Topography and Architecture of Visual and
Somatosensory Areas of the Agouti
I.A. Dias,1C.P. Bahia,1J.G. Franca,2J.C. Houzel,3R. Lent,4A.O. Mayer,2L.F. Santiago,5L.C.L. Silveira,6 C.W. Picanc¸o-Diniz,5and Antonio Pereira7*
1
Laboratory of Neuroplasticity, Institute of Health Sciences, Universidade Federal do Para, 66075-110, Belem (PA) Brazil
2
Programa de Neurobiologia, Instituto de Biofısica Carlos Chagas Filho, Universidade Federal do Rio do Janeiro, 21941-902, Rio de Janeiro (RJ), Brazil
3
Laboratory of Neuroscience Frontiers, Institute of Biomedical Sciences, Universidade Federal do Rio de Janeiro, 21941-902 Rio de Janeiro (RJ), Brazil
4
Laboratory of Neuroplasticity, Institute of Biomedical Sciences, Universidade Federal do Rio de Janeiro, 21949-900, Rio de Janeiro (RJ), Brazil
5
Laboratory of Investigations in Neurodegeneration and Infection, Institute of Biological Sciences, Universidade Federal do Para, 66075-110 Belem (PA), Brazil
6
Eduardo Oswaldo-Cruz Laboratory of Neurophysiology, Institute of Biological Sciences, Universidade Federal do Para, 66075-110 Belem (PA), Brazil
7
Brain Institute, Universidade Federal do Rio Grande do Norte, 59056-450, Natal (RN) Brazil
ABSTRACT
We analyzed the organization of the somatosensory and visual cortices of the agouti, a diurnal rodent with a rel-atively big brain, using a combination of multiunit microelectrode recordings and histological techniques including myelin and cytochrome oxidase staining. We found multiple representations of the sensory periphery in the parietal, temporal, and occipital lobes. While the agouti’s primary (V1) and secondary visual areas seemed to lack any obvious modular arrangement, such
as blobs or stripes, which are found in some primates and carnivores, the primary somatosensory area (S1) was internally subdivided in discrete regions, isomorphi-cally associated with peripheral structures. Our results confirm and extend previous reports on this species, and provide additional data to understand how variations in lifestyle can influence brain organization in rodents. J. Comp. Neurol. 522:2576–2593, 2014.
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INDEXING TERMS: rodents; somatosensory cortex; visual cortex; cortical evolution; comparative neuroanatomy
Since the first electrophysiological mapping studies of the cerebral cortex, multiple representations of the sensory periphery have been demonstrated in visual and somatosensory areas of every mammal, including those with the smallest brains (Krubitzer et al., 1986; Beck et al., 1996; Slutsky et al., 2000; Rocha et al., 2007; Santiago et al., 2007; Naumann et al., 2012). It has been proposed that the increase in the number of cortical fields is an evolutionary trend towards greater brain complexity (Krubitzer et al., 1986; Beck et al., 1996; Slutsky et al., 2000; Rocha et al., 2007; San-tiago et al., 2007). In primates, for instance, some sensory modalities such as vision and audition pos-sess tens of cortical areas, most performing mid-level sensory processing (Krubitzer et al., 1986; Beck et al., 1996; Slutsky et al., 2000; Rocha et al., 2007; San-tiago et al., 2007).
Since nervous tissue does not fossilize, there are cur-rently two major options for studying mammalian brain evolution: the evo-devo program, which focuses on the molecular processes responsible for brain segmentation during development as the targets of evolutionary pres-sure, and the comparative analysis of brain organization in extant mammals (Krubitzer et al., 1986; Beck et al.,
Grant sponsor: CAPES/PROCAD; Grant number: 0024/01–5, CNPq/ PROSET-MCT 350106/2007-0, CNPq 306722/2009-7, CNPq (476627/ 2011-7), CNPq (474933/2012-1), CNPq (311253/2009-1), CNPq (474933/2012-1), FAPERJ (E-26/111.721/2012), CNPq (483404/2013-6).
*CORRESPONDENCE TO: Antonio Pereira, Brain Institute, Universidade Federal do Rio Grande do Norte, 59056-450 Natal (RN), Brazil.
E-mail: [email protected]
Received March 18, 2013; Revised January 17, 2014; Accepted January 21, 2014.
DOI 10.1002/cne.23550
Published online January 29, 2014 in Wiley Online Library (wileyonlinelibrary.com)
VC2014 Wiley Periodicals, Inc.
1996; Slutsky et al., 2000; Rocha et al., 2007; Santiago et al., 2007). The latter approach has been very suc-cessful in producing valuable insights about brain evolu-tion. Its usefulness, however, depends on the right choice of brains from animals with different body plans and adapted to different environments (Krubitzer et al., 1986; Beck et al., 1996; Slutsky et al., 2000; Rocha et al., 2007; Santiago et al., 2007).
Rodents, the largest mammalian order, with more than 2,000 species inhabiting different ecosystems around the planet, are an ideal model group for the study of brain evolution. Detailed cortical maps have been produced for both the visual and somatosensory cortices of several rodents (Krubitzer et al., 1986; Beck et al., 1996; Slutsky et al., 2000; Rocha et al., 2007; Santiago et al., 2007), even though there has been much disagreement about the specifics of the proposed anatomical and functional parcellation schemes. For instance, there are discrepant interpretations concern-ing the number of extrastriate visual areas in the mouse brain (Krubitzer et al., 1986; Beck et al., 1996; Slutsky et al., 2000; Rocha et al., 2007; Santiago et al., 2007).
