THE JOURNAL OF COMPARATIVE NEUROLOGY 420:233–243 (2000)

Thalamic Projections From the Whisker- Sensitive Regions of the Spinal Trigeminal Complex in the

PIERRE VEINANTE,1 MARK F. JACQUIN,2 AND MARTIN DESCHEˆ NES1* 1Centre de Recherche Universite´ Laval-Robert Giffard, Hoˆpital Robert Giffard, Que´bec G1J 2G3, Canada 2Department of Neurology, Washington University School of Medicine, St. Louis, Missouri 63110

ABSTRACT This study investigated the axonal projections of whisker-sensitive cells of the spinal trigeminal subnuclei (SP5) in rat oral, interpolar, and caudal divisions (SP5o, SP5i, and SP5c, respectively). The labeling of small groups of trigeminothalamic axons with biotin- ylated dextran amine disclosed the following classes of axons. 1) Few SP5o cells project to the : They innervate the caudal part of the posterior group (Po) and the region intercalated between the anterior pretectal and the medial geniculate nuclei. These fibers also branch profusely in the tectum. 2) Two types of ascending fibers arise from SP5i: Type I fibers are thick and distribute to the Po and to other regions of the midbrain, i.e., the prerubral field, the deep layers of the , the anterior pretectal , and the ventral part of the zona incerta. Type II fibers are thin; branch sparsely in the tectum; and form small-sized, bushy arbors in the ventral posterior medial nucleus (VPM). Accordingly, a statistical analysis of the distribution of antidromic invasion latencies of 96 SP5i cells to thalamic stimulation disclosed two populations of neurons: fast-conducting cells, which invaded at a mean latency of 1.23 Ϯ 0.62 msec, and slow- conducting cells, which invaded at a mean latency of 2.97 Ϯ 0.62 msec. 3) The rostral part of SP5c contains cells with thalamic projections similar to that of type II SP5i neurons, whereas the caudal part did not label thalamic fibers in this study. A comparison of SP5i projections and PR5 projections in the VPM revealed that the former are restricted to ventral-lateral tier of the nucleus, whereas the latter terminate principally in the upper two tiers of the VPM. These results suggest a functional compartmentation of thalamic barreloids that is defined by the topographic distribution of PR5 and type II SP5i afferents. J. Comp. Neurol. 420:233–243, 2000. © 2000 Wiley-Liss, Inc.

Indexing terms: barrels; barreloids; vibrissa; ventral posterior medial nucleus; posterior group nucleus; biotinylated dextran

Brainstem nuclei that receive vibrissal primary afferents At each level of the neuroaxis, the peripheral arrange- in include the principal trigeminal nucleus (PR5) and all ment of the vibrissae is maintained in arrays of cellular subdivisions of the spinal trigeminal complex (SP5). Each of aggregates referred to as barrelettes (), barre- these (sub)nuclei contributes axons to the trigeminothalamic tract, but the main stream of ascending fibers arises from PR5 and from the interpolar division of SP5 (SP5i; Smith, Grant sponsor: Medical Research Council of Canada; Grant number: 1975). Bulk labeling with anterograde tracers has shown MT-5877; Grant sponsor: National Institutes of Health; Grant numbers: that both PR5 and SP5i axons innervate the ventral poste- DE07662 and DE07734; Grant sponsor: UMDNJ Foundation; Grant spon- rior medial (VPM) and the posterior group (Po) nuclei (Pe- sor: American Osteopathic Association. schanski, 1984; Chiaia et al., 1991a; Williams et al., 1994). *Correspondence to: Martin Descheˆnes, Centre de Recherche Universite´ Laval-Robert Giffard, Hoˆpital Robert Giffard, 2601 de la Canardie´re, Que´- For the moment, the axonal distribution of whisker-sensitive bec G1J 2G3, Canada. E-mail: [email protected] cells of the oral and caudal divisions of SP5 (SP5o and SP5c, Received 11 October 1999; Revised 27 December 1999; Accepted 30 respectively) remains ill defined. December 1999

