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THE ANATOMICAL RECORD 291:960–973 (2008) Functional Connectivity of the Secondary Somatosensory Cortex of the Rat CHIA-CHI LIAO AND CHEN-TUNG YEN* Institute of Zoology, National Taiwan University, Taipei, Taiwan ABSTRACT The hierarchical relationship of the rat primary somatosensory cortex (S1) and secondary somatosensory cortex (S2) is controversial. The existence of a direct thalamocortical projection from ventral posterolateral thalamic nucleus (VPL) to S2 is a key factor in determining the relative position of S2 in the processing flow. In this study, the inter-connections of forepaw and hindpaw representations in VPL, S1, and S2 were examined by neuroanatomical tracing and electrophysiological approaches. In the tracing experiments, VPL, S1, and S2 were electrophysiologically identified, and then iontophoretically injected with biotinylated dextran amine (BDA, a bi-directional tracer). In the double-labeling experiments, two of the following retrograde tracers—BDA, Rhodamine dextran (RD), and/or Fluoro-Gold (FG)—were injected into homotypical S1 and S2 forepaw representations simultaneously. In the electrophysiological studies, paired somatic evoked multiunit responses in S1 and S2 were compared. Our results revealed that: (1) VPL forepaw and hindpaw neurons projected to corresponding S1 and S2 areas in a parallel and somatotopic manner; (2) very low percentage of double projecting VPL neurons were found, indicating parallel and independent pathways from forepaw VPL to S1 and S2; (3) forepaw S1 and S2 were symmetrically and reciprocally connected; (4) response latencies of the S1 and S2 multiunits to forepaw stimulation were in accordance with a direct and parallel pathway. This study provides further evidence to support the equivalent hierarchy of S1 and S2 in processing sensory information of the rat. Anat Rec, 291:960–973, 2008. Ó 2008 Wiley-Liss, Inc. Key words: parallel; primary somatosensory cortex; serial; ventral posterolateral thalamic nucleus The peripheral somatosensory information is known to convey to the contralateral primary somatosensory cortex (S1) and secondary somatosensory cortex (S2). Although extensive studies have been carried out on the thalamocortical and corticothalamic circuitry of the somatosensory system, whether S1 and S2 process Abbreviations used: Ang 5 angular thalamic nucleus; BDA 5 biotinylated dextran amine; CPu 5 caudate putamen; eml 5 external medullary lamina; ic 5 internal capsule; ml 5 medial lemniscus; POm 5 posterior medial nucleus; RD 5 rhodamine dextran; Rt 5 reticular thalamic nucleus; str 5 superior thalamic radiation; VA 5 ventroanterior thalamic nucleus; VL 5 ventrolateral thalamic nucleus; VM 5 ventromedial thalamic nucleus; VPL 5 ventral posterolateral thalamic nucleus; VPM 5 ventral posteromedial thalamic nucleus; ZId 5 zona incerta, dorsal part. Grant sponsor: National Science Council, Taiwan; Grant number: NSC 95-2311-B-002-017-MY3. *Correspondence to: Chen-Tung Yen, Institute of Zoology, National Taiwan University, No. 1, Roosevelt Road, Sec. 4, Taipei 106, Taiwan. E-mail: [email protected] Received 23 January 2008; Accepted 11 February 2008 DOI 10.1002/ar.20696 Published online 30 April 2008 in Wiley InterScience (www.interscience.wiley.com). Ó 2008 WILEY-LISS, INC. CONNECTIVITY OF RAT S2 peripheral information in parallel or serially remains an issue of contention (Jones, 2007). Previous animal studies have reported that S2 receives topographic projections from ipsilateral S1 in monkeys (Jones et al., 1975, 1978; Friedman and Murray, 1986), cats (Jones and Powell, 1968; Manzoni et al., 1979; Burton and Kopf, 1984), tree shrews (Weller et al., 1987), squirrels (Krubitzer et al., 1986), mice (Carvell and Simons, 1987), and rats (Koralek et al., 1990; Li et al., 1990; Fabri and Burton, 1991). Based on this line of evidence, and in addition to the welldescribed thalamic ventral posterior complex (VP) projecting densely to S1 (Jones and Powell, 1969b, 1970; Burton and Jones, 1976; Lin et al., 1979), a hierarchical scheme of somatosensory transmission pattern was constructed, in which peripheral information is processed sequentially from VP thalamus to S1 and then to S2. This hypothesis was supported further by lesion studies, that is, removal of an S1 representation abolished the somatic evoked response in the corresponding S2 region in macaques and rhesus monkeys (Pons et al., 1987, 1988; Burton et al., 1990). In contrast, S1 responsiveness was not affected by the elimination of the homotypical S2 region (Pons et al., 1988). Electrical studies further supported the notion that S1 provided sensory information to S2 because S2 neurons responded to peripheral stimulation at a longer latency than S1 neurons in rats (Brett-Green et al., 2003, 2004; Benison et al., 2007). In contrast, other studies support a parallel processing of somatic information. Retrograde tracing investigations have shown that a single neuron in the ventral posterolateral thalamic nucleus (VPL) project to both S1 and S2 in the cat (Andersson et al., 1966; Spreafico et al., 1981; Fisher et al., 1983). Retrograde degeneration methods also indicated that VP neurons connect directly to both S1 and S2 in cats and squirrel monkeys (Jones, 1975; Stevens et al., 1993). Finally, cooling