However, different research groups seem to agree that most mammals share five somatosensory areas in the parietal lobe, including a primary somatosensory area (S1), a secondary somatosensory area (S2) and a parietal ventral area (PV) located immediately rostral to S2, plus two narrow bands of somatosensory cortex along the rostral and caudal borders of S1 (Krubitzer et al., 1986; Beck et al., 1996; Slutsky et al., 2000; Rocha et al., 2007; Santiago et al., 2007). Each of these somatosensory fields contains a complete repre-sentation of the animal body and their boundaries can be visualized with different histological methods, espe-cially those revealing myelin staining and cytochrome-oxidase (CO) activity (Land and Simons, 1985; Krubitzer et al., 1986; Elston and Manger, 1999; Slutsky et al.,
2000; Elston et al., 2006; Campi et al., 2007; Rocha et al., 2007; Anomal et al., 2011). In some of these areas, especially in S1, an internal modular organization composed of peripheral isomorphs can also be revealed with those techniques. The posteromedial barrel sub-field (PMBSF) in S1 of both rats and mice is one of the best known examples of this organization. In this region, each cortical barrel receives primary thalamic afferents related to a single whisker on the snout (e.g., Woolsey and Van der Loos, 1970; Lent, 1982; Dawson and Killackey, 1987; Wallace, 1987). Other examples include isomorphs of hand digits in primate area 3b (Jain et al., 1998) and the nasal appendices of the star-nosed mole in S1 area (Catania and Kaas, 1995).
The agouti is a mid-sized rodent which is prevalent in the Amazon region and possesses a, mid-sized brain (Fig. 1A,B) weighing 18 g on average, with over 800 mil-lion neurons, 15% of which reside in the cerebral cortex (Catania and Kaas, 1995; Herculano-Houzel et al., 2006). The agouti’s lissencephalic brain allows detailed mapping of cortical areas and also facilitates compari-son between electrophysiological and histochemical maps (Rocha et al., 2007). Previous descriptions of the somatosensory cortex of the agouti revealed a number of anatomically and functionally distinct areas (Pimen-tel-Souza et al., 1980; Rocha et al., 2007; Santiago et al., 2007). The localization and organization of the agouti areas S1 and S2 are similar to those described for other rodents (Campos and Welker, 1976; Krubitzer et al., 1986; Remple et al., 2003). As for the visual cor-tex, a study of its cyto- and myeloarchitecture, associ-ated with microelectrode mapping, revealed five subdivisions corresponding to distinctive visuotopic rep-resentations (Picanco-Diniz et al., 1989, 1991).
In the present work we expand on those earlier stud-ies and use both morphological and microelectrode techniques in an attempt to better describe the visual and somatosensory areas of the agouti’s brain and establish this mid-size rodent as a suitable model for the comparative analysis of brain evolution.
MATERIALS AND METHODS
Multiunit recording maps
Eight adult agoutis (Dasyprocta prymnolopha) weigh-ing 1.5–3.0 kg were separated into two groups of four subjects for the visual and somatosensory studies. All experimental procedures were approved by the Ethics Committee on Experimental Animal Research of the Fed-eral University of Para. Animals were donated by the Emilio Goeldi Zoo-Botanic Museum, under license of the Brazilian Institute of the Environment and Renewable Natural Resources (IBAMA, 207419-0030/2003) and
Abbreviations
A Auditory primary area AL Anterolateral area D Digit F Face FL Forelimb HL Hindlimb LI Lower incisor LL Lower lip LS Lateral sulcus PT Posterior temporal area S1 Primary somatosensory area S2 Secondary somatosensory area
T Trunk
UP Up lip
V Vibrissae
V1 Primary visual area V2 Secondary visual area V3 Tertiary visual area
VS Visual
maintained in the central animal facility of the Federal University of Para. Procedures for electrophysiological mapping of the visual cortex have been described in detail elsewhere (Picanco-Diniz et al., 1991). Briefly, 1 day before the recording session, animals were treated with dexamethasone (Decadron, Prodome, Brazil; 1.0 mg/kg, i.m.) to prevent brain edema, and vitamin K (Kanakion, Roche, Nutley, NJ; 1.0 mg/kg, i.m.) to avoid excessive bleeding. On the next day, animals were deeply anesthetized with an intramuscular injection of a mixture of xylazine (Rompun, Bayer, Elkhart, IN; 1.0 mg/kg, i.m.) and ketamine chloridrate (Ketalar, Parke-Davis, Ann Arbor, MI; 10 mg/kg, i.m.). The electrocar-diogram was recorded throughout the experiment, and additional doses of the anesthetics were administered if necessary. Body temperature was kept around 37C with a thermal blanket.