© 2000 WILEY-LISS, INC. 234 P. VEINANTE ET AL. loids (thalamus), and barrels (cortex). Whereas the and Descheˆnes, 1999). In the current study, we sought to whisker-like patterning of the terminal fields of PR5 ax- determine the single-cell make-up of SP5 projections to ons in rat VPM has been well documented both at the the thalamus by labeling small groups of axons that arise ensemble level and the single-cell level (Williams et al., from the whisker-responsive regions of the three SP5 sub- 1994; Veinante and Descheˆnes, 1999), the topographic nuclei. distribution of SP5i projections remains unclear. Previous studies reported either a complete overlap with PR5 ter- minal fields (Peschanski, 1984) or a patchy, scattered dis- MATERIALS AND METHODS tribution lacking a clear, barreloid-like arrangement Anterograde labeling experiments (Chiaia et al., 1991a; Williams et al., 1994). Whatever the case, SP5i terminals should be relatively abundant across Experiments were carried out in 52 adult rats (Sprague- the field of barreloids, because electron microscopic stud- Dawley) in accordance with federally prescribed and uni- ies consistently reported SP5i profiles presynaptic to VPM versity animal care and use guidelines (Olfert et al., 1993). cells dendrites (Chiaia et al., 1991a; Wang and Ohara, Rats were anesthetized with ketamine (75 mg/kg) and 1993; Williams et al., 1994). In addition, large numbers of xylazine (5 mg/kg), and injections of biotinylated dextran VPM neurons exhibit an overt multiwhisker responsive- amine (BDA; molecular weight, 10,000; Molecular Probes, ness in PR5 lesioned rats (Rhoades et al., 1987) that Eugene, OR) were made with small-sized micropipettes disappears after subsequent ablation of the SP5i (see also (5–8 ␮m) into the three SP5 subnuclei. The stereotaxic Freidberg et al., 1999). For the moment, these ultrastruc- coordinates of the atlas of Paxinos and Watson (1986) tural and physiologic results are difficult to reconcile with were used to target the injections in SP5o and SP5i. The those provided by tract-tracing studies. subnucleus caudalis and the caudal part of SP5i were A confusing issue with most studies that used massive reached through the opening of the cisterna magna by tracer injections to map SP5 projections to the thalamus using the obex as a landmark. After recording vibrissae- relates to their lack of specificity with regard to the dif- evoked responses to ascertain the correct placement of the ferent populations of orofacial afferents. This makes it injections, BDA (2% in 0.5 M potassium acetate) was hard to reach strong conclusions about the projections of ejected by iontophoresis (positive current pulses of 300– whisker-sensitive trigeminothalamic cells. A second prob- 1,000 nA, 500 msec duration, half-duty cycle for 30 min- lem concerns the heterogeneity of this cellular population: utes). After a survival period of 5–7 days, animals were Intracellular staining of cells antidromically invaded from perfused with saline followed by a fixative containing 4% the thalamus revealed various morphologic types of paraformaldehyde and 0.5% glutaraldehyde in 0.1 M vibrissa-responsive neurons across the SP5 subnuclei phosphate buffer (PB), pH 7.4. were postfixed in (Jacquin et al., 1986a, 1988, 1989; Renehan et al., 1986; the same fixative for 2 hours, cryoprotected overnight in Jacquin and Rhoades, 1990). A similar diversity was high- 30% sucrose, and cut at 75 ␮m on a freezing microtome lighted by parvalbumin and calbindin immunostaining of along the horizontal plane. Sections were processed for retrogradely labeled trigeminothalamic cells (Bennett- cytochrome oxidase and BDA histochemistry according to Clarke et al., 1992). It appears likely that these different previously described protocols (Wong-Riley, 1979; populations of neurons give rise to different patterns of Horikawa and Armstrong, 1988). Finally, sections were axonal projections. When studied at a single-cell level, for mounted on gelatin-coated slides, dehydrated in alcohols, instance, the axonal projections from the PR5 demon- cleared in toluene, and coverslipped without counterstain- strate far more complexity than what is apparent imme- ing. Labeled trigeminothalamic fibers were drawn with a diately in conventional tract-tracing studies (Veinante camera lucida by using ϫ25 or ϫ40 objectives. Photomi- crographs were taken with a digital camera (Agfa-Gevaert NV, Montsel, Belgium) and were processed with Photo- shop software (version 3.0; Adobe Systems, Mountain Abbreviations View, CA). Apart from brightness and contrast adjust- ments, they were printed without modifications. 3v third ventricle An anterior group nuclei Control experiments Ang angular nucleus APT anterior pretectal nucleus Control experiments were made to address anatomic AV anterior ventral nucleus issues raised by the anterograde labeling experiments. fr fasciculus retroflexus These included calbindin immunostaining of sections con- LG lateral geniculate nucleus MG medial geniculate nucleus taining labeled fibers to determine whether some of these Po posterior group nucleus fibers terminate in the anterior intralaminar nuclei. After PR5 principal trigeminal nucleus bilateral injections of BDA into SP5i, one was cut at PRF prerubral field 50 ␮m along the frontal plane, and alternate sections were RT reticular thalamic nucleus SC superior colliculus processed either for cytochrome oxidase and BDA reac- sm stria medullaris tions or for revealing BDA and calbindin immunoreactiv- SP5 spinal trigeminal nucleus ity. Once the BDA reaction was terminated, sections were SP5c caudal division of the spinal trigeminal nucleus rinsed three times in 0.01 M phosphate-buffered saline SP5i interpolar division of the spinal trigeminal nucleus SP5o oral division of the spinal trigeminal nucleus (PBS, pH 7.4), and incubated overnight in a solution con- TR thalamic reticular nucleus taining 5% normal horse serum, 0.2% Triton X-100, and VL ventral lateral nucleus anticalbindin D-28K antiserum (Sigma; St. Louis, MO; VPL ventral posterior lateral nucleus dilution, 1:2,500). After three rinses in PBS, sections were VPm ventral posterior medial nucleus ZI zona incerta incubated for 1 hour in the secondary antibody (biotinyl- ZId dorsal division of the zona incerta ated horse immunoglobulin G; Vector Laboratories, Bur- SP5 PROJECTIONS IN THE RAT 235