of S1 does not change S2 responsiveness to peripheral stimulation in rabbits, opossums, rats, and marmoset monkeys (Murray et al., 1992; Coleman et al., 1999; Heppelmann et al., 2001; Zhang et al., 2001b). These results provide an alternative view, that is, S2 received direct thalamic inputs rather than through a serially organized path by means of S1. One hypothesis to explain these divergent findings is the possibility that serial or parallel processing types vary among species and/ or different body regions. The present study used neuroanatomical and electrophysiological approaches to study the connectional patterns between forepaw and hindpaw VPL, S1, and S2, an important body regions less well studied in the commonly used animal model, rat, to address this parallel vs. serial controversy. MATERIALS AND METHODS A total of 29 adult Long-Evans rats (250–350 g) of either sex were conducted in accordance with guidelines approved in the Codes for Experimental Use of Animals of the Council of Agriculture of Taiwan, based on the Animal Protection Law of Taiwan. All experimental protocols were approved by the Institutional Animal Care and Use Committee of National Taiwan University. 961 Anatomical Tracing Study Anesthesia was induced Surgical procedure. with an intraperitoneal injection of sodium pentobarbital (50 mg/kg). During surgery, the depth of anesthesia was verified by periodically testing for the absence of pinch reflex. Supplementary doses were given as necessary. The animal was mounted on a stereotaxic apparatus and its core temperature was maintained at 37.58C by a feedback-controlled heating pad. While referring to the atlas of Paxinos and Watson (1998), unilateral craniotomies were made to expose the parietal cortical areas immediately above the electrode penetration sites. In the sections that follow, all stereotaxic coordinates are given relative to bregma. Single injections of bi-directional tracer. A bidirectional tracer, biotinylated dextran amine (BDA: 2% in distilled water, molecular weight [MW] 10,000, Molecular Probes, Eugene, OR) was used in the tracing experiments. To avoid damaging the S1, glass micropipettes (tip size: 25–50 mm, #602500, A-M system, Carlborg, WA) were filled with BDA solution and lowered at a 30-degree angle toward the VPL (forepaw region: 3 mm lateral, 7 mm posterior, n 5 4 rats; hindpaw region: 3.5 mm lateral, 7 mm posterior, n 5 4 rats) and S2 (forepaw region: 6.0 mm lateral, 4.7 mm posterior, n 5 4 rats). In contrast, the microelectrodes were oriented perpendicularly to the brain surface when targeting S1 areas (forepaw region: 4 mm lateral, 0.5 mm anterior, n 5 4 rats). To ensure the precise location of the injection sites, forepaw responsiveness was identified by standard electrophysiological multiunit recording and tested with light tapping and brushing. In all cases, BDA was deposited iontophoretically (5 mA, positive current; 5 sec on, 5 sec off) for 20 min. To reduce backflow after injection, the micropipettes were maintained in the injection sites for 30 min before withdrawing. The wound was sealed by surgical staples and the animal was allowed to recover in its home cage. Double injections of retrograde tracers. For the retrograde studies, two additional tracers were used: Fluoro-Gold (FG: 10% in distilled water, Fluorochrome, LLC, Denver, CO), and Rhodamine dextran (RD: 10% in distilled water, MW 10,000, Molecular Probes). The forepaw areas of S1 and S2 were identified physiologically and tracers were then injected per the scheme in Table 1. To deposit these tracers, BDA and FG were injected iontophoretically, and RD was injected mechanically. After each injection, micropipettes were kept in place for 30 min to minimize backflow. Fixation and immunohistochemistry. After a period of 4–10 days, animals were anesthetized with an overdose of sodium pentobarbital (75 mg/kg) and perfused transcardially with 0.9% normal saline followed by 4% paraformaldehyde in 0.1 M phosphate buffer at pH 7.4. The brains were stored overnight at 48C in cryoprotectant buffer (25% sucrose, 0.1 M phosphate buffer, pH 7.4), and then sectioned serially in the coronal plane at 50 or 100 mm on a freezing microtome. In the BDA tracing cases, brain sections were rinsed three times in phosphate-buffered saline (PBS) and reacted sequentially with avidin-labeled peroxidase 962 LIAO AND YEN TABLE 1. Tracer injection schemes for double-labeling of forepaw representation areas S1 tracer and locationa Scheme 1 Scheme 2 Scheme 3 S2 tracer and locationa N 0A, 4.0L 1.7P, 6.0L 2 1 3 Rhodamine dextran (RD) Fluoro-Gold (FG) Rhodamine dextran (RD) Fluoro-Gold (FG) Rhodamine dextran (RD) Biotinylated dextran amine (BDA) a Locations are millimeters relative to bregma (A, anterior; P, posterior; L, lateral). S1, primary somatosensory cortex; S2, secondary somatosensory cortex. (ABC kit, PK-6100, Vector Laboratories, Burlingame, CA) for 1 hr and a diaminobenzidine-30% H2O2 solution for 10–15 min at room temperature. After three rinses in PBS, sections were placed on gelatin-coated glass slides, air-dried, mounted in DPX (Fluka, Seelze, Germany), and coverslipped. In double-labeling experiments with BDA injections, sections were incubated with the streptavidin-conjugated cyanine dyes Cy2 or Cy3 (1:200 dilution, Jackson ImmunoResearch Lab, West