopic organization

1. Introduction Cutaneous processing in the rodent thalamus is mediated largely by two regions, the ventrobasal (VB) complex and the medial part of the posterior complex (POm). Abundant anatomical evidence indicates that both of these thalamic regions receive ascending afferent inputs from the brainstem [8,16,35], and that they both have reciprocal connections with the primary (SI) and secondary (SII) somatosensory cortical areas [1,2,6,10,14,19,28,31,33,34,36]. Consistent with these anatomical findings, neurons in both

*Corresponding author. Tel.: 11-717-531-6413; fax: 11-717-5315184. E-mail address: [email protected] (K.D. Alloway).

VB and POm exhibit short latency responses to cutaneous stimulation [12,13,32]. The fact that two thalamic regions are involved in processing somatosensory information raises many questions about how these structures differ and whether they play complementary roles in sensory discrimination. Comparisons of the neuronal connections and response properties of VB and POm have revealed distinctions between these nuclei that probably reflect important functional differences. In response to whisker stimulation, for example, neurons in the ventroposteromedial (VPM) subdivision of VB are highly responsive to stimulation of a single whisker, but whisker-sensitive neurons in POm are not as responsive unless multiple whiskers are stimulated simultaneously [12]. Furthermore, a comparison of neuronal responses to different stimuli indicates that neurons in

0006-8993 / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. doi:10.1016 / S0006-8993(02)04265-8

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POm are more sensitive to stimulation frequency than neurons in VPM, and this suggests that POm is more important for encoding temporal information [32]. Finally, thalamocortical projections from the barreloid region of VPM terminate precisely in layer IV of the corresponding SI barrels [23,27], whereas projections from POm terminate more broadly in all layers of the septal zones surrounding the SI barrels [10,22,27]. These differences in neuronal connections and response properties suggest that spatial information is probably encoded with more precision in VB than in POm. But the few studies that have directly compared the somatotopic organization within VB and POm have produced contradictory findings. Thus, while some tracing experiments indicate that POm has a well-defined somatotopic organization that matches the level of organization observed in VB [14], other tracing experiments suggest that POm lacks the topographic organization that is readily apparent in VB [38]. In view of these divergent findings, we used a dual or triple anterograde tracing paradigm to quantify the amount of overlapping labeled projections to POm and VB from different somatosensory cortical areas. This experimental design allowed us to determine within each injected animal whether the amount of tracer overlap in POm was equivalent to that found in the VB complex. If variations in tracer overlap indicate the relative precision of topographic organization in different nuclei, then injecting a pair of different tracers into neighboring, non-overlapping SI and SII cortical representations should produce low amounts of labeled overlap in those thalamic nuclei that have a precise topographic organization. By comparison, a thalamic region that does not have a precise topographic organization should display substantially greater amounts of tracer overlap in response to the same pair of tracer injections.

2. Materials and methods All procedures were approved by the institutional animal welfare committee and followed NIH guidelines for the care and use of laboratory animals. Data were obtained from 57 male Sprague–Dawley rats (350–650 g) that had received dual or triple tracer injections in somatosensory cortex to characterize the relative topography of corticostriatal, corticopontine, and corticocortical projections. Most of the results concerning these projection systems have already been reported in papers or as an abstract [1,2,18,20,25].

2.1. Surgery and electrophysiological mapping Surgical procedures have been reported previously [1,2]. Briefly, each rat was anesthetized with an intramuscular injection of ketamine (20 mg / kg) and xylazine (6 mg / kg), and then it was intubated through the oral cavity. Each

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animal received atropine sulfate (0.05 mg / kg, i.m.) to reduce bronchial secretions, chloromycetin sodium succinate (50 mg / kg, i.m.) to prevent infection, and dexamethasone (0.5 mg / kg, i.m.) to reduce inflammation. Subsequently, each rat was placed in a stereotaxic frame and artificially ventilated with a 2:1 mixture of nitrous oxide and oxygen containing either halothane or isoflurane (0.5–1.0%). Ophthalmic ointment was applied to prevent corneal drying. Heart rate, end-tidal CO 2 , and body temperature were monitored continuously. After exposing the skull, craniotomies were performed over the right hemisphere. Holes were drilled at stereotaxic coordinates overlying the whisker, forelimb, or hindlimb representations of SI or over the whisker representation of SII as indicated by published maps [7,15]. A tungsten microelectrode (300–700 kV) was inserted through each cranial opening and lowered 800–1000 mm below the pial surface. Extracellular recordings of multiunit activity were amplified, displayed on an oscilloscope, and monitored over an acoustic speaker. Receptive fields (RFs) were determined by stimulating the whiskers or hairy skin with a slender rod, and then plotting the regions of stimulusevoked activity on figurine drawings of the vibrissae and limbs. The coordinates of the cortical recording sites were recorded with respect to bregma and were also noted on a hand-drawn map of the cortical vascular pattern that was visualized by means of an operating microscope. These records were subsequently used to guide tracer injections into specific body part representations.