Fig. 1. Photomicrographs of biotinylated dextran amine (BDA) injection sites in the oral (A), inter- polar (B), and caudal (C) divisions of the spinal trigeminal nucleus (SP5o, SP5I, and SP5c, respectively). All photomicrographs were taken from horizontal sections. 7n, Seventh nerve. Scale bar ϭ 500 ␮m.

lingame, CA), rinsed again three times in PBS, and re- RESULTS acted with the avidin-biotin-peroxidase complex (ABC; Data base Vector Laboratories). Bound peroxidase was revealed by using 3,3Ј-diaminobenzidine tetrahydrochloride (DAB; In horizontal sections of the brainstem, cytochrome ox- Sigma) as a substrate. idase staining clearly delineated SP5 subnuclei. Six sites Finally, four experiments were conducted to map in were located in SP5o, 16 were located in SP5i, and 17 were frontal sections the terminal fields of PR5 and SP5i located in SP5c (see Fig. 1). The thalamic projections of 51 afferents. Rats were anesthetized with ketamine/ darkly stained trigeminothalamic fibers were recon- xylazine, and BDA was injected iontophoretically into structed totally; then, proceeding backward, the main the whisker-sensitive division of both subnuclei by trunks and collateral branches were mapped to the other means of coarse micropipettes (tip diameter Ϸ 15 ␮m) terminal sites in the mesencephalon. An additional group and large-current pulses (3–4 ␮A, 7 seconds on/7 sec- of 65 axons was reconstructed in part to verify whether onds off). After a survival period of 6 days, animals were their projections conformed to those of the fully recon- perfused, and the tissue was processed as described structed fibers. above. Projections from SP5o Electrophysiological experiments Previous anatomic studies reported few labeled cells in SP5o after the injection of retrograde tracers into the Data pertaining to the conduction velocity of thalamic thalamus (Smith, 1975; Silverman and Kruger, 1985; projecting SP5i neurons were obtained in the course of a Bruce et al., 1987; Mantle-St.-John and Tracey, 1987; separate series of experiments carried out previously by Bennett-Clarke et al., 1992). Accordingly, large injections one of the authors (for a description of the methods used, of BDA (1 ␮A) made with relatively coarse micropipettes see Jacquin et al., 1989). In these experiments, the laten- (Ϸ15 ␮m) labeled very few SP5o trigeminothalamic fibers cies of antidromic invasion to VPM stimulation were mea- (typically, 0–3 axons per injection site). The reconstruc- sured for 96 SP5i neurons. Most of these cells responded to tion of these axons (n ϭ 7) revealed a common pattern of whisker stimulation, and some were injected intracellu- innervation in the caudal-ventral part of the Po and in the larly with horseradish peroxidase. These data were used crescent-shaped region adjacent to the anterior pretectal in conjunction with the anatomic data base to assess the nucleus (Fig. 2). In matching horizontal sections that were hypothesis that SP5i projections to the thalamus arise stained for calbindin, this later region stained positively from two populations of neurons that have different con- and displayed a sieve-like appearance. It comprised the duction velocities. ethmoid nucleus, the medial part of the medial geniculate 236 P. VEINANTE ET AL.