Grove, PA) for 1 hr at room temperature. Data analysis. A 6A Zeiss microscope (Axioplan 2) equipped with a Nikon digital camera (Coolpix 5000) was used to capture brightfield BDA images. An Olympus DSU spinning disk confocal microscope configured with a BX61 fluorescent microscope and an Olympus FluoView1000 confocal microscope with an IX81 fluorescent microscope were used to investigate fluorescently labeled sections. Labeled fibers and boutons were inspected and digitally acquired. Labeled profiles were also examined from line drawing with camera lucida attachment, and then scanned into Photoshop 7.0 for three-dimensional reconstructions. To determine the projection patterns, BDA, RD, or FG were examined with appropriate filters. Single-labeled and double-labeled cells were counted and summed across every sixth section to determine the total number of projecting cells in the VPL. Thus, the ratio or percentage of dual-projecting VPL neurons was calculated as the number of double-labeled cells divided by the total number of S1- or S2-projecting cells. Electrophysiological Experiments Rats (n 5 7 rats) were Surgery preparation. anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneally). The femoral vein was cannulated for supplemental doses of the same anesthetic (diluted 1:4) throughout the experiment as needed. Animals were mounted on a stereotaxic frame and the body temperature was maintained at 37.58C by a feedback-controlled heating pad. A unilateral craniotomy was performed to expose the forepaw area of the S1 (0 mm anterior, 4 mm lateral) and S2 (1.7 mm posterior, 6.0 mm lateral). A stainless steel screw was implanted into the skull above the cerebellum as a reference electrode. Stimulation and recording procedures. Electrical stimulation was achieved by needle-electrodes inserted subcutaneously into the contralateral forepaw pad. The needle electrodes were connected to a constantcurrent stimulator (Grass, S48, Warwick, RI). Multiunit response in S1 and S2 were evoked by electrical stimulation (120 rectangular pulses, 500 mA, 2 msec duration, 1 Hz). The recording period was 3 min in total, including 30 sec before and 30 sec after applying the current stimulation. Responses in S1 and S2 were recorded simultaneously by using two tungsten microelectrodes (#573400, A-M systems, Carlsborg, WA). Microelectrodes were lowered into S1 and S2 areas independently, after both recording sites were first identified by gentle tapping responses to the same receptive field in the contralateral forepaw region. Neuronal activities were transmitted into a multichannel acquisition processor system (MAP, Plexon, Dallas, TX). Multiunit spike waveforms exceeding 1.5 times the peak-to-peak noise were collected into 1,000msec-long wavelets and further analyzed by using OfflineSorter and NeuroExplorer (Plexon, Dallas, TX). Peristimulation histograms were generated with a bin size of 1 msec. A ‘‘boxcar’’ filter (with a width of three bins) was used for postprocessing. Multiunit responses were normalized as Z scores (Tsai et al., 2004). Briefly, the 50msec period before stimulation was used as the baseline period. The mean firing rate and standard deviation of the 50 bins in this baseline period were calculated. All bin values were transformed to Z scores according to the mean and standard deviation of the baseline period. A 99% confidence level (Z > 2.33) was used for identifying the responsiveness. The initial latencies, peak latencies, and half-maximum durations of the evoked multiunit responses in S1 and S2 were compared by a paired t-test. Histology. At the conclusion of the experiment, S1 and S2 recording sites were marked with electrolytic lesions (100 mA, 15 sec). The animal was deeply anesthetized with an overdose of pentobarbital (75 mg/kg) and then perfused through the ascending aorta with 0.9% saline followed by 10% formalin. The brains were stored overnight at 48C in a cryoprotectant buffer (25% sucrose, 0.1 M phosphate buffer, pH 7.2) and then sectioned at 100 mm thicknesses. Serial sections were mounted on gelatin-coated slides, followed by Nissl staining. The lesion sites and recording tracks were documented with camera lucida drawings. RESULTS Thalamocortical Projection From VPL When BDA was injected into the VPL forepaw area (n 5 4), labeled processes and terminals were easily found in ipsilateral cortical areas (Fig. 1). In S1, fine-caliber thalamocortical fibers and terminals with small boutons form two dense plexuses in vertical cortical columns: one in layer IV, and the other in layer VI (Fig. 1B). Intermixed spinous (terminaux) and bead-like (en passant) terminals were attached to these labeled afferent 963 CONNECTIVITY OF RAT S2 Fig. 1. An example of BDA injected in the VPL forepaw area. A: Photomicrograph of a coronal section showing the injection site. B: Photomicrograph of BDA-labeled terminals in the ipsilateral S1. Amplified terminal boutons are shown in the insert. C: Photomicrograph of BDA-labeled terminals in ipsilateral S2. D: Coronal planes depicting the distribution of BDA-labeled terminals in S1 and S2. Arrowheads indicate the demarcation between layers four and five of the cortex. BDA, biotinylated dextran amine; S1, primary somatosensory cortex; S2, secondary somatosensory cortex; VPL, ventral posterolateral thalamic nucleus. Scale bar 5 