2.2. Tracer injections Different anterograde tracers were injected into different body part representations of somatosensory cortex. We used 10–15% solutions of Fluoro-Ruby (FR), Alexa Fluor (AF), or biotinylated dextran amine (BDA) (Molecular Probes, Eugene, OR). The fluorescent tracers, FR or AF, were pressure injected through glass micropipettes (tip diameters of 75– 100 mm) cemented to 1.0 ml Hamilton syringes. In each case, the pipette was lowered into the cortex at an angle perpendicular to the cortical surface. Injections were typically placed at depths of 1400, 1200, and 1000 mm below the pia. At each depth, 25–50 nl of tracer was injected over the course of 3 min; an additional 3–5 min elapsed before the pipette was raised to the next depth. The volume and rate of tracer delivery was controlled with the aid of a Kopf (model 5000) microinjection unit. In some cases, the pipette was also reinserted into another local cortical site (within 50–200 mm of the first injection) for a second set of injections in the same somatotopic representation. Biotinylated dextran amine was injected iontophoretically through glass micropipettes (tip diameters545–75 mm) using positive current pulses of 3–6 mA and a duty cycle of 7 s. Iontophoresis was applied for 9–18 min at two or

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more cortical depths (1400, 1200, 1000 mm). In some instances, this tracer was injected at one or two neighboring cortical sites (separated by 100–300 mm) to insure that many corticofugal neurons at the injection site were labeled. After completing the injections, the wound margins were sutured and treated with a topical antibiotic, and the rat was returned to its home cage.

2.3. Histology and microscopy After a survival period of 7–14 days, each rat was deeply anesthetized with sodium pentobarbital (50 mg / kg, i.p.). Fiduciary marks at known stereotaxic coordinates were placed in the brain using a tungsten wire coated with India ink. The animal was then transcardially perfused using a three-step procedure. The perfusates included: heparinized physiological (0.9%) saline, 4% paraformaldehyde in 0.1 M phosphate buffer (PB; 4 8C, pH 7.4), and 4% paraformaldehyde with 10% sucrose. Following the perfusion, the brain was removed and post-fixed overnight in cold 4% paraformaldehyde with 30% sucrose. In most cases, the cortex was dissected from the injected hemisphere, flattened between glass slides, and post-fixed for an additional 24 h. Tangential sections through the cortex were cut at 25–75 mm intervals; the subcortical hemisphere was cut in the coronal plane at 50 mm intervals. Cortical (tangential) and subcortical (coronal) sections were saved in serial order and processed as three separate series. For both cortical and subcortical sections, a one-inthree series was processed for BDA and a second one-inthree series was left unprocessed for fluorescent microscopy. The remaining one-in-three cortical series was processed for CO to reveal the SI barrels. The remaining one-in-three subcortical series was stained with thionin to visualize the borders of thalamic nuclei and other brain structures. Anatomic borders were determined using a published atlas [30]. Sections were processed for BDA labeling as described previously [3,21]. Briefly, sections were washed in 0.1 M PB with 0.3% Triton-X 100 (pH 7.4), incubated in an activated avidin–biotinylated horseradish peroxidase solution (Vector Novocastra Laboratories, Burlingame, CA) for 2 h, washed in PB, and then incubated with 0.05% diaminobenzidine (DAB), 0.005% H 2 O 2 , and 0.04% NiCl 2 in 0.1 M Tris buffer (pH 7.1) for 11–13 min. After the reaction product appeared, the sections were rinsed in PB, mounted on gel-coated slides, and dried overnight. The sections were subsequently defatted in xylene and coverslipped with Cytoseal. BDA-labeled terminals and processes were visualized with bright field illumination. Neuronal processes labeled with FR or AF were visualized with epifluorescent illumination in both the BDAprocessed and unprocessed sections. Unprocessed sections were mounted on gel-coated slides, dehydrated in alcohol, defatted in xylene, and coverslipped with Cytoseal. FRlabeled terminals and processes were observed with the aid

of a TRITC filter set (Chroma Technology, Brattleboro, VT) that permitted excitation from 540 to 580 nm and emission from 590 to 660 nm. AF-labeled terminals and processes were observed with the aid of a FITC filter set (Olympus, Lake Success, NY) that permitted excitation from 455 to 490 nm and emission from 535 to 540 nm. Serially-ordered tangential sections through SI cortex were processed for CO using published techniques [24,37]. Briefly, sections were washed in PB and incubated at 35 8C for 7–8 h in PB with 0.05% DAB, 0.05% Cytochrome C (Sigma, St. Louis, MO), and 4% sucrose. The sections were then washed in PB again, mounted on gel-coated slides, and dried. Mounted sections were post-fixed in neutral formalin for 30 min, dehydrated in a graded series of alcohol, defatted in xylene, and coverslipped with Permount. Tangential sections through SI were then examined to reveal the location of tracer injections with respect to the CO-labeled subfields (i.e. whisker barrels, limb representations). Measurements of the separation between injections were made from tangential sections through layer V of SI cortex. The effective zone of tracer transport was considered to be the dense core of the injection site, where tracer filled the neuropil. The separation between injection sites was measured as the minimum distance between their perimeters (i.e. edge-to-edge). We also verified that tracer injections were localized to SII or SI, the latter of which was defined by the pattern of CO labeling. Instances in which tracers were deposited outside these representations were excluded from analysis, as were cases in which the tracer injections failed to produce labeling in both the POm and the VB thalamus. Digital photomicrographs were obtained with a Coolsnap HQ CCD digital camera. Although BDA labeling was unaffected by light exposure, FR and AF labeling were quenched during the prolonged light exposure that was needed to plot these fluorescent tracers. Hence, digital images of the fluorescent tracers were often taken from an adjacent unplotted section that had not been processed for BDA. Each raw digital image was acquired in a tagged image file format (TIF) and was subsequently imported into Canvas 7.0 (Deneba Systems Inc., Miami, FL) where the intensity of the gray-scale and color levels were adjusted to produce an image that resembled what was seen through the microscope. In photomicrographs of fluorescent material, we also observed the presence of brightly-fluorescent perivascular cells (i.e. pericytes). In addition to their location around blood vessels, these cells had spindle-shaped processes and a discontinuous granular appearance that allowed them to be distinguished from the continuous labeling seen in densely impregnated thalamocortical relay cells or corticothalamic terminals. Because photomicrographs at low magnification do not permit visualization of this granular appearance, we reduced the brightness and size of these cells in cases where their appearance interfered with accurate depiction of corticothalamic labeling. The presence or absence of

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autofluorescent perivascular labeling is noted in each figure legend.