The animal was positioned in a stereotaxic apparatus and a wide craniotomy was performed. The dura mater was reflected and the exposed cortical surface was pro-tected with warm mineral oil. For the somatosensory mapping experiments, we used a standard headholder with ear bars; mechanical stimulation was performed by gently touching or scratching both the animal’s skin and hair with the help of wooden sticks or brushes, and multiunit somatosensory receptive fields were drawn on
a schematic representations of the animal’s body. For visual mapping experiments, animals were secured in a headholder especially designed to minimize obstruction of the visual field in animals with lateral eyes such as the agouti (Silva-Filho et al., 1991). The eyes were aligned with reference to an occulocentric equatorial azimuthal coordinate system implemented on a transparent plastic hemisphere. In this system, the vertical meridian roughly corresponds to the nasotemporal retinal decussation and the horizontal meridian corresponds to the visual streak (Gallyas, 1979; Silveira et al., 1989). Cycloplegia and mydriasis were obtained by administration of 1% atropine sulfate to the eyes. The cornea was protected with a thin layer of silicone fluid (Dow Corning, Midland, MI; 200/350). To prevent eye movements, muscle paralysis was induced with trimethyl-gallamine (Flaxedil, Rhodia, Spain; diluted 1:3 in 0.9% saline) while artificial ventila-tion was provided through an endotracheal cannula. The projection of the optic disk onto the visual field was regularly checked throughout the experiment with a reversible ophthalmoscope. For both somatosensory and visual experiments, the activity of small neuronal clusters was recorded with varnish-insulated tungsten microelectrodes (impedance 1; at 1 kHz, FHC, Bowdoin-ham, ME) held by a micromanipulator. The multiunit
Figure 1. The brain of the agouti (Dasyprocta prymnolopha). A,B: Photomicrographs showing the dorsal and lateral aspects of the brain, respectively. C: The agouti is shown manipulating food. Notice that the animal sits on its hindlimbs while uses the forelimbs to manipulate and bring food to the mouth.
signal was differentially amplified, bandpass-filtered between 1 and 3 kHz (ME04011, FHC) and fed to a dual-beam storage oscilloscope (1476A, BK Precision, Yorba Linda, CA) and an audio monitor (SR771, Sansui, Japan). At the end of the experiment, several electro-lytic lesions were performed at select cortical sites to allow the reconstruction of topographic maps.
Perfusion and histology
At the end of the mapping experiments the animals were perfused through the aorta with warm 0.9% saline followed by aldehyde fixatives (4% paraformaldehyde in 0.1 M, pH 7.4 phosphate buffer or 10% formaldehyde-saline solution). The brains were removed from the skull and processed for histological analysis. Alternate 50-lm-thick parassagital sections were stained for Nissl substance (with cresyl violet) or myelin (Gallyas, 1979). Alternatively, the cortical sheet was separated from subcortical structures, flattened overnight between two glass slides, and cut tangentially in 50 lm sections with a vibratome. Alternate sections were then proc-essed for myelin and cytochrome-oxidase histochemis-try (Wong-Riley and Welt, 1980). In one case, both hemispheres were cut parasagittaly (50-lm-thick) and alternate sections were stained with cresyl violet, mye-lin (Heindenhain method, after Hutchins and Weber, 1983), or immunostained for neurofilaments (SMI-32 monoclonal antibodies) (Sternberger and Sternberger, 1983; Campbell and Morrison, 1989; Hof and Morrison, 1995).
For immunohistochemistry against SM1–32, free-floating sections were initially washed three times in phosphate-buffered saline (PBS) 0.1 M for 10 minutes, and subsequently incubated with 2% bovine serum albu-min (BSA) in a solution of 0.3% Triton X-100 in PBS (PBS-Tx), for 1 hour at room temperature. After three rinses in PBS, sections were gently and continuously shaken overnight at room temperature in a solution of mouse monoclonal SMI-32 antibody (Covance, Denver, PA; 1:5,000) in 2% BSA diluted in 0.3% PBS-Tx. The sec-tions were then washed three times in PBS, incubated in biotinylated secondary horse antimouse antibody (1:200, Vector Laboratories, Burlingame, CA) for 2 hours at room temperature, washed again (3 3 10 minutes in PBS), and incubated for 1 hour in the Vec-tastain ABC Standard System (1:500 Vector Laborato-ries) at room temperature. Immunoreactivity was revealed with 0.05% 3,30-diaminobenzidine (DAB) and 0.1% nickel ammonium sulfate. Sections were then mounted on bi-gelatinized slides, dehydrated in increas-ing alcohol concentrations (75–90–100–100%, 1 minute each), defatted with xylene (2 3 3 minutes) and cover-slipped with DPX.
Data analysis
Location of recording sites was reconstructed from coronal sections using microelectrode tracts and elec-trolytic lesions as landmarks, and projecting those onto a representation of the cortical surface (see an example in Fig. 3). We first projected an enlarged view of each coronal section on a flat surface using a photographic enlarger. Then we drew the fundus of the lateral sulcus as a reference point for the measurements. This region corresponds to the medial border of area 17, as previ-ously described (Picanco-Diniz et al., 1991). A digitizing tablet was then used to measure the lateral distance of each penetration in relation to the fundus of the lateral sulcus, generating a flat reconstruction of the recording sites. In flattened brains, the cortical surface was reconstructed by superimposing different sections of the same or different subjects using various anatomical landmarks. Since tangential sections of CO and myelin stains originated from different subjects, we used the lateral sulcus and the contour of histological sections as fidutiary marks for alignment with the flat recon-structions of the recording sites. We did not perform any correction for tissue shrinkage in the present work.
For the anatomical analysis, photomicrographs of parasagittal sections processed for myelin, cresyl violet, or immunoreacted for SMI-32 were obtained using a Zeiss Axioplan microscope with an X/Y motorized stage and a CCD camera connected to a computer running the Neurolucida software (MBF Bioscience, Williston, VT). Neurolucida has an application called Virtual Tissue (MBF Bioscience) that scans the sections, and then pro-vide a reconstructed unified image of the entire section. Before image acquisition, color balance was adjusted to eliminate background from the glass slides. Images of the whole section were collected using a 53 (Zeiss Plan-Neofluoar) objective. To minimize background noise and improve visualization of anatomical features, brightness and contrast were corrected in the image as a whole.