500 mm in A–C, 10 mm in both inserts. TABLE 2. Projections from bidirectional tracers labeling to ipsilateral brain regionsa Injection site Representation Projection S1 location S2 location VPL location Fig. 1 (N 5 1) Fig. 2 (N 5 1) Fig. 3 (N 5 8) VPL VPL VPL TC TC TC 0.7A-1.3P 0.4A-1.3P 0.7A-2.2P 1.3P-1.8P 2.6P 1.3P-2.8P ---- Fig. 4 (N 5 1) S1 Forepaw Hindpaw Forepaw and hindpaw, summary Forepaw Fig. 5 (N 5 1) S2 Forepaw Fig. 6 (N 5 8) S1 & S2 CC CT TC CC CT TC CT TC CT TC ---1.0A-2.0P ------ 1.3P-2.5P --------- -2.8P-3.3P 2.6P-3.5P -2.56P-3.6P 3.1P-3.6P 2.3P-3.6P 2.3P-3.6P 2.6P-4.1P 3.1P-4.1P S1 forepaw summary S2 forepaw summary a Locations are millimeters relative to bregma (A, anterior; P, posterior). TC, thalamocortical projections; CT, corticothalamic projections; CC, corticocortical projections; S1, primary somatosensory cortex; S2, secondary somatosensory cortex; VPL, ventral posterolateral thalamic nucleus. axons. A comparable labeling was also observed in S2 (Fig. 1C): two clusters composed of fine axons and small varicosites were also located in layers IV and VI. The distribution of labeled terminations in cortical region was depicted using camera lucida drawings (Fig. 1D) and was summarized in Table 2. In a second series of experiments, BDA was deposited into the hindpaw region of VPL (n 5 4) and a similar cortical projection pattern was observed (Fig. 2). Labeled fibers extending from thalamus terminated in ipsilateral S1 and S2 regions, at locations summarized in Table 2. As in the VPL forepaw injection experiments, labeled processes and terminals were divided into two clusters located in both layers IV and VI. Although the labeled terminals in S2 (shown in Fig. 2C) were less dense than in the forepaw experiments, labeled boutons could still be clearly discerned and characterized by fine-caliber fibers with boutons both terminaux and en passant. 964 LIAO AND YEN Fig. 2. An example of BDA injected in the VPL hindpaw area. A: Photomicrograph of a coronal section showing the injection site. B: Photomicrograph of BDA-labeled terminals in the ipsilateral S1. Amplified terminal boutons are shown in the insert. C: Photomicrograph of BDA-labeled terminals in ipsilateral S2. D: Coronal planes showing the distribution of BDA-labeled terminals in S1 and S2. Arrowheads indicate the demarcation between layers four and five of the cortex. Abbreviations as in Figure 1. Scale bar 5 500 mm in A,B, 10 mm in C (and insert). The thalamocortical fibers ascending from VPL to either S1 or S2 were examined. They first passed through caudate putamen, and then reach the S2. These labeled thalamocortical fibers can further be traced toward S1. Projection destinations in ipsilateral cortical areas arising from VPL forepaw and hindpaw neurons are summarized in Figure 3 and Table 2. Two somatosensoryrelated regions were heavily labeled, corresponding to the traditionally defined S1 and S2 (Fig. 3A). When the distribution of the ascending terminals in the ipsilateral cortical area were plotted on the two-dimension plane (Fig. 3B), both S1 and S2 are characterized by somatotopic organization of VPL forepaw and hindpaw neuron terminations. However, in addition to S1 and S2, a third area located caudally was also noted to be labeled with VPL projecting terminations. All three regions received direct inputs from VPL forepaw and hindpaw neurons. ranging from 2.6P to 3.2P (Fig. 4D). In the dorsal thalamus, a dense plexus of small labeled axons and varicosites were located in the medial VPL and BDA-labeled VPL cells were mingled with those terminals ranging from 2.6P to 3.3P. Corticothalamic terminals were also found in the posterior medial nucleus and the reticular thalamic nucleus. Two types of terminals were found in the posterior medial nucleus: one of large size (3–10 mm), and the other of smaller size (0.5–0.8 mm). Furthermore, the S1 forepaw representation also projected to contralateral S1 and S2 regions. Thalamocortical and Corticocortical Connections of S1 Input and output connections of the S1 forepaw region were traced by BDA injections (Fig. 4). After injection, labeled terminals and cells were easily found in ipsilateral S2 and VPL (Fig. 4B–D). In S2, labeled axons were noted in every layer, but concentrated more in layer II/ III. Several retrogradely labeled cells were distributed in layer II/III and VI. Comparable termination was observed in another cortical area, located at 3.5 mm posterior (3.5P) and 6.5 mm lateral (6.5L). In addition, labeled terminals were found around the rhinal sulcus Thalamocortical and Corticocortical Connections of S2 When BDA was injected into S2 forepaw region (Fig. 5), BDA-labeled cells and fibers with both terminaux and en passant terminals were observed in ipsilateral S1, particularly in layers II/III and VI (Fig. 5B). In addition to S1, labeled profiles were also found at approximately 3.6P and 8.0L, and perirhinal cortex ranging from 3.1P to 3.6P. The medial VPL contained a labeled plexus composed of very thin axons with irregularly arranged terminals (both terminaux and en passant), analogous to the S1 forepaw axons (Fig. 4C). It is important to note that retrogradely labeled cells existed within VPL and were intermixed with the labeled corticothalamic fibers. These S2-projecting VPL cells were located in areas ranging 3.1P to 