2.4. Statistical analyses To reconstruct the topography of corticothalamic projections, sections through the thalamus were inspected at 2003 or 4003 magnification (203 or 403 objectives with a 103 eyepiece) using epifluorescent (FR and AF) or bright field (BDA) illumination. Anatomical landmarks and the locations of labeled corticothalamic processes were plotted with the aid of a computerized charting system that uses optical encoders to detect x–y movements (resolution52 mm) of the microscope stage (AccuStage, St. Paul, MN). Using the multiple symbol and color features available in the software, the coordinate positions of FR-, AF-, and BDA-labeled cell bodies and beaded terminals were recorded for each section and saved to a computer file. Each file was analyzed with a custom program that subdivided each section into an array of 35 mm 2 bins, and the number of labeled terminal varicosities in each bin was counted. Bins that contained two or more FR- or AF- or BDA-labeled terminals were coded with the colors red, green, or blue, respectively. Bins that contained at least two or more beaded varicosities for each of two different tracer combinations (e.g. 2 FR- and 2 BDA-labeled terminals) were coded white. Thus, for animals that received triple tracer injections, it was necessary to run the analysis three times so that we could compare each combination of tracers separately. For each animal, we summed the number of labeled bins across sections to determine the total number of bins in the POm or VB that were occupied by labeled terminals. The proportion of labeled bins that contained overlapping projections from different tracer injection sites was expressed as the ratio of the white bins to the sum of the relevant colored bins (e.g. FR-BDA total overlap (%)5(white bins /(white1red1blue bins))3100). The amount of tracer overlap is influenced by bin size and threshold criteria. As discussed extensively in previous reports [1,20], small bins and high thresholds minimize the amount of overlap, whereas large bins and low thresholds maximize the amount of overlap. We have generally used 35 mm 2 bins and a threshold of two labeled varicosities per bin [1,2,20], and we continued to use these criteria in the present study so that we could compare the results with our previous work. The main rationale for using 35 mm 2 bins is that larger bins often yield overlapping distributions in the thalamus even when the labeled terminals are clearly segregated [1]. We did not use bins smaller than 35 mm 2 because the dendritic arbors of thalamic neurons are capable of integrating information over larger areas [9,17,29]. Using the overlap criteria described above (i.e. 35 mm 2 bins and two labeled varicosities of each tracer per bin), we performed several statistical analyses to test whether the amount of tracer overlap in POm was equivalent to the

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amount of tracer overlap measured in VB. The underlying assumption was that tracer overlap reflects the precision of topographic organization in these thalamic regions. If this assumption is correct, then injections of different tracers into neighboring, non-overlapping SI and SII cortical representations should produce low amounts of tracer overlap in thalamic regions that have a precise topographic organization. By comparison, thalamic regions that do not have a precise topographic organization should display substantially greater amounts of tracer overlap. The amount of tracer overlap should also be related to the size and proximity of the injection sites, but since both POm and VB are labeled in each animal, a matched-subject comparison of the amount of overlap in these two regions should indicate whether they possess the same degree of topographic organization. To assess this hypothesis, we used a matched-sample t-test to directly compare the amount of tracer overlap in POm and VB of the injected animals. Furthermore, we also performed correlation analysis and linear regression to characterize the relative amounts of overlap in these thalamic regions.

3. Results Corticothalamic labeling was analyzed in 57 animals that received multiple tracer injections in somatosensory cortex. These animals were also used to characterize the relative topography of corticostriatal, corticopontine, and corticocortical projections from two or more parts of somatosensory cortex. Many results from these studies have already been reported in several papers [1,2,25], and these papers provide a complete list of the somatotopic locations of tracer injection sites, as well as representative illustrations of the tracer injections. Since the publication of those reports, two additional studies have examined the topography of SI projections to the neostriatum [20] or to motor cortex [18]. Table 1 indicates the cortical locations of the injection sites for 23 cases in these latter studies in which corticothalamic projections to POm and VB had also been plotted and were available for the analysis presented here. To compare the pattern of corticothalamic projections to POm and VB, we subdivided the cases into three groups of animals based on the combinations of SI and SII body part representations that received tracer injections. The first group consisted of cases (n519) in which all tracers were placed in SI and at least one of the injection sites included a limb representation. The largest subcategory of animals in this group involved cases in which two or three tracers were placed in neighboring parts of the forelimb representation (n57). For the remaining animals in this group, different combinations of tracers were placed either in the forepaw and hindpaw representations (n55), the forepaw and whisker representations (n53), the hindpaw and whisker representations (n52) or in the forepaw, hindpaw,

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58 Table 1 Somatotopic location of SI injection sites a Case