RESULTS
Architectonic organization of agouti visual
and somatosensory cortical areas
Analysis of tangential sections through cortical layer IV revealed at least three fields that consistently stood out in contrast due to intense CO reactivity and myelin stain (Fig. 2). Two of these fields were electrophysiolog-ically characterized as primary somatosensory (S1) and primary visual (V1) areas (see below). We identified the third field as auditory cortex (A), because its location was similar to that of primary auditory area (A1) in other rodents (Pimentel-Souza et al., 1980; Rocha
et al., 2007; Santiago et al., 2007) and because some auditory responses were recorded next to this region (see below). In the lateral and posterior occipital cortex, a fourth field was also characterized by strong CO reac-tivity, but myelin stain was comparatively less intense in this region than in the primary sensory areas described above (Fig. 2A,B). Our mappings suggested that visual areas V3 and the temporal posterior area (TP) are usually found in this region (see below).
Immunohistochemical processing for SMI-32 revealed a clear areal parcellation in the cortex due to different patterns of laminar immunostaining in each cortical region, corroborating what was observed with CO, Nissl, and myelin stains (Fig. 3). In all cortical areas, SMI-32 labeling was more intense in layers 3, 5, and 6. In some cortical areas like V1 and V2, neuropil
immunore-activity for SMI-32 in layers 5 and 6 was more homoge-neous, conferring a "bilaminar" labeling appearance to these areas. In other areas such as S1, V3, and TP, layer 6 was subdivided into a superficial region (sub-layer 6a) that was less immunoreactive for SMI-32 than deeper sublayers (6b), thus conferring a "trilaminar" appearance to those cortical areas given the intense staining of layers 3, 5, and 6b.
Located at the occipital pole, area V1 lay caudal to the somatosensory cortex, medial to area V2, and lat-eral to the cingulate cortex, from which it is separated by the lateral sulcus, and extends caudally to the tento-rial surface of the brain, where it reaches the retrosple-nial agranular cortex (Picanco-Diniz et al., 1989). In tangential sections, area V1 showed denser myelin stain and more intense CO reactivity than other visual
Figure 2. Photomicrographs of tangential sections of the agouti’s cerebral cortex stained for cytochrome oxidase (two cases, A and C,D), and for myelin (one case, B), showing the main architectonic subdivisions of the cortex. Notice the heterogeneous CO labeling pattern across the section, closely associated with borders of sensory areas in A, and the dense myelination of primary areas in B. D is an enlarged view of the rectangular area in C. Notice that the pattern of CO activity reveals subdivisions within S1, corresponding to different parts of the body surface. Arrow in A,B points to the lateral sulcus. Scale bar5 5 mm.
areas, especially at its medial border (Fig. 2A,B). Area V1 also stood out in parasagittal sections, displaying two thick bands of myelin corresponding to the deeper portion of layer 3 and layers 4 and 6 that were intensely stained but separated by the less myelinated layer 5. Additionally, supragranular layers in V1 tended to be more intensely stained when compared to other
areas (Fig. 4). At higher magnification, radially oriented myelinated fibers ascending from the white matter to layer IV could also be observed (Fig. 4E). Fibers running horizontally could also be observed, especially in infra-granular layers (Fig. 4E). Both in tangential and parasag-ittal preparations, the anterior half of area V1 was more myelinated than the posterior half. Such difference in
Figure 3.Architectonic organization of primary somatosensory area and visual areas of the agouti brain immunostained for SMI-32. A–I: Adjacent parasagittal sections of the left hemisphere, positioned from medial to lateral, with A being the most medial. Arrows indicates cortical boundaries. The level o section is indicated on a schematic drawing of a dorsal view of the left hemisphere (top right). Black boxes in the sections indicate the location of higher-magnification photomicrographs of (J, K) areas SI, and V1 of section C; (L, M) areas AL and V2 of section G; and (N, O) areas V3 and TP of section H.
Figure 4.Architectonic organization of primary somatosensory and visual areas revealed by Nissl and myelin staining. A–C: Representative myelin-stained parasagittal sections of the left hemisphere, positioned from medial to lateral, with A being the most medial. These sections are adjacent to sections C, G,H. The level of the section is indicated on schematic drawing of a dorsal view of the left hemisphere (top right). Black boxes in the sections indicate the location of higher-magnification photomicrographs of (E,G,I) areas S1 and V1, of section A; (I, K) areas V2 and AL of section B; and (M,O) areas V3 and TP of section C. High-magnification photomicrographs (D, F, H, J, L, N) of these cortical areas in adjacent Nissl-stained sections.
myeloarchitecture were symultaneous to differences in SMI-32 distribution, suggesting that V1 architecture is not uniform along its anterior to posterior extent. In more anterior portions, SMI-32-positive neuropil was more intense in all cortical layers than in the posterior half of V1, where layers 1, 2, and 4 were less reactive, similar to what was observed in other cortical areas (Fig. 4B–D). Additionally, area V1 presented an intense SMI-32 immunoreactive labelling of pyramidal cells in layers 3, 5, and 6. Many medium-sized pyramidal neu-rons were stained in layer 3, whereas in layer 5 SMI-32-positive pyramidal neurons were larger but less numerous. In layer 6 pyramidal neurons were small and even less numerous than in layers 3 and 5. In area V1, many SMI-32-reactive apical dendrites of pyramidal neurons from layers 5 were seen traversing to layer 4.
Area V2 was characterized by homogeneously weak CO reactivity when compared to V1 medially and to other visual areas laterally (Fig. 2A). Additionally, V2 was less myelinated, especially when supragranular layers were compared with those in V1. The general pattern of SMI-32 staining in V2 was similar to that in V1, but not identical. Compared with V1, neuropil in supragranular layers was less stained; layer 5 SMI-32-positive pyramidal neurons were smaller; and in layer 6 SMI-32 neuropil was evident, but neuronal cell bodies were not labeled. Vertically oriented SMI-32 labeled dendrites in layer 5 and 6 were not as apparent as they were in area V1.