3.6P. The ipsilateral posterior medical nucleus and reticular thalamic nucleus were labeled simultaneously, with the axons in the Fig. 3. Summary of the labeled terminal distributions after BDA was injected into VPL forepaw (arrowhead, green asterisk, n 5 4) and hindpaw (arrow, red asterisk, n 5 4) regions. A: Anteroposterior plane depicting the thalamocortical terminations in the ipsilateral S1 and S2. B: Two-dimensional organization of the labeled terminations in the ipsilateral cortical region. The green squares represent thalamocortical terminations extending from VPL forepaw neurons, and the red squares symbolize the projecting terminations of VPL hindpaw neurons. Note that both ipsilateral S1 and S2 were topographically labeled. BDA, biotinylated dextran amine; A, anterior; POm, posterior medial nucleus; L, lateral; S1, primary somatosensory cortex; S2, secondary somatosensory cortex; VPM, ventral posteromedial thalamic nucleus. 966 LIAO AND YEN Fig. 4. An example of BDA injected into S1 forepaw area. A: Photomicrograph depicting the injection site. B: Photomicrograph of labeled corticocortical terminations in ipsilateral S2. C: Photomicrograph of labeled corticothalamic terminations in ipsilateral VPL. D: Camera lucida drawings of the terminal fields in ipsilateral S2 and dorsal thalamus. Solid circles represent the retrogradely labeled cells, and small dots mark the labeled terminations. Inserts in B and C are enlarged views of labeled axons and boutons. BDA, biotinylated dextran amine; CPu, caudate putamen; ic, internal capsule; Rt, reticular thalamic nucleus; S1, primary somatosensory cortex; S2, secondary somatosensory cortex; VL, ventrolateral thalamic nucleus. Scale bar 5 1 mm in A–C, 20 mm in inserts. former carry both large and small terminals, and axons in the latter are with exclusively small boutons. Furthermore, contralateral S2, but not S1, was labeled with callosal-extending axons mainly in layer VI. The distribution of labeled cells and terminations within dorsal thalamus with BDA injected into S1 and S2 forepaw regions was summarized in Figure 6. Corticothalamic terminations originating from S1 forepaw neurons within VPL were mainly located in areas ranging from 2.3P to 3.6P, and the S2 axons terminated in the medial VPL ranging from 2.6P to 4.1P. Concerning the retrogradely labeled VPL cells, S1-projecting VPL cells were chiefly distributed from 2.3P to 3.6P, while most S2-projecting cells were located at more posterior level, from 3.1P to 4.1P. These data suggested that S1 and S2 share comparable thalamocortical linking pattern, even though the S1- and S2-projecting VPL cells were arranged in an antero–posterior manner. the existence of double-labeled VPL cells were examined in six rats. Among S1-projecting VPL cells (48–105 neurons per rat), 8.3 6 1.5% (mean 6 SEM) also project to S2, while 17.4 6 5.4% of S2-projecting cells (22–67 neurons per rat) were double-labeled with S1 projecting neurons. In total, 5.2 6 0.4% (23 of 443 neurons) of the labeled thalamocortical neurons projected to both S1 and S2. It is important to note that these retrogradely labeled cells, despite S1- or S2-projecting cells, were roughly distributed in either the central part or the ventral fringe of the VPL. One example—with RD injected into S1 and BDA injected into S2—is shown in Figure 7. Merged RD and BDA/ Cy2 staining views show neurons double-labeled inside the VPL region (arrows and arrowheads). Furthermore, the distribution of S1- and S2-projecting VPL cells along the antero–posterior axis were shown in Figure 7G. Most S1-projecting cells were located from 2.3P to 3.3P, while S2-projecting cells were mainly situated around 3.1P to 3.7P. Among these projecting VPL cells, few S1- and S2-dual projecting cells were distributed in the intermediate zone, from 3.2P to 3.4P. Although double-labeled cells represent a low proportion within the total labeled population, the presence of these cells demonstrate that dual projections from VPL cells to ipsilateral homotypical S1 and S2 regions do exist. Double Projection From VPL to S1 and S2 The above tracing results revealed that VPL neurons project directly to both S1 and S2. However, whether a single VPL neuron can provide collateral branches to both S1 and S2 was yet unknown. To explore this issue, retrograde tracers were injected into physiologically identified S1 and S2 forepaw areas in the same rat, and CONNECTIVITY OF RAT S2 967 Fig. 5. An example of BDA injected into S2 forepaw region. A: Photomicrograph of the injection site. B: BDA-labeled cells and corticocortical terminals in the ipsilateral S1. C: BDA-labeled corticothalamic termination in ipsilateral VPL. D: Coronal planes showing the distribution of the labeled cells and terminals in ipsilateral cortex and dorsal thalamus. Solid circles represent the retrogradely labeled cells, and small dots mark the labeled terminations. Inserts in B and C are enlarged view of axons and boutons. BDA, biotinylated dextran amine; S2, secondary somatosensory cortex; VA, ventroanterior thalamic nucleus. Scale bar 5 1 mm in A–C; 20 mm in inserts. Electrophysiological Comparison of S1 and S2 Thalamic Coactivation of S1 and S2 Stimulation-evoked multiunit responses in S1 and S2 were simultaneously recorded by electrical stimulation applied to contralateral forepaw (n 5 7). All recording sites in the S1 and S2 were histologically confirmed at locations in layer V or VI. An example of simultaneous recorded S1 and S2 evoked multiunit responses are shown in Figure 8A. The average initial latencies of the evoked multiunit responses in the S1 and S2 from the seven cases were 8.6 6 0.5 msec and 6.9 6 0.7 msec, respectively (Fig. 8B, P 5 0.045*, pair t-test). Furthermore, the average peak latencies were 12.0 6 1.1 msec and 11.9 6 0.9 msec (P 5 0.685), while the half-maximum durations were 7.1 6 0.9 msec and 8.7 6 0.9 msec (P 5 0. 