Fig. 2. Terminal fields of three SP5o axons in the thalamus and tectum. All reconstructions were made from horizontal sections. For abbreviations, see list. Scale bar ϭ 500 ␮m. nucleus (MGm), and the caudalmost extension of Po in- these fibers, some also innervate the dorsal part of the tercalated between the pretectal and the medial genicu- ventral-lateral nucleus (VL) and/or give off branches that late nuclei. Axons from the SP5o formed loose clusters of make a distinct cluster of terminations in a dorsomedial terminations in caudal Po and more dispersed clumps in region of Po situated behind the anterior ventral nucleus. the rest of their terminal fields. As a rule, SP5o axons also This later region is not an actual part of the intralaminar gave off a number of branches in the superior colliculus thalamus, as evidenced by its lack of calbindin immuno- (SC), and some innervated the dorsal division of the zona reactivity in double-stained sections. Rather, it seems to incerta (ZId). correspond to the angular nucleus in the atlas of Paxinos and Watson (1986). Finally, most axons distribute scat- Projections from SP5i tered terminals throughout caudal Po between the In agreement with previous electrophysiological studies parafascicular and the lateral geniculate nuclei. Although (Woolston et al., 1982; Jacquin et al., 1986a, 1989), record- many type I axons give off branches that ascend through ings made in SP5i prior to the injections disclosed almost VPM, none gives off terminations in the field of barreloids. exclusively units that responded to the movement of mul- Type II axons (n ϭ 15) are observed more frequently tiple . On the basis of the distribution of termi- after injections made into the caudal, barrelette-patterned nations in the upper mesencephalon and thalamus and of region of SP5i or the rostral part of SP5c. Injections made the shape of terminal arbors, two types of axons make up into the rostral SP5i label few of these axons, and, when it the ascending projections from this subnucleus. is present, the staining is often faint. Most type II axons Type I axons (n ϭ 23) have diameters of 2–4 ␮m and are of small diameter (1–2 ␮m), they branch very sparsely consistently showed up after all injections made into SP5i. in the tectum and MGm, and none terminates in ZI. In Collectively, these axons project to a number of sites in the VPM, they make bushy terminal fields containing one to thalamus and upper brainstem. The most robust projec- three clumps of medium-sized boutons (2–4 ␮m; see Figs. tions are to the superior colliculus, the pretectum, the 4, 5B). In horizontal sections, terminal fields form thin ventral division of the zona incerta (ZIv), and the pre- (Ϸ80 ␮m), narrow sagittal bands (Ϸ100 ϫ 250 ␮m) that rubral field, including the parvocellular division of the red most often are present in sections cut deep through the nucleus. Terminal fields in these regions often are so thalamus. dense that even the labeling of only two or three fibers makes their complete reconstruction hazardous. Tracing Projections from SP5c the main branches of single axons clearly shows, however, Whereas recordings made in SP5o and SP5i prior to the that most distribute terminals to each of these targets. In injections disclosed almost exclusively units that re- the thalamus, terminal fields are less dense and are con- sponded to the movement of multiple whiskers, the re- centrated principally in Po (Figs. 3, 5A). After giving off a cordings made in SP5c yielded a number of single-whisker series of collaterals in the mesencephalon and ZIv, type I cells; however, very few of these cells seem to project to the axons enter the thalamus and divide in a number of sec- thalamus. Of 17 injections made into this subnucleus, only ondary branches that form one or multiple clusters of those located rostrally (n ϭ 6 injections), within Ϸ0.8 mm medium-sized terminations (2–4 ␮m) concentrated prin- from the caudal border of SP5i, produced anterograde cipally in a shell-like region over the dorsal aspect of VPM labeling in the thalamus. Other injection sites resulted in and/or the caudal part of VPM lacking barreloids. Among heavy staining of intersubnuclear axons that formed a SP5 PROJECTIONS IN THE RAT 237