Lateral to area V2, we identified two additional vis-ual areas using both electrophysiological (next sec-tion) and architectonic criteria (see also Kaas et al., 1972; Picanco-Diniz et al., 1989). Both area V3 and the anterior lateral visual area (AL) shared the lateral border of V2 and were identified by reversions in the progression of receptive fields (see below) that was also coincident with changes in architectonic features. V3 was labeled slightly more intensely for CO and myelin than area V2, especially at its caudal third (Fig. 2A,B). In parasagittal sections immunostained for SMI-32, V3 and AL presented very distinct features both from each other and from V2. Compared with V2, AL had less intense neuropil labeling, especially in layers 4 and 6; thinner layers 3 and 6; and higher density of labeled neurons in layer 5. In the SMI-32 preparation, V3 could be easily identified by a clear transition at the caudal border of area V2. V3 was characterized by the weak labelling associated with labeling in layers 3 and 6 and no labelling in layers 2, 4, and 5. Small and weakly labeled neurons were found in layer 5. In layer 3, labeled cell bodies were arranged in a more compact fashion than in V2. In parasagittal sections stained for myelin, V3 was
asso-ciated with intense labelling of horizontal fibers in layer 6 and very weak staining in layer 4 and 5.
Lateral to V3, TP (Kaas et al., 1989) had an interme-diate level of CO activity (albeit more intense than in V3), particularly in its caudal pole (Fig. 2A,B). Myelin intensity in TP tended to be higher than in V3 (Fig. 4I). In SMI-32 preparations, TP displayed large but weakly stained neurons in layers 5 and 6, with apical dendrites that extended upwards, giving a striped appearance to this area, especially when compared with neighboring V3. Additionally, in spite of a clear (but weak) neuropil label, no SMI-32-positive neurons were found in layer 3. Anterior to the visual cortical areas of the occipital lobe and posterior to the somatosensory cortex, we found a strip of cortical tissue characterized by a reduced level of CO activity, myelination, and SMI-32 immunoreactivity. This field extended in a lateral and posterior direction from the anterior tip of the lateral sulcus, separating the auditory cortex (A) from visual areas V3 and TP. This region might be homologous to medial parietal (Pm), lateral parietal (Pl), and intermedi-ate temporal (Ti) areas which were previously described in the squirrel (Kaas et al., 1989). Our recordings indi-cated that this transitional region was responsive to dif-ferent sensory modalities. In the region separating the anterior pole of areas V1 and V2 from S1 (putative area Pm) we recorded neurons responsive to both visual and somatosensory stimulation (see Santiago et al., 2010). Additionally, in the more caudal and posterior parts of this field (putative area Ti), visual and auditory responses were also simultaneously elicited in some sites (see below).
In the parietal cortex, area S1 was characterized by both high CO reactivity and dark myelin staining. In tan-gential sections, regions that were both intensely myelinated and strongly reactive for CO were separated by lightly stained septa (Figs. 2, 9). One of those septa corresponded to an anterior and lateral extension of the anterior tip of the lateral sulcus. Another more medial septum merged posteriorly with the anterior tip of the lateral sulcus, as seen in some sections. In para-sagittal sections, architectonic features revealed by both Nissl and myelin staining were typical of primary sensory areas and similar to area V1 (see above). With Nissl staining, S1 presented a well-defined layer 2 with compact medium-sized cell bodies. Layer 3 was very thick, with medium-sized cell bodies that were less densely packed than in layer 2. Layer 4 displayed neu-rons with a typical granular appearance. Layer 5 had large neuronal cell bodies, most of them pyramidal-shaped. Layer 6 was characterized by small neurons that were less packed than those found in layer 4. From layer 6 to layer 4, bodies are arranged in cellular
columns with the same orientation as the myelinated axons identified in myelin preparations. Myelinated fibers were oriented radially from layers 4 to 6. Myelin was more concentrated in layer 6, where horizontally oriented fibers could also be observed.
In SMI-32 immunoreacted sections, S1’s had a layer 5 had the largest pyramidal neurons. These layer 5 neu-rons possessed apical dendrites that extended through layer 4 and 3. Layers 1, 2, and 4 were characterized by the absence of labeled neurons and weak neuropil reac-tivity. Layer 3 displayed intense neuropil reactivity, with small and densely compacted SMI-32 reactive neurons.
Somatosensory cortex lateral to S1 showed more variable levels of staining both for CO and myelin, usu-ally weaker than in S1. In this region, multiunit record-ings confirmed the previous finding of at least one additional somatosensory field, called area S2 (Pimen-tel-Souza et al., 1980). In tangential sections stained either for myelin or CO, S2 did not have sharply defined borders like S1. A more detailed description of the agouti lateral somatosensory cortex can be found else-where (Santiago et al., 2010).
The lateral somatosensory cortex was separated from the temporal auditory cortex by a region of weak CO and myelin staining. In the temporal region, the auditory cortex ("A" in Fig. 2A,B) had strong CO reactivity and intense myelin staining. Cortical regions moderately stained for both myelin and CO surround the auditory
cortex at its caudal, rostral, and medial edges (Fig. 2). As mentioned above, we also recorded multimodal responses in this region.