091). Direct connections between VPL, S1, and S2 have been reported in mice, rats, cats, macaques, squirrel monkeys, and gray squirrels (Rowe and Sessle, 1968; Jones and Powell, 1969a; Manson, 1969; Jones, 1975; Spreafico et al., 1981; Fisher et al., 1983; Burton and Kopf, 1984; Carvell and Simons, 1987; Krubitzer and Kaas, 1987; Aldes, 1988; Stevens et al., 1993; Pierret et al., 2000). In the present study, both S1 and S2 were anterogradely labeled after BDA was injected into forepaw or hindpaw regions. In addition, VPL cells were labeled after retrograde tracers were deposited into either S1 or S2 forepaw fields, further suggesting the possibility of coactivation. Although both the S1- and S2-projecting VPL cells were found in the medial division of VPL (forepaw area), S2-projecting neurons were distributed at a more posterior level than S1-projecting cells, forming a mosaic pattern in the intermediate zone. The phenomenon suggested that VPL simultaneously activates S1 and S2 through segregative streams, even there are only a few double-labeled VPL cells (5%). Our results are in accordance with previous cat studies in that S1- and S2-projecting neurons were colocalized in the VPL, with a low percentage of the neurons providing bifurcate branches to S1 and S2 (Spreafico et al., 1981; Fisher et al., 1983; Burton and Kopf, 1984). However, the distribution of these projecting cells remained controversial among those investigations. DISCUSSION Anatomical and electrophysiological methods were used to investigate the flow of information processing to S2. Our major findings were as follows: (1) VPL neurons provided parallel projections to S1 and S2, (2) very low percentage of S1- and S2-dual projecting VPL cells were found, (3) S1 and S2 were reciprocally connected, and (4) S1 and S2 neurons responded to electrical stimulation of the forepaw with comparable latencies and response patterns. These results suggest that S1 and S2 participated in the processing of paw sensory information in parallel rather than serially. 968 LIAO AND YEN Figure 6. CONNECTIVITY OF RAT S2 Fisher et al. (1983) stated that S1- and S2-projecting cells coexisted within VPL, and 10% of them activated S1 and S2 by means of collateral axons. However, Spreafico et al. (1981) found that most S1-projecting cells were located in the VP core region, while S2-projecting neurons were found in the ventral fringe, the shell division, of the VPL. Among these projecting VPL cells, 13–20% had dual projections and were found only within the ventral shell region, where they were intermixed with VPL cells that projected solely to S2. Although no obvious demarcation of core and shell divisions has been reported in the rat, double-labeled cells in the present study were mainly positioned in two locations: one is in the central VPL, and the other is in the ventral border. This phenomenon may suggest the possibility of anatomical segmentation of the VPL in the rat. In this study, when the contralateral forepaw was stimulated, the average initial latency of evoked multiunit response of S2 neurons was significantly shorter than S1 neurons, but no significant difference existed between peak latencies and half-maximum duration of S1 and S2 responsiveness. To rationalize this result, we observed the path taken by the BDA-labeled thalamocortical axons stemming from the VPL to cortical areas. The extending fibers ascended through caudate putamen and then entered either S1 or S2 directly. The distance between VPL neurons and their S2 destination is shorter than the path taken by VPL-S1 axons. Our result is consistent with several previous studies (Woolsey and Wang, 1945; Heppelmann et al., 2001; Kwegyir-Afful and Keller, 2004), which showed that S1 and S2 neurons responded to peripheral stimulation with similar peak latencies in rabbits and rats. However, Brett-Green et al. (2003, 2004) and Benison et al. (2007) examined epipial-evoked potentials when stimulating a rat’s whisker and other body regions, and found a much longer latency of evoked response in S2 than in S1. The discrepancy may be due to variations in stimulation modality or recording methods. In the current study, electrical stimulation was applied to the forepaw, while studies by Brett-Green et al. (2003, 2004) and Benison et al. (2007) used mechanical stimulation. However, Kwegyir-Afful and Keller (2004) examined S1 and S2 responses evoked by whisker deflection, and found that S1 and S2 responded with similar peak latencies. In addition, we investigated the evoked S1 and S2 responses by multiunit recording, whereas Brett-Green et al. used epipial-evoked potential. Although the disagreement may result from different recording methods, Heppelmann et al. (2001) also examined S1 and S2 response latencies by measuring cortical surface potentials, and found similar electrical-evoked response latencies in S1 and S2. The effect of recording methods or stimulation modalities on measurements of relative latencies needs further investigation. Fig. 6. Summary distribution of labeled cells and terminations with BDA injected into S1 (n 5 4) and S2 (n 5 4) forepaw regions. The green color represents results from S1 injections and red color symbolizes the results from S2 injections. The solid circles indicate the distribution of retrogradely labeled cells and small dots mark the projecting terminations. Note that the labeled cells and terminals were observed within the medial VPL and POm after S1 and S2 injections. 