Fig. 3. A–D: Terminal fields of type I SP5i axons in the posterior posterior medial nucleus (VPM) and the presence of distinct clusters group nucleus (Po) and the upper brainstem. Thalamic arborizations in the angular and ventral lateral nuclei. For other abbreviations, see are reconstructed fully, and arrows indicate the other terminal sites list. Scale bar ϭ 500 ␮m. in the midbrain. Note the absence of terminations in the ventral thin, sagittal band across the full length of the trigeminal give rise to thalamic projections are characterized by the complex; however, the thalamus and upper brainstem presence of multiwhisker cells. Trigeminothalamic axons were completely free of labeling in these cases. Sites that arising from these sites (six fibers were reconstructed) are 238 P. VEINANTE ET AL.

Fig. 4. A–D: Terminal fields of type II SP5i axons in the VPM. Note the small sizes and elongated aspect of terminal fields, which are Ϸ80 ␮m thick. Drawings were made from horizontal sections. For abbreviations, see list. Scale bar ϭ 200 ␮m. of small diameter (1–2 ␮m) and form bushy terminal fields these fibers was drawn completely, partial reconstruction similar to those made by type II SP5i fibers. Likewise, revealed that they also give off terminal branches in MGm terminal fields are distributed principally in the ventral and tectum. part of VPM (Fig. 4). Comparative distribution of PR5 and SP5i Small numbers of thin-diameter axons labeled from the rostral part of SP5c projected to both VPM and Po. They projections in VPM arborized in the caudal part of VPM that lacks barreloids A remarkable feature of SP5i axons that terminated in and in the neighboring region of Po. Although none of VPM was their preferential distribution in horizontal sec- SP5 PROJECTIONS IN THE RAT 239

Fig. 5. Photomicrographs of SP5 terminal fields in the thalamus. A: Terminal field of type I axons in the rostral Po (shell region over the VPM). B: A single type II axon in the VPM. Note the clumped terminations of type II axons. Scale bars ϭ 100 ␮minA;50␮minB. tions cut deep through the thalamus. Therefore, we cut ent axonal conduction velocities. The histogram of Figure two brains along the frontal plane to visualize better the 7 shows the distribution of the antidromic invasion laten- location of terminal fields. After a large BDA injection into cies of 96 SP5i units that were backfired from the con- the whisker-responsive division of SP5i (two cases), the tralateral VPM. At first glance, this distribution looks anterograde labeling in VPM was found exclusively in the neither clearly unimodal nor bimodal. Thus, a test of bi- ventral-lateral tier of the nucleus that is contiguous to the modality was carried out to determine whether the distri- ventral posterior lateral nucleus (VPL). Figure 6A shows bution contains one population or a mixture of two popu- the distribution of darkly labeled terminal fields in a rep- lations of neurons (McLachlan and Basford, 1988). This resentative frontal section passing through VPM. After a test compared the likelihood of two hypothesis: The ob- similar injection made into PR5 (two cases), labeled ter- served distribution of latencies arises either from a single minal fields were found in a more restricted zone of VPM population of normally distributed latencies (hypothesis corresponding to the size of one or two barreloids. This P ) or from a mixture of two populations of normally zone contains dense clusters of terminations, principally 1 distributed latencies (hypothesis P ). In the latter case, it in the upper two tiers of the nucleus, with a sparser 2 distribution of boutons in the lower tier adjacent to VPL was assumed that the variance within the two populations (Fig. 6B,C). Thus, these results show that the terminal was the same, but the mean latency was allowed to differ. fields of PR5 and SP5i axons form complementary projec- The likelihood ratio of the two hypothesis (L2/L1) indicates tion patterns in VPM, except in the lower tier of the which of hypothesis is more likely and twice its logarith- 2 nucleus, where both projections seem to overlap. mic value follows a ␹ distribution that can be used to test the null hypothesis of a single population (hypothesis P ) Conduction velocities of thalamic projecting 1 against the alternative hypothesis (P2). This test clearly SP5i cells ϭ favors the P2 hypothesis, with a likelihood ratio 518 The anatomic results described above show that SP5i (␹2 ϭ 12.5; 2 degrees of freedom; P ϭ 0.002). Accordingly, projections to the thalamus arise from two classes of fibers the histogram would contain two populations of thalamic that have different diameters. One would expect this fea- projecting cells consisting of 74% of fast-conducting cells Ϯ ture to be related to cellular populations that have differ- invaded at a mean latency of 1.23 0.62 msec (P1) and 240 P. VEINANTE ET AL.