Tracer locations BDA

Fluoro-Ruby

Alexa-Fluoro

H5 H6 H7 H8 H9 H12 H15 H18 H19 H20 H21 H22 H23 H24 H25 H26 H28 H29 H34 CC28 CC29 CC31 CC33

Whiskers Forepaw Whiskers Upper forelimb Forepaw Forepaw Wrist Forepaw Hindpaw Forepaw Whiskers Forepaw Forepaw Upper forelimb Upper forelimb – Hindpaw Forepaw Forepaw Whiskers D2 Whiskers E4 / D4 Whiskers E3 / D3 Whiskers E6 / E7

Forepaw Whiskers Forepaw Forepaw Wrist Hindpaw Forepaw Hindpaw Forepaw Hindpaw Hindpaw Wrist Upper forelimb Wrist Wrist Whiskers Forepaw Hindpaw Hindpaw Whiskers D6 Whiskers C4 Whiskers D6 Whiskers E3 / E4

– – – – – – – Whiskers – Whiskers – Upper forelimb Wrist Forepaw Forepaw Hindpaw – – – – – – –

a

Additional injection sites in SI and SII are listed in Refs. [1,2].

and whisker representations (n52). All of the animals in this first group have been reported in a study that examined the relationship between somatotopic continuity and corticostriatal overlap [20], and all of the injection sites for this group are listed as ‘H’ cases in Table 1. In the second group of animals, two tracers were placed in adjacent parts of the SI barrel cortex, either in barrels residing in the same row (n511) or in barrels residing in different rows (n510). Most of the cases in this group (n517) were used in a study that analyzed the topography of corticostriatal projections [1]. The four additional cases were obtained from a study analyzing the relative topography of SI barrel field projections to primary motor cortex [18], and the injection sites for these four cases are listed as ‘CC’ cases in Table 1. For the third group of animals (n517), one tracer was placed in SI barrel cortex and another tracer was placed in a corresponding part of the SII whisker representation. All cases in this group were reported in a study that analyzed the topography of corticostriatal projections from the SI and SII whisker representations [2].

3.1. Cortical tracer injections and corticothalamic labeling An example of neighboring tracer injections in somatosensory cortex is illustrated in Fig. 1. Although tracer

injections varied in size, they rarely exceeded a diameter of 1000 mm when viewed in the tangential sections. In addition to extracellular diffusion away from the injection site, tracers were transported by neuronal processes to surrounding regions of cortex. As shown in Fig. 1, much of the transported tracer could be distinguished from tracer diffusion because it was separated from the dense injection core by a region of relatively sparse labeling. Using these criteria, we were able to place tracers into neighboring cortical regions and be confident that the two tracers were not diffusing into overlapping cortical sites. Cases in which different tracers appeared to diffuse into overlapping parts of cortex were not included in our analysis. Labeled corticothalamic axons and their terminals were marked by the presence of beaded enlargements that appeared to form en passant synapses as the labeled axons traversed the thalamic neuropil. These enlargements or axon varicosities have been observed in both POm and VB [4,34,38]. As these reports indicate, many corticothalamic terminals in POm contain varicosities that are much larger than those in VB. Thus, as shown in Fig. 2, varicosities in POm varied in size, and many are considerably larger than those seen in VB. Consistent with reports that POm and VB are reciprocally connected with somatosensory cortex [6,10,19,23,33], we frequently observed retrogradely labeled soma and dendrites as well as anterogradely labeled axon terminals. These processes were intermingled in compact, densely labeled regions that were 200–500 mm in diameter and extended rostrocaudally for 1–2 mm. Often these regions were so dense that it was impossible to distinguish axon terminals from other neuronal processes. To mark the spatial extent of this labeling, we moved the microscope stage along one axis and plotted labeled processes as they moved under the eyepiece crosshairs. The stage was then shifted orthogonally 25–30 mm, and the process was repeated as the stage moved in the reverse direction. Around the edges of these densely-labeled regions, the labeled terminals and their beaded varicosities could be distinguished from labeled soma, and we used a different symbol to mark the location of the retrogradely labeled cell bodies. Although the labeled cell bodies had a similar if not identical distribution, only the labeled terminals were used for the analyses presented here. We found that both POm and VB had a topographic organization. Because of this topographic organization and the fact that tracers were usually injected into non-corresponding representations of somatosensory cortex, overlapping projections from different injection sites almost always occurred in regions where the labeling was not densest. This meant that most of the labeled overlap appeared where the perimeters of the densely labeled regions abutted one another. As shown by Fig. 2C, such results permitted easy identification of the beaded terminal varicosities that were labeled by different tracers in the same thalamic region.

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Fig. 1. Representative example of Fluoro-ruby (FR) and biotinylated dextran amine (BDA) injections in the SI whisker barrel field of animal CC33. (A) Photomicrograph of a tangential section through cortical layer V indicates the location of an FR injection site that is visualized during fluorescent microscopy. Scattered fluorescent labeling associated with blood vessels far away from the injection site represents autofluorescent labeling in pericytes. (A9) Another view of the same section in panel A under bright field illumination indicates the extent of the BDA injection site. (B) Photomicrograph of a tangential section through layer IV shows the locations of SI whisker barrels marked by high concentrations of cytochrome oxidase (CO). (C) Outline drawing of the CO-labeled barrels shown in B. Horizontal and vertical hatching indicate the location of the BDA and FR injections, respectively. All panels are illustrated at the same scale; arrowheads and filled circles indicate identical blood vessels in each panel.