969 S1 and S2 hierarchies have been studied by inactivation strategies in many species. Functionally, S2 seemed to be equivalent with S1, because it maintained its responsiveness when S1 was reversibly inactivated in rabbits, opossums, prosimian primates, tree shrews, marmoset monkeys, and rats (Woolsey and Wang, 1945; Garraghty et al., 1991; Murray et al., 1992; Coleman et al., 1999; Heppelmann et al., 2001; Zhang et al., 2001b). When the treatment was reversed in cat and marmoset monkey studies, S1 was similarly found to maintain its responsiveness after S2 inactivation (Turman et al., 1995; Zhang et al., 2001b). Somatic evoked responses of most neurons were unaffected, though a portion of neurons showed some reduction. The reduction may be induced by the reciprocal connections between S1 and S2 (Burton et al., 1990; Krubitzer and Kaas, 1990; Schwark et al., 1992; Barbaresi et al., 1995; Cauller et al., 1998; Karhu and Tesche, 1999; Zhang et al., 2001b). The investigations suggest that S1 and S2 occupy comparable position in the somatosensory information flow in most mammals, including some primates. Cortical Connection Between Somatosensory-Related Fields Reciprocal corticocortical connections between homotypical S1 and S2 regions have been well established (Burton, 1984; Alloway and Burton, 1985; Krubitzer and Kaas, 1990; Schwark et al., 1992; Catania and Kaas, 2001; Disbrow et al., 2003). Despite the mutual connection, S2 was argued to be superior to S1 in hierarchy, based on the anatomical relationship. In marmoset and cat, S1 projects to middle layers in S2, while S2 projects to superficial layers of S1 (Krubitzer and Kaas, 1990; Schwark et al., 1992; Burton et al., 1995). This projecting pattern has been established as hierarchical conveyance across cortical regions in the visual system, a serial processing model (Felleman and Van Essen, 1991). However, Barbaresi et al. used light and electron microscopy to study the laminar distribution of corticocortical projecting terminations between S1 and S2 in cats (Barbaresi et al., 1994, 1995). They stated that the origin and termination of the two corticocortical projections reciprocally connecting S1 and S2 in cats showed similar, although not identical, laminar organization: both corticocortical axons terminated mostly in the supragranular layers. In our study, a symmetrical laminar arrangement of reciprocal projections between S1 and S2 was found, with analogous anatomical organization of the horizontal connection between S1 and S2. No clear hierarchical relationship was seen between the S1 and S2 of the rat paw regions. In addition to S1 and S2, the parietal ventral (PV) has been known to be the third cortical region in processing somatosensory information (Jones and Powell, 1968; However, only terminals were found in Rt. BDA, biotinylated dextran amine; Ang, angular thalamic nucleus; eml, external medullary lamina; ml, medial lemniscus; Rt, reticular thalamic nucleus; S1, primary somatosensory cortex; S2, secondary somatosensory cortex; str, superior thalamic radiation; VM, ventromedial thalamic nucleus; ZId, zona incerta, dorsal part. 970 LIAO AND YEN Fig. 7. A representative example of labeled thalamic neuronal fibers and terminals with RD injected into S1 forepaw region and BDA injected into S2 forepaw region. A: RD-labeled, S1 projecting cells within the VPL. B: BDA/ Cy2-labeled, S2 projecting cells within the VPL. C: Merged photo showing the double-labeled cells. Note that the double-labeled cells were located within the central VPL (arrowheads), or the ventral fringe (arrows). D–F: Amplified RD-, BDA/ Cy2-labeled VPL cells and merged photo (arrowhead). G: Numbers of RD-labeled VPL cells (open bars) and BDA/ Cy2-labeled VPL cells (gray bars). The black bars represent the numbers of RD- and BDA/ Cy2 double-labeled VPL cells. BDA, biotinylated dextran amine; VPL, ventral posterolateral thalamic nucleus; RD, rhodamine dextran; Cy2, streptavidin-conjugated cyanine dyes; S1, primary somatosensory cortex; S2, secondary somatosensory cortex; P, posterior. Scale bar 5 200 mm in A–C, 30 mm in D–F. CONNECTIVITY OF RAT S2 971 Fig. 8. Electrical-evoked multiunit responses recorded in S1 and S2 when peripheral stimulation was applied at time 0 to contralateral forepaw (500 mA, 2 msec, 1 Hz, 2 min). A: An example of initial latencies of evoked response: 8 msec in S1 and 7 msec in S2. Peak latencies were 14 msec in S1 and 11 msec in S2. Half-maximum durations were 11 msec in S1 and 9 msec in S2. B: Comparison of evoked mul- tiunit responses in S1 and S2 (n 5 7). The average initial latencies were 