Fig. 6. A–C: Anterograde labeling of principal trigeminal nucleus and C. Note that terminal fields are concentrated mainly in the upper (PR5) and SP5i afferents in the VPM. A shows the distribution of SP5i two tiers of the VPM. All photomicrographs were taken from frontal type II terminal fields in the ventral lateral tier of the VPM. Scattered sections, and asterisks delineate the VPM. Scale bars ϭ 500 ␮minA; black dots in the VPM and Po are fragments of labeled axons. The 250 ␮m in B,C. distribution of PR5 afferents is shown in two consecutive sections in B

26% of slow-conducting cells invaded at a mean latency of cells, we demonstrated the existence of two main types of Ϯ 2.97 0.62 msec (P2). ascending fibers to the thalamus: thick fibers that distrib- ute to the Po and to other regions of the midbrain and thin fibers that arborize principally in the VPM. In addition, DISCUSSION the SP5 projection to VPM was shown to be restricted to Two principal findings resulted from this study. By us- the lower tier of the nucleus, where PR5 projections are ing BDA to label small groups of whisker-sensitive SP5 sparse but present. SP5 PROJECTIONS IN THE RAT 241

collicular and incertal cells to whisker displacements (Tiao and Blakemore, 1976; Chalupa and Rhoades, 1977; Yamasaki and Krauthamer, 1990; Chiaia et al., 1991b; Diamond et al., 1992; Nicolelis et al., 1992) also matches with the projection patterns observed in the current study. Thus, it seems unlikely that our anatomic data base was contaminated much by nonwhisker-related axons. Comparison with previous studies Thalamic projections from the SP5 have been described previously in studies using anterograde degeneration and a variety of neuronal tracers (Lund and Webster, 1967; Smith, 1975; Erzurumlu and Killackey, 1980; Peschanski, 1984; Chiaia et al., 1991a; Williams et al., 1994). Because most studies used massive lesions or injections that likely involved many types of second-order orofacial afferents, there exist some minor mismatches between prior results and the current set of whisker-related data. For instance, we did not observe any SP5 projection to the submedius or Fig. 7. Distribution of antidromic invasion latencies of 96 SP5i cells backfired from the thalamus. For explanation of statistics, see text. to the anterior intralaminar nuclei, likely because these regions receive input from afferents of other submodalities (Ma et al., 1988). Labeled terminal fields were not ob- served in the ventral-medial wing of VPM, which contains Technical comments the upper lip and lower mandibular representations. How- BDA is a very efficient and stable anterograde tracer ever, the other projection sites to the thalamus and the that stains cell processes in a Golgi-like manner. This midbrain were confirmed again by the current study. tracer seemingly enters into the cells through the small- Although it can never be said that the current sample of sized axonal and dendritic branches that are severed by SP5 axons is representative of all fiber types that make up the micropipette (Pinault, 1996; Chen and Aston-Jones, the ascending projections from this division of the trigeminal 1998). This is probably the reason why labeling appears complex, it came rather as a surprise that none of the labeled solid and uniform instead of granular, as with tracers that fibers actually innervated both the field of barreloids and the can be taken up actively (e.g., lectins or cholera toxin). Po. Such axons were expected from prior double-retrograde Thus, after extracellular deposits, the labeling of fibers of labeling studies, which estimated that they constitute Ϸ8% passage is unavoidable. This drawback, which is common of the ascending contingent from SP5i (Chiaia et al., 1991a). to most tracers, can be minimized by the use of small-sized Our negative result hardly can be ascribed to the small size micropipettes. The fact that injections made into the dif- of the injections and, thus, to a sampling bias, because in- ferent trigeminal subnuclei labeled axons with different jections of similar size made into the PR5 showed that 4% of projection patterns is a good indication that BDA uptake ascending fibers from that nucleus terminate in both VPM through axon collaterals is not a critical issue in the cur- and Po (Veinante and Descheˆnes, 1999). Interpolaris cells rent study. However, when large pipettes are used to with a dual thalamic projection, thus, may be absent or very pressure inject tracers, as in most previous studies, the few in number. The double-retrograde labeling observed af- probability of breaking fine collaterals increases as the ter injections of tracers into VPM and Po likely resulted from square of the pipette diameter. Large injections into PR5, tracer uptake by damaged branches of SP5i axons