3.2. Group 1: SI limbs and whiskers The basic somatotopic organization in POm and VB is illustrated by a case in which three different tracers were separately deposited into the SI forepaw, hindpaw, and whisker representations. As illustrated by Figs. 3 and 4, deposits of BDA, FR, and AF into these SI representations produced well-defined patches of segregated labeling in both VB and POm. The labeling in the limb and whisker representations of POm and VB produced a pattern of mirror images that was described previously [6,11,14,36]. Within the VB complex, the distal limb representations were located ventrally and the whisker representation appeared more dorsally. Within POm, which was located dorsomedial to the VB complex, labeling for the whisker representation was located lateral to the distal limb repre-

sentations. Because the boundary between these mirror images had an oblique orientation, in VB the forelimb representation was located medial to the hindlimb representation, but in POm the forelimb representation was located ventral to the hindlimb representation. Consistent with these segregated patches of labeling, quantitative analysis revealed very little overlap in thalamic labeling in either VB or POm when the tracers were deposited in different limb representations. Thus, as shown in Fig. 3B, the number of white bins denoting tracer overlap was very small. Labeled projections from the whisker representations terminated in adjoining parts of VB and POm, and these terminals sometimes formed a continuous patch of labeling in the most rostral sections of these nuclei. Although labeling of the whisker representations was observed

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Fig. 2. Representative examples of labeled corticothalamic terminals in the thalamus. (A) Photomicrograph showing an isolated cluster of FR-labeled corticothalamic terminals in the medial posterior nucleus (POm). Note the enlarged beaded varicosities that are typically observed in this thalamic nucleus. Bright pericytes appear near two blood vessels, one in the upper right quadrant of the image and another in the lower left corner. (B) Photomicrograph showing an extensive plexus of BDA-labeled corticothalamic terminals and beaded varicosities in the ventrobasal complex (VB). (C and C9) Photomicrographs showing both FR- and BDA-labeled corticothalamic terminals in overlapping parts of the POm. These views in the thalamus were taken from animal CC33, the injection sites for which are shown in Fig. 1. Grids indicate the area contained within a single 35 mm 2 bin. Arrowheads indicate the same blood vessel appearing in both photomicrographs; slight differences in the position of the blood vessels with respect to the grids are due to differences in the focal plane of the two views.

straddling the boundary between VB and POm at sections located 3.2–3.6 mm caudal to bregma (see Fig. 3), sections through more caudal parts of the thalamus revealed separate patches of labeled whisker representations located away from the boundary between VB and POm. In sections where labeling of the whisker representations crossed the POm and VB borders, the nuclear boundary was determined by inspecting adjacent sections stained for thionin (sections not shown). Furthermore, as reported previously [36], terminal labeling in POm usually appeared less dense than the corresponding terminal labeling in VB (see Figs. 2 and 4). Labeled corticothalamic projections were much more likely to occupy overlapping parts of both POm and VB when the tracers were deposited in adjoining regions of somatosensory cortex. As shown in Fig. 5, injections of AF, FR, and BDA into contiguous regions of the SI forepaw, wrist, and proximal arm representations produced

a densely-packed topographically-organized strip of labeling in VB. While the forepaw representation appeared ventromedially in VB, the proximal arm representation appeared dorsolaterally, and the wrist representation appeared between these regions and around the AF-labeling of the paw representation (Fig. 5A). Similarly, in POm of the same animal, the BDA-labeled region of the proximal arm representation was located dorsolaterally, the AFlabeled region of the forepaw representation was located ventromedially, and the FR-labeled region of the wrist representation was located both between these regions and around the AF-labeled processes that represented the paw. Consistent with the contiguous locations of these SI forelimb representations, the edges of the labeled regions overlapped with each other in both POm and VB. Quantitative analysis revealed that tracer overlap in POm of this case involved as much as 20% of the labeled bins in some of the coronal sections (Fig. 5B). Tracer overlap was also

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Fig. 3. Topography of labeled corticothalamic projections to POm and VB following anterograde tracer injections in the SI forepaw, hindpaw, and whisker representations of animal H20. (A) Reconstruction of selected coronal sections showing the relative pattern of projections from the forepaw, hindpaw, and C4 whisker barrel representations of SI. Blue, red, and green dots mark the location of beaded varicosities labeled with BDA, FR, and AF, respectively. Terminal labeling shown with respect to the internal capsule (ic); boxed regions indicate the location of the photomicrographs appearing in Fig. 4. Dashed lines represent the border between POm and VB as determined from an adjacent section stained with thionin. (B) Overlap analysis of the reconstructed sections shown in panel A. Bins containing BDA-, FR-, or AF-labeled terminals are colored blue, red, or green, respectively. Bins containing overlapping projections from more than one tracer injection site are colored white. Percentages indicate the proportion of the 35 mm 2 bins in POm or VB that contain overlapping projections from the different tracer injection sites in SI.

present throughout the rostrocaudal extent of VB and sometimes involved as many as 15% of the labeled bins in some sections (Fig. 5B). Statistical analysis of our first group of animals (SI limbs and sometimes whiskers) indicated that the amount of labeled overlap was similar in VB and POm. Mean tracer overlap was 2.2560.61% (mean6S.E.M.) in VB and 2.7360.69% in POm. Although the mean overlap was slightly higher in POm than in VB, this result was not consistently observed across all cases, and a matchedsample t-test indicated that the differences between these thalamic regions was insignificant (paired t51.11, P. 0.10, one-tailed). Furthermore, as shown by the scatter plot in Fig. 6 (left panel), tracer overlap in POm was highly correlated with the amount observed in VB (r50.79, P, 0.0001). A major factor associated with the variations in tracer overlap was the cortical distance between the SI tracer injections. Thus, as shown in Fig. 7 (left panel), tracer overlap for cases involving the SI limb representations was inversely correlated with the injection site separation for both VB (r520.585, P,0.001) and POm (r520.498, P,0.01).