Visuotopic organization of the agouti
occipital cortex
In the occipital lobe, the different compartments revealed by both cytochrome oxidase and myelin stain (last section, Fig. 2) corresponded to different represen-tations of the contralateral visual field. Figure 5A illus-trates the results of one mapping experiment showing the correspondence between the distribution of multiu-nit recording sites and the location of respective recep-tive fields. Projection of the recording sites onto a tangential section reacted for CO activity is illustrated in Figure 5B. When the microelectrode was positioned in different sites along the cortical surface, the location of multiunit receptive fields moved continuously on the visual field.
As previously described by (Picanco-Diniz et al., 1991), a precise point-to-point representation of the contralateral hemifield was the hallmark of V1 record-ings (Fig. 5). The caudalmost recording sites in V1 (sites #1–5) clearly demonstrate a regular visuotopic progression towards the vertical meridian located at approximately the same elevation. The lateral border of V1 represented the nasal edge of the visual field (i.e., the vertical meridian; sites #4–5, 14–15, 30–31, 42,
Figure 5.Topographic reconstruction of microelectrode penetrations in the visual cortex. A: Localization of multiunit receptive field centers across the contralateral visual hemifield. B: Histological reconstruction of the sequence of microelectrode penetrations shown in A, super-imposed on the tangential pattern of CO activity through layer IV. Abbreviations as in Figure 2.
52–53). The rostral portion of V1 represented the lower visual field (sites #31–31, 42, 52–53) while the caudal region represented the upper visual field (sites #1–5, 14–15).
Area V2 also had a topographically organized repre-sentation of the visual field that mirrored that observed in V1. The vertical meridian corresponded to the V1/ V2 border. Similar to V1, the anterior part of V2 repre-sented the inferior visual field (sites #43–46, 54–56), and the caudalmost part of V2 corresponded to the superior visual field (sites #6–9, 16–19). The two halves were separated from each other by the repre-sentation of the horizontal meridian (sites #31–38) that crossed V2 from the medial to lateral border. Record-ing sequences from the V1/V2 border (i.e., the vertical meridian representation) towards the CO/myelin-defined lateral border of V2 revealed a progression of multiunit receptive fields towards the temporal edge of the visual field (sites #6–9, 15–19, 32–38, 43–47). Also, in the caudal part of V2, medial-to-lateral sequen-ces corresponded to receptive field progressions from upper visual field towards the horizontal meridian (sites #7–9, 17–19).
Similar to the progression of V1 receptive fields towards the lateral margin of V2, two particularities in
the location of multiunit receptive fields could be observed in V2: 1) In the caudal part of V2 there was a reversion in the progression of receptive fields which turned away from the temporal-most part of the visual field (in V2), and went back to the vertical meridian, thus indicating a transition into V3 (sites #8–11, 18– 21); 2) In the rostral part of V2, receptive fields moved towards the lowest part of the inferior visual field, sug-gesting proximity to the anterolateral area (AL), where this portion of the visual field is represented (sites #43–49, 54–57). Also, a reversion in receptive field progression could be observed after crossing the V2/ AL border (sites #55–58).
Visual field representation in area V3 appeared to be mirror-reversed in relation to that in neighboring V2. The V2/V3 border represented the temporal visual field (sites #9–10, 19–20, 38–40). Additionally, the lateral margin of V3 seemed to house another representation of the vertical meridian (sites #13 and 24). Most of area V3 space represented the superior visual field. Only sites #22–24 had receptive fields located below the horizontal meridian. But even those did not cross the215 elevation line in the inferior visual field.
Finally, recording sequences crossing the architec-tonically defined lateral margin of V3 revealed receptive
Figure 6.Point-image size (PIS) values across the visual streak (68 meridian) (A) and along the visual streak (0 latitude) (B). C,D: Multiu-nit receptive field areas and PIS values along the visual streak in V2, respectively.
fields moving away from the vertical meridian towards the temporal periphery, as would be expected when another visual area is reached (area TP, sites #23–27).
In the region located between visual and auditory cortices we obtained bimodal (auditory and visual) responses (penetration #50), whereas purely auditory responses were also recorded in the auditory cortex (penetration #51). This suggests that a multimodal association cortical region lies between higher-order vis-ual cortical areas (AL, V3, and TP) and the auditory cor-tex (Fig. 5).
Point-image size in V1 and V2 cortical areas
Point-image size (PIS) is defined as the product of the magnification factor and receptive field size and is an estimate of the amount of tissue activated by a sin-gle point of light in the visual scene (Capuano and McIlwain, 1981; Chaplin et al., 2013). Figure 6A, B illus-trates the PIS values across the visual streak (68 meridian) (A) and along the visual streak (0 latitude) (B). Notice that the size of the region activated by a punctiform stimulus in V1 is larger when the 68 merid-ian crosses the 0 horizontal meridian (Fig. 6A) and that two other peaks of PIS values were observed in the extremities of the visual streak representation (nasal and temporal) (Fig. 6B). Figure 6C,D depicts mul-tiunit receptive field areas (C) and PIS values (D) along the visual streak in V2. Although the multiunit receptive
field size did not change very much along the visual streak representation in V2 (different from V1) the larg-est PIS in V2 are also concentrated in the intersection of the visual streak with the optical axis (LON: 68, LAT: 0) (Fig. 6C). We found that PIS in V1 varied from 0.5–7 mm, with the highest value located at the nasal extremity of the horizontal axis, whereas in V2 the high-est values lay around 9 mm from the intersection of LON 68with LAT 0.