that as- for instance, in combination with the use of sensitive his- cend through VPM to reach Po. tochemical methods, are likely to yield false-positive re- All electrophysiological studies agree on the fact that sults, because many trigeminothalamic axons from SP5i whisker-sensitive SP5i cells that project to the thalamus send collaterals to this nucleus (Jacquin et al., 1986a, have multiwhisker receptive fields (Woolston et al., 1982; 1990).Consequently, labeling in the VPM may appear Jacquin et al., 1986a, 1989). In one of these studies (Jac- more extensive than what we report in this study and may quin et al., 1986a), SP5i neurons were backfired from the be more abundant in the lower tier of the nucleus, where thalamus and labeled intracellularly. Although the num- we found only a sparse projection. This contention is sup- bers of neurons studied were small, there was a clear ported by the spatial distribution of the terminal fields of dichotomy among this cellular population (see Table 1 in single PR5 axons that were labeled in a previous series of Jacquin et al., 1986a). Half of the sample consisted of cells experiments (Veinante and Descheˆnes, 1999). Reexamina- with thick axons (2–5 ␮m) that were invaded antidromi- tion of these data confirms the preferential arborization of cally at latencies shorter than 1 msec, whereas the rest of PR5 fibers in the two dorsal-medial tiers of VPM. the sample consisted of cells with thinner axons (1–2 ␮m) Tracer injections were made into the whisker- that were backfired at latencies longer than 1.5 msec. responsive regions of SP5. It remains possible, however, These results are fully in line with the two types of axons that some labeled fibers may have receptive fields on that were labeled by our BDA injections and with the neighboring regions of the mystacial pad (i.e., guard hairs, statistical analysis of the distribution of antidromic laten- intervibrissa fur). This possibility actually is impossible to cies. Thus, it seems that two populations of neurons relay dismiss; however, insofar as the projections to the barre- vibrissae information from SP5i to the thalamus: fast- loids are concerned, there is no doubt that they arise from conducting cells that project to Po and slow-conducting the whisker-sensitive populations of the trigeminal com- cells that project to VPM. In all likelihood, these two plex. The well-documented responsiveness of Po and of classes of neurons correspond to the large and small tri- 242 P. VEINANTE ET AL. geminothalamic cells that were identified previously in these factors add up to favor a faster transmission through the SP5i by Phelan and Falls (1991). the PR5 than the SP5i channel. Thus, these crude anatomic Ultrastructural and electrophysiological studies of the tri- considerations give full support to the conclusions reached geminal projections in the rat VPM provided evidence that by Lee et al. (1994b), who provided clear physiological evi- both PR5 axons and SP5i axons make synapses with the dence that a late-arriving, multiwhisker input to the VPM same relay cells (Chiaia et al., 1991b; Wang and Ohara, arises from the SP5i. 1993; Freidberg et al., 1999). In light of the current data, it is The above-described anatomic organization is consis- clear that both types of prethalamic afferents should contact tent with and, indeed, clarifies previous functional studies a fair proportion of cells in a thalamic barreloid. Only cells of vibrissa-sensitive cells in rat VPM. On striking a single situated dorsally in barreloids may have dendrites that are vibrissa, relay cells in the corresponding barreloid first out of reach of type II SP5i axons. However, if their dendrites will be activated by the firing of fast-conducting PR5 af- extend across the VPM/Po border, then these neurons may ferents. Activation will be followed by inhibition arising be the target of PR5 axons and type I afferents from the SP5i from reticular thalamic cells that will prevent the late- that terminate in the shell region of the VPM. For the mo- arriving, multiwhisker input from the SP5i to reach ment, this remains an open issue, and additional labeling threshold. The critical point here is how effective is the studies will be required to settle the question. The segrega- inhibition. Strong inhibition will confer on thalamic cells a tion of SP5i axons in the ventral-lateral tier of the VPM single-whisker responsiveness, whereas inhibition that is suggests a functional compartmentation within barreloids. not so strong will allow these cells to manifest a multi- Such a notion has been introduced and discussed previously whisker sensitivity. Because the inhibition induced by by Land et al. (1995) on the basis of the different cytochrome reticular thalamic cells is strongly state-dependent (Ste- oxidase