3.3. Group 2: adjacent SI whisker representations The topographic organization of corticothalamic projections was also apparent in animals in which both tracers were placed in SI barrel cortex. As shown by a representative example in Fig. 8, the vibrissal representation within POm possessed a topographic organization that mirrored the topography of labeling in VB. In this example, FRlabeled projections from the barrel columns in row E terminated in a region of VB that was ventrolateral to the BDA-labeled projections from row C, and the relative configuration of these projection patterns was reversed in POm. Although the tracer injections were separated by a region of SI cortex that represented the D row of whiskers, many BDA- and FR-labeled projections terminated in adjoining or partially overlapping regions of VB and POm. One section through POm, for example, displayed tracer overlap that involved as many as 23.45% of the labeled bins (see Fig. 8B). When all sections were considered, however, only 9.9% of the total number of labeled bins in POm of this animal contained both tracers. By comparison, analysis of tracer overlap in VB of this animal revealed

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Fig. 4. Photomicrographs depicting the topography of the labeled terminals that were illustrated in the boxed areas of Fig. 3. (A) Light microscopy reveals a dense plexus of BDA-labeled terminals in the ventral part of the VB complex. (A9) Fluorescent microscopy of a section adjacent to the one shown in panel A reveals the segregated locations of corticothalamic terminals and retrogradely-labeled cell bodies containing FR (red) or AF (green). Panels A and A9 appear at same scale. (B) Light microscopy reveals part of a BDA-labeled plexus of terminals in POm; note the presence of large beaded varicosities that characterize the neuropil within POm. (B9) Fluorescent microscopy of a section adjacent to the one shown in panel B reveals separate patches of FRand AF-labeled terminals and cell bodies. Panels B and B9 appear at same scale. Arrowheads indicate the same blood vessels in adjacent sections. Fluorescent pericytes, which represent artifactual labeling around blood vessels, were selectively reduced in size and brightness in panels A9 and B9.

that 6.6% of all labeled bins contained projections from both cortical injection sites. Statistical analysis of the second group of animals (SI whiskers) revealed similar amounts of labeled overlap in POm and VB. Thus, when both tracers were placed in neighboring parts of the SI barrel field, the mean amount of tracer overlap was 3.1460.64% in VB and 3.2660.87% in POm. A matched-sample t-test indicated that this difference was not statistically significant (t50.26, P. 0.39, one-tailed). The scatter plot in Fig. 6 (middle panel) indicates a high degree of correlation in the amount of tracer overlap observed in POm and VB of different animals (r50.85, P,0.0001). As with the first group of animals, Fig. 7 (middle panel) suggests that one factor responsible for tracer overlap in these cases was the proximity of the cortical injections. Thus, for cases in which both tracers were placed in the SI barrel field, the

amount of overlap was inversely correlated with injection site separation for both VB (r520.577, P,0.001) and POm (r520.568, P,0.01).

3.4. Group 3: SI and SII whisker representations Tracer overlap in POm and VB was also measured in animals in which different tracers were injected into the SI and SII whisker representations. Both POm and VB are reciprocally connected with SI and SII [2,6,10,14,19,34], and the amount of tracer overlap in these regions should reflect the degree of functional correspondence at the cortical injection sites. Furthermore, to the extent that both POm and VB have a precise topographic organization, the amount of labeled overlap in these thalamic nuclei should be similar in each animal. Corticothalamic overlap in these cases was often ob-

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Fig. 5. Topography of labeled corticothalamic projections to POm and VB of rat H25. Selected reconstructed sections show the relative pattern of projections from the forepaw, wrist, and proximal arm representations of SI. Reconstructions presented as in Fig. 3.

served in both POm and VB, and the proportion of labeled bins that showed overlap was greatest in those cases in which the SI and SII injection sites involved corresponding whisker representations. It should be noted, however, that injection sites in SI usually represented only one or two whiskers whereas the SII injection site invariably represented as many as three or four whiskers in the same row. This is consistent with findings that SII neurons often represent multiple whiskers [5]. Nonetheless, as shown by Fig. 9, a pair of tracer injections that involved the same

whisker representations in both SI and SII produced high amounts of tracer overlap. In this case, different tracers were deposited into the D2 whisker representations of SI and SII, and the number of labeled bins that contained both tracers exceeded 30% in selected sections through POm or VB. Across all thalamic sections in this case, projections from the two injection sites overlapped in 8.57% of the labeled bins in VB and in 12.36% of the labeled bins in POm. Statistical analysis failed to reveal any consistent differ-

Fig. 6. Relative amounts of tracer overlap in POm and the ventrobasal complex as a function of different injection site combinations. In each scatter plot, a point represents the conjoint overlap in POm and the ventrobasal complex for a pair of tracer injections; animals that received three tracer injections are represented by three points. The correlation coefficient and best linear fit are presented in each scatter plot. Left panel: Cases in which two or three tracers were injected into combinations of different SI representations including different parts of the forelimb, forelimb and hindlimb, forelimb and whiskers, and hindlimb and whiskers. Middle panel: Cases in which two tracers were injected into adjacent parts of the SI barrel field. Right panel: Cases in which two tracers were injected into the SI and SII whisker representations.