Organization of the somatosensory
cortex of the agouti
The most remarkable feature of the primary somatosen-sory cortex of the agouti was the heterogeneous pattern of both CO and myelin staining (see Fig. 2), revealing sub-compartments related to different body parts. These subdi-visions are usually regions of higher CO reactivity and higher myelin content, separated by unstained septa or broader sectors of light staining (Fig. 2).
Area S1 had a consistent topographic organization in all animals, corresponding to an upside-down represen-tation of the contralateral half of the body surface (Figs. 7, 8), with caudal body parts (e.g., tail and hind-paw) located more medially, while progressively more anterior regions of the body (like forepaw and face) were found in more lateral parts of S1 (Figs. 7, 8). Fig-ure 8 shows a sequence of recording sites (Fig. 8B) and the corresponding multiunit receptive fields (RFs) (Fig. 8C).
In the medial-most part of S1, a region of high CO activity represented the contralateral hindlimb (Figs. 7B, 8B). This region is separated from the forepaw repre-sentation by a septum that could be identified in both myelin (Fig. 2B) and CO (Figs 2C, 7B, 8B) preparations. Depending on the level of the section this septum sometimes appeared to be coextensive with the ante-rior tip of the lateral sulcus, thus separating completely the representation of the hindpaw from the forepaw, the latter being located more laterally (see Fig. 2C). In other sections, separation between the forepaw and hindpaw isomorphs was incomplete. In this case, they were connected to each other in their posterior part by the representation of the trunk, which was also intensely stained (Fig. 2C,D). Therefore, hindlimb, trunk, and forelimb representations appeared as a continuous and well-delineated architectonic region, evidenced by both CO and myelin.
Lateral to the representation of the forelimb, both CO (Figs. 2C, 9) and myelin stains (Fig. 10) revealed another clear septum that separated the representation of the forelimb from the representation of the face, correspond-ing to a "hand–face border" which has already been char-acterized in other species (Jain et al., 1998). This septum
Figure 7.Topography and chemoarchitecture of S1 (animal SMPV 06). A: Localization of S1 in tangential sections. B: Topographic map reconstructed from 56 recording sites. The continuous lines indicate CO architectonic borders. The maps were aligned with the help of electrolytic lesions (asterisks). Notice that the regions of greater CO activity correspond to different parts of the body surface. Scale bar5 2 mm.
gives rise to a bifurcation leading to two densely stained regions (Figs. 2D, 9). Laterally, the forepaw representa-tion is followed by representarepresenta-tions of the lower lip and vibrissae (Figs. 9, 10).
Outside S1, in a region that is both lightly myelinated and weakly reactive to CO, located caudal to the vibris-sae representation, we were able to elicit visual responses (Figs. 7B, 8B, 9B). In some instances visual and somatosensory bimodal multi-unit responses were also obtained.
As described above, the septum separating the hand– face regions bifurcates and extends, on its turn, an ante-rior and lateral direction, in practice isolating the region representing the inferior lip (located in the anteriormost CO/myelin patch of S1) from that representing the rest of the face. The latter corresponded to another CO/mye-lin-rich region located more lateral and caudal to the infe-rior lip representation both bordering area S2 laterally and containing the representation of the superior lip, rhi-narium, snout, and vibrissae (Figs. 9, 10). Representation of vibrissae occupied the most caudal and lateral parts of S1. CO and myelin-stained sections through layer IV revealed barrel-like structures in this region that may cor-respond to vibrissae isomorphs (Figs. 9, 10).
The region lateral to area S1 also contained neurons responsive to the cutaneous stimulation of the contra-lateral body surface, with large receptive fields. Pimentel-Souza and coworkers (1980) associated this region with the second somatosensory area (S2) in the agouti. S2 has an upright topographic representation of the contralateral body surface, smaller than the one found in S1, and with the S1/S2 border corresponding to the representation of the face, as illustrated by the reversion in receptive field progressions along penetra-tion tracks 1–11 (Fig. 8B).
DISCUSSION
Several comparative studies have explored the diver-sity of mammalian cortical plans in search of shared organizing principles that could provide clues to the evo-lution of the neocortex (Choudhury, 1978; Kuljis et al., 1979; Olavarria and Mendez, 1979). There had been sig-nificant breakthroughs along the way. For instance, stud-ies of neocortical organization have confirmed that both primary and secondary sensory areas are present in all living representatives of mammalian lineages (protother-ians, metather(protother-ians, and eutherians), suggesting that this layout was present in a common ancestor (Choudhury, 1978; Kuljis et al., 1979; Olavarria and Mendez, 1979). Another regular motif in sensory studies is the way the sensory periphery is represented through topographically organized maps. Although all mammals display some degree of columnar organization in sensory areas, the presence of more elaborate modules spanning cortical layers is not a universal characteristic (Picanco-Diniz et al., 1991). Thus, the elucidation of common principles behind cortical mammalian organization depends on the comparative analyses of the brains of animals of different sizes and degrees of phylogenetic separation.
Visual areas of the agouti
Very little is known about the organization of the vis-ual cortex of Histricomorph rodents, such as the agouti
Figure 8.Reversal of receptive field sequences across the border between S1 and S2. A: Localization of S1 in tangential sections. The gray areas show the architectonic borders defined by CO activity. B: Topographic map obtained from 48 microelectrode penetrations. C: The localization of multiunit receptive fields responsive to light touch on the body’s surface. Notice the rever-sal of receptive fields progression at the border between S1 and S2 and receptive fields become enlarged in S2 (compare penetra-tion 5 with 10 or 11). Asterisks indicate electrolytic lesions. Dark gray areas indicate CO modules. Scale bar5 2 mm.