reactivity of the dorsal and ventral aspects of barre- riade and Descheˆnes, 1984), it becomes obvious that the loids and of their differential retrograde labeling after horse- weight of SP5i inputs will increase as the level of anes- radish peroxidase injections made at different depths of a thesia diminishes (Freidberg et al., 1999). After lesion of barrel column. Here again, a single-cell study may reveal an the reticular nucleus, most relay cells will respond to as yet unsuspected specificity of connections between differ- multiple whiskers (Lee et al., 1994a). Lesion of the PR5, ent regions of a thalamic barreloid and its corresponding however, will prevent the early induction of reticular inhibi- cortical barrel. tion and confer on relay cells the multiwhisker responsive- ness that characterizes SP5i afferents. In contrast, SP5i Projections from SP5i to barreloids lesion will reduce the receptive field to one or two whis- In sections cut in an oblique sagittal plane through the kers (e.g., see Rhoades et al., 1987; Freidberg et al., 1999). VPM, cytochrome oxidase-rich barreloids form curved, ta- pering cylinders that extend through the thickness of the Projections from SP5i to Po nucleus (Land et al., 1995). In the dorsal-medial part of The SP5 projection to Po is extensive, with some clusters, the VPM, the long axis of barreloids lies normal to the and it is particularly dense in the shell region over the VPM VPM/Po border but bends horizontally in the ventral- and in the caudal ventral part of the ventrobasal complex, lateral part adjacent to VPL. Most PR5 trigeminothalamic where it is difficult to distinguish Po from the nonbarreloid fibers that project to VPM have single-whisker receptive region of VPM. This latter region demonstrates heavy retro- fields and form small-sized, bushy arbors that are re- grade labeling after tracer injections into the second somato- stricted to the dimension of a single barreloid (Williams et sensory cortical area (Spreafico et al., 1987). Vibrissal infor- al., 1994; Veinante and Descheˆnes, 1999). Although the mation processed in this cortical area likely transits through shape of individual barreloids could not be visualized in this zone of the posterior thalamus, which receives input our material, the terminal field of single type II axons from all subnuclei of the trigeminal complex. from the SP5i appears isomorphic with the size and The distinct clusters of terminations in the angular and curved structure of barreloids in the ventral lateral part of ventral lateral nuclei also clearly were present in the VPM. Thus, it seems reasonable to propose that single photomicrographs published by Williams et al. (1994; see type II axons from the SP5i convey multiwhisker informa- Fig. 2). It remains unclear whether these thalamic regions tion to a single thalamic barreloid. project to the barrel field and/or to other whisker-related areas of the neocortex. Because these two regions receive Receptive field of VPM cells a robust collateral input from layer 5 cells of the barrel The PR5 and SP5i are innervated by the same first-order field, whose axons also project to the deep layers of the vibrissal afferents that branch repeatedly throughout the superior colliculus (unpublished observations), they may trigeminal column (Hayashi, 1980, 1985; Jacquin et al., be involved in the motor control of whiskers. The same 1986b). The PR5 is situated next to the entry of the trigem- comment also applies to the shell region over the VPM, inal nerve and at Ϸ7 mm from the VPM, whereas the bar- which receives a layer 5 corticothalamic input and con- relette region of the SP5i lies Ϸ2.5 mm more caudally. In tains cells that project to both the somatic sensory and the addition, single-whisker PR5 neurons are much smaller motor cortices (Descheˆnes et al., 1998). Thus, the hodology than the multiwhisker units of the SP5i (Jacquin et al., 1988, of prethalamic and corticothalamic connections in Po sug- 1989; Veinante and Descheˆnes, 1999), indicating that they gests that this nucleus may be more involved in the pro- likely possess a higher input resistance that would reduce cessing of information related to active whisking than to the rise time of synaptic potentials. Assuming a factor of passive displacements of the vibrissae. proportionality of 6 m/second per micrometer of axon diam- eter (Hursh, 1939; Rushton, 1951), the conduction velocity of ACKNOWLEDGMENTS PR5 axons would be about twice that of type II SP5i fibers (PR5 axons diameter, 2–3 ␮m, as measured from a previous This work was supported by grant MT-5877 from the data base; Veinante and Descheˆnes, 1999). Together, all of Medical Research Council of Canada to M.D., by National SP5 PROJECTIONS IN THE RAT 243

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