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Fig. 7. Relative amounts of tracer overlap in POm and VB as a function of injection site separation. Best linear fit illustrated for POm or VB as indicated by legend. Each panel illustrates different combinations of injection sites as shown in Fig. 6.

ence in the proportion of labeled overlap that was observed in VB and POm of animals that received SI and SII tracer injections. For these cases, the mean proportion of labeled bins that contained overlapping projections was 3.8360.65% in VB and 4.7360.76% in POm. Although the average proportion of labeled overlap was higher in POm than in VB, a matched-sample t-test indicated that this difference was not significant (t51.27, P.0.10, onetailed). The proportion of labeled overlap in POm and VB of this group of animals was moderately correlated (r5 0.505, P,0.05) as illustrated by the scatter plot in Fig. 6 (right panel). In contrast to the first two groups of animals, Fig. 7 (right panel), indicated that the proximity of the injection sites in SI and SII was not strongly correlated with tracer overlap in either VB (r50.224, P.0.35) or

POm (r520.254, P.0.30). Cortical proximity was probably not a factor because the SI and SII whisker representations have adjacent mirror image representations, and corresponding whisker representations in these areas are not located at constant distances from each other.

3.5. Comparison of labeled areas in POm and VB Within each animal, the number of labeled bins was almost always higher in VB than in POm. This is consistent with the fact that much more thalamic volume is devoted to representing the body in VB than in POm. As shown by Fig. 10, the amount of thalamic labeling was greatest in the first group of animals, largely because some of those animals received all three tracers (BDA, FR, and

Fig. 8. Topography of labeled corticothalamic projections to POm and VB in rat D42, which was previously reported in Ref. [1]. Selected reconstructed sections show the relative pattern of projections from the E1-2 and C4-5 whisker representations of SI. All reconstructions presented as in Fig. 3.

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Fig. 9. Topography of labeled corticothalamic projections to POm and VB from specific whisker barrel columns in SI and SII of rat SS49, which was previously reported in Ref. [2]. Selected reconstructed sections show the relative pattern of corticothalamic projections from the D2 and D2-4 whisker representations in SI and SII, respectively. All reconstructions presented as in Fig. 3.

AF) in SI. Although the number of labeled bins in POm and VB varied because of these differences or because of differences in injection sizes, a matched-sample analysis clearly indicated that the amount of corticothalamic labeling produced by a given injection was significantly greater in VB than in POm. In the first group of animals (SI limbs and whiskers), the number of labeled bins in VB was 85%

greater than in POm and this difference was statistically significant (paired t57.06; P,0.001). Similarly, when both tracers were injected into the SI barrel field, 50% more labeled bins were observed in VB than in POm and this difference was also significant (paired t56.34, P, 0.001). When different tracers were injected into the SI and SII whisker representations, the number of labeled bins was 31% greater in VB than in POm, but most of this difference was due to inequalities in corticothalamic projections from the SI barrel field. Thus, in this group of animals (SI and SII whiskers), corticothalamic projections from SI occupied 48.1% more bins in VB than in POm and this difference was statistically significant (paired t55.00; P,0.001). By comparison, corticothalamic projections from SII occupied only 14% more bins in VB than in POm, and this difference was not significant (paired t51.45, P.0.05).

4. Discussion

Fig. 10. Relative comparison of the amount of labeled area in POm and VB following different combinations of anterograde tracer injections into somatosensory cortex. Each bar represents the mean number of total labeled bins in the POm or VB as indicated by the legend. Brackets represent S.E.M.

We directly compared the precision of corticothalamic projections to POm and VB across a series of animals in which two or three anterograde tracers were placed in different combinations of somatotopic representations in the SI and SII cortical areas. Our results confirm other reports indicating that POm has a somatotopic organization that mirrors the topography of the VB complex [6,11,14,36]. Furthermore, our statistical analyses failed to

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detect significant differences in the amount of labeled corticothalamic overlap in POm and VB. Based on our analysis of corticothalamic projections, the topographic organization of POm appears to have a spatial resolution that is roughly equivalent to that observed in VB.

4.1. Somatotopic organization in POm and VB Most anatomical studies of POm indicate that this structure has a somatotopic organization [14,19,28,36], but the evidence concerning whether it matches the precision of the topography in VB has not been conclusive. While some investigators state that the somatotopic organization in POm is similar to that seen in VB [14], others have stated that POm is less precisely organized [19], or that it has no discernable topography [38]. Studies using physiological methods to compare the topographical precision of POm and VB have also differed in their assessment of these thalamic regions. Thus, one study reports that whisker-sensitive neurons in POm have receptive fields that are considerably larger than those recorded in VPM [9], while another indicates that neurons in these nuclei have similar receptive field sizes [12]. In the latter study by Diamond and colleagues, a series of electrode penetrations in POm revealed a complete topographic representation of the body, including a detailed representation of individual whisker rows within the ventrolateral portion of this nucleus. It is noteworthy that this physiological map corresponds with the topography observed by us and by other investigators [12,14].