(Choudhury, 1978; Kuljis et al., 1979; Olavarria and Mendez, 1979). Previous systematic electrophysiologi-cal mapping of the agouti primary visual cortex revealed that each hemisphere contains a complete representa-tion of the contralateral visual field (Picanco-Diniz et al., 1991). Similar to most mammals, the retinotopic map is arranged in a way such that the upper visual field is represented in the posterior half of the visual cortex and the frontal visual field projects onto the lateral, bin-ocular part of the visual cortex (Picanco-Diniz et al., 1991). In the agouti, the medial border of area 17 is located within the depths of the lateral sulcus, contigu-ous with the cingulate cortex. Previcontigu-ously, we did not record visually-evoked responses from the cortex medial to V1 (Picanco-Diniz et al., 1991). A similar result has been described in the gray squirrel (Hall et al., 1971), but not in other rodents, such as the
guinea pig (Choudhury, 1978), the degu (Kuljis et al., 1979; Olavarria and Mendez, 1979), the rat (Montero et al., 1973a,b; Zilles et al., 1980; Wree et al., 1981; Olavarria and Van Sluyters, 1982; Espinoza and Thomas, 1983), the mouse (Caviness, 1975; Drager, 1975; Wagor et al., 1980; Olavarria and Van Sluyters, 1982), and the hamster (Tiao and Blakemore, 1976; Lent, 1982), where there is experimental evidence for the existence of visual areas located medial to area 17.
In this report we confirmed and extended the previ-ous description of the agouti visual cortex originally described by Picanc¸o-Diniz and coworkers (1989). All five visual areas previously described in this species— V1, V2, V3, AL, and TP—were consistently identified using morphological criteria such as CO reactivity and myelin staining, but could also be distinguished electro-physiologically by multiunit receptive field mappings
Figure 9.Modular organization in S1 (animals SMPV 10 and SMPV 15). The gray areas show architectonic borders defined by the superpo-sition of several sections reacted for cytochrome oxidase. Notice the presence of reactive clusters similar to barrels within the region of vibrissae representation. Asterisks and arrowheads depict electrolytic lesions. Scale bars5 2 mm.
(Figs. 2A,B, 5). We also used SMI-32 immunohistochem-istry, which has been described as a reliable tool for identification of visual areas in the mammalian cortex (Hof and Morrison, 1995; Homman-Ludiye et al., 2010).
The parcellation scheme for the visual cortex of the agouti is similar to that provided for the gray squirrel (Hall et al., 1971; Kaas et al., 1972), with the possible exception of area AL. In this respect, even though our results with CO histochemistry, SMI-32, and myelin, together with previous cytoarchitectonic descriptions (Picanco-Diniz et al., 1989), suggest that AL is a distinct area it could be argued that, in reality, AL belongs to area V3, since it represents only the inferior portion of visual field. More recently, the presence of three tectonic visual areas (17, 18, and 19) and three archi-tectonically distinct regions in the temporal cortex of both gray and ground squirrels has been confirmed (Wong et al., 2008; Wong and Kaas, 2008). These par-cellation schemes are very much similar to the ones in the present report and has been described befotre for the agouti (Picanc¸o Diniz et al., 1989). On the other hand, architectonic distinction between the binocular and monocular fields representations in the agouti’s
area 17 is not as conspicuous as in both the gray and ground squirrels, with the amount of cortex dedicated to representation of the binocular field being larger than in the agouti (Picanc¸o Diniz et al., 1989; Wong and Kaas, 2008). Since the gray squirrel has a binocu-lar field wider than both the agouti and the ground squirrel, it is not surprising to find that the amount of cortex devoted to represent the binocular field in the former is proportionally larger than in the latter species (Picanc¸o Diniz, 1987; Wong and Kaas, 2008; Picanc ¸o-Diniz et al., 2011). It is interesting to note that the analysis of myelin and SMI-32 staining patterns revealed that V1 was not homogeneously stained along its anterior to posterior extent. This was consistent with previous findings by Picanc¸o-Diniz and coworkers (1989) (see their fig. 5A). Van der Gucht and coworkers (2007) also found in the mouse that sections immuno-stained for SMI-32 the anterior and posterior parts of V1 were clearly different. Although they attributed this difference to the "progressive curvature of the brain" (which was not the case for the agouti), they also observed a difference "between the monocular and binocular segment of V1 along the posterior–anterior
Figure 10.Myelin-defined modules in the region of face representation in S1 (animal SMPV 13). A: Reconstruction of electrode penetra-tions superimposed on myelin secpenetra-tions. B: Photomicrograph of a 50-lm tangential section through S1. C: Localization of receptive fields in the animal face. Notice the isomorphic relationship between the modules and the contralateral facial whiskers. In A,B, notice that the barrels are surrounded by lightly myelinated regions. Asterisks and arrowheads depict electrolytic lesions. Scale bars5 2 mm.
extent of the visual cortex" (Van der Gucht et al., 2007).
Point-image size in V1 and V2
The present results confirm previous assessments that PIS in V1 is higher in the nasal field and decreases towards the temporal direction (Picanc¸o Diniz et al., 1989). In V1, close to the intersection of the vertical and horizontal meridians, there was a significant decrease in receptive field size but a striking increase in the cortical magnification factor (Picanco-Diniz et al., 1991). Thus, the PIS in V1 seems to follow this tend-ency mainly due to the fact that the representation of the nasalmost 20 of the visual field, located between parallels6 10, occupies around 40% of V1 (Picanco-Diniz et al., 1991). However, higher values of the PIS as seen in V2 seem to provide emphasis to region around the optical axis in the visual field (LON: 68). Because