8.6 6 0.5 and 6.9 6 0.7 msec, the peak latencies were 12.0 6 1.1 and 11.9 6 0.9 msec, and the half-maximum durations were 7.1 6 0.9 and 8.7 6 0.9 msec in S1 and S2. *, P < 0.05; n.s., no significant difference. S1, primary somatosensory cortex; S2, secondary somatosensory cortex. Burton and Kopf, 1984; Koralek et al., 1990; Krubitzer and Kaas, 1990; Li et al., 1990; Fabri and Burton, 1991; Krubitzer et al., 1995; Disbrow et al., 2000; Qi et al., 2002; Disbrow et al., 2003). The PV was reported to link reciprocally with ipsilateral VPL, S1, and S2 (Krubitzer and Kaas, 1990; Disbrow et al., 2000; Qi et al., 2002; Disbrow et al., 2003). A similar organizational arrangement occurred in rats (Koralek et al., 1990; Li et al., 1990; Fabri and Burton, 1991; Brett-Green et al., 2004; Benison et al., 2007). In the rat, PV was described as a complete, mirrorsymmetric area of the body surface, with the limb representations joining neighboring S2 (Fabri and Burton, 1991; Remple et al., 2003). Therefore, the relative organization of PV is difficult to identify by the present neuroanatomical method due to the adjacent arrangement of S2 and PV. However, Benison et al. (2007) recently mapped the rat somatosensory area using epipial potential recording, and claimed that the S2 field is somatotopically organized, with the body area located caudal to the S1 barrel fields. Forepaw is represented in S2 at 3.5 mm posterior and 6 mm lateral to bregma, and in PV (lateral to S1 barrel field) at 1.5 mm posterior and 9 mm lateral to bregma. This arrangement is inconsistent with the previous view that S2 is an upright rattunculus positioned just lateral to S1 (Chapin and Lin, 1984; Carvell and Simons, 1987; Fabri and Burton, 1991; Remple et al., 2003). In our study, three locations were labeled after anterograde tracers were injected into VPL forepaw area. One was located in the traditionally defined S1 forepaw area, another at 1.5–2.0 mm posterior and 9 mm lateral, identified as S2 in the current and previous studies (Carvell and Simons, 1987; Fabri and Burton, 1991; Remple et al., 2003), and the third at 3.5 mm posterior and 7 mm lateral. When compared with the arrangement reported by Benison et al. (2007), our ‘‘S2 forepaw area’’ is located in their ‘‘PV forepaw area,’’ while the caudal labeling area corresponds to their ‘‘S2 region’’. Nevertheless, we found that the den- sity of labeled thalamocortical terminations in the rostral ‘‘S2’’ is much higher than the caudal ‘‘S2’’, therefore, it might be reasonable to recognize the rostral one as ‘‘S2 forepaw area,’’ despite that the labeled region may be composed of S2 and PV as previously described. In addition to the thalamocortical terminations, the caudallabeled region also received corticocortical inputs from S1 and S2 neurons. The results suggest that the area may also be involved in processing somatosensory information, although the definite role remains to be proven. Hierarchical View of Somatosensory Flow Observations favoring serial hierarchical processing came from a sparse projection from VPL to S2, and a dense projection to S1 in macaques (Kaas, 1983; Manzoni et al., 1984; Friedman and Murray, 1986; Krubitzer and Kaas, 1990), coupled with observations of thick corticocortical projections from S1 to S2 in many mammals (Akers and Killackey, 1978; Kunzle, 1978; Wise and Jones, 1978; Friedman et al., 1980; Jones and Friedman, 1982; Jones, 1983; Manzoni et al., 1990; Fabri and Burton, 1991; Burton et al., 1995). The assumption was strengthened by ablation experiments in macaques, which demonstrated that the removal of the posterior parietal cortex (area 3a, 3b, 1, and 2) eliminated responsiveness in the S2 representation, although there was no detectable effect on S1 responsiveness when the operation was reversed (Pons et al., 1987; Burton et al., 1990). However, these results conflicted with the inactivation findings described above (Woolsey and Wang, 1945; Garraghty et al., 1991; Murray et al., 1992; Coleman et al., 1999; Heppelmann et al., 2001; Zhang et al., 2001b), perhaps because the latter studies used reversible cooling instead of irreversible aspiration, or it could be due to the differences between species. 972 LIAO AND YEN A recently developed evolutionary hypothesis suggests that a prominent change of somatosensory organization occurred when the Old World macaques diverged from other primates along the evolution path (Kaas, 2004). In the higher-primates, S2 lost the direct thalamic afferents and depend on the corticocortical inputs from ipsilateral S1 for activation. Summing the various aforementioned studies, results favoring serial transmission were performed mostly on macaque and rhesus monkeys (Pons et al., 1987, 1988, 1992; Burton et al., 1990), while investigations supporting the parallel view used nonprimitive primates and other mammals, including rats, cats, marmoset monkeys, tree shrews, and opossums (Spreafico et al., 1981; Fisher et al., 1983; Murray et al., 1992; Turman et al., 1995; Coleman et al., 1999; Heppelmann et al., 2001; Zhang et al., 2001a,b; Kwegyir-Afful and Keller, 2004). Our results from rats further support the distinction between serial somatosensory processing in higher primates and parallel somatosensory processing in other mammals. 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