4.2. Methodological issues Differences in the methods used to characterize somatic topography in the thalamus might explain why some studies suggest that VB is more precisely organized than POm. Some studies, for example, used only a single anatomical tracer across a sample of animals to make inferences about the relative resolution of the somatotopic organization in POm and VB [28,36]. By contrast, a dual or triple tracing paradigm, as in the present study, allows a comparison of POm and VB topography within each animal. Furthermore, our quantitative methodology enabled the use of statistical techniques to compare the relative somatotopic organization of these two regions. Another experimental variable concerns whether topographic comparisons are based on thalamocortical or corticothalamic projections. While some studies focused primarily on the locations of thalamocortical relay cells in POm and VB [28,31], our study used tracers that were transported primarily in the anterograde direction so that we could analyze the relative patterns of corticothalamic terminals. Although we did not systematically analyze labeled cell bodies, visual inspection of our labeled material did not reveal any striking differences in the labeling patterns of the cell bodies and terminals. We

focused on the relative patterns of labeled corticothalamic terminals because this allowed us to use small bins (i.e. 35 mm 2 ) to characterize the distribution of these projections. Quantitative analysis of labeled cell bodies, which are much larger than the beaded varicosities of terminals, requires much larger bin sizes to detect tracer overlap, and this would lower the resolution for depicting labeled distributions in POm and VB. By using small bins to measure tracer overlap, we minimized the probability of detecting overlap among terminals that are unlikely to innervate neurons with similar receptive fields. Another source of experimental variation concerns the size of cortical tracer injections. While many studies have injected quantities of tracers that diffuse across representational boundaries, very few studies have placed limited quantities of tracer in restricted cortical regions. In one study in which iontophoresis was used to deposit different anterograde tracers into adjacent SI barrel columns, the authors report that corticothalamic projections from these neighboring sites terminated in overlapping parts of POm [38]. In other studies, iontophoresis of biocytin was used to impregnate a very small number of corticothalamic neurons in SI or SII [4,26]. When an extremely small quantity of this tracer was deposited in cortex, these studies showed that individual corticothalamic fibers terminate more loosely and widely in POm than fibers that terminate in VB. It is important to note, however, that when slightly larger quantities of the tracer labeled more corticothalamic projection fibers, the increased number of fibers led to an increase in terminal density, not an increase in the size of the area that was innervated [4]. Hence, comparing the relative tightness of individual corticothalamic fibers that terminate in VB or POm leads to the conclusion that individual corticothalamic fibers innervate broader somatotopic representations in POm than in VB. When comparing the distribution of local populations of corticothalamic fibers, however, a focal cortical representation appears to innervate a similar somatotopic representation in POm or VB. With respect to these issues, we used iontophoretic and pressure injection parameters that generally produced dense tracer deposits within a limited cortical representation. This view is corroborated by the fact that, among cases in which tracers were injected into the SI barrel fields, 43% of our injections were confined to a single barrel column and 46% of the injections invaded no more than two barrel columns [1,2].

4.3. Analysis of corticothalamic labeling In each animal we measured the total number of labeled bins and the amount of overlapping corticothalamic labeling throughout POm and VB. Regardless of whether tracers were placed in different whisker representations or in a combination of whisker and limb representations, the proportion of bins that contained corticothalamic labeling from both tracers appeared similar in both thalamic

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regions. Furthermore, a matched-sample statistical analysis failed to detect significant differences between these thalamic structures on this particular parameter. On average, however, the proportion of labeled overlap was always slightly higher in POm than in VB for each of the three groups of animals that we studied. If a larger sample of animals were analyzed, it is possible that our statistical analysis might have detected small, but significant differences in the amount of overlap in POm and VB. With respect to the total number of labeled bins, we consistently found more extensive labeling in VB than in POm. This difference could be attributed to differences in the size and volume of these thalamic regions. Most anatomical comparisons indicate that VB is larger than POm [11,14] and, hence, it was not surprising to find that projections from the same SI cortical representations terminated more extensively in VB than in POm. If VB has more neurons and a larger volume than POm but both thalamic nuclei represent the same body surface, then reciprocal corticothalamic connections should have differential spatial distributions. Contrary to this view, however, we found that tracer injections in SII cortex produced virtually the same number of labeled bins in POm and VB. These results suggest fundamental differences in the integrative functions that are served by the corticothalamic projections from the SI and SII cortices. Differences in the spatial distribution of SI and SII projections to POm and VB raises a potential issue concerning the use of corticothalamic distributions to indicate the functional topography of these nuclei. While a direct comparison of the relative distribution of neuronal response properties in POm and VB would seem to be the most appropriate parameter, this measure can be biased by anesthetics and other technical problems [11]. Connectional data, by contrast, are not subject to these difficulties, and it is relatively easy to compare the spatial distribution of projections to POm and VB within the same animal where all other variables are identical. Finally, while physiologic and anatomic measures have different strengths and weaknesses, data from both types of studies suggest that POm has a topographic organization that is comparable, if not equivalent, to the topographic precision seen in VB [11,12,14,19,28,36].

[2]

[3]

[4]

[5]

[6]

[7]

[8]

[9]

[10]

[11]

[12]

[13]

[14]

[15]

[16]

Acknowledgements [17]

The authors thank Henry Arantes for helping with some of the tracer injections and histological procedures. This work was supported by NIH Grant NS-37532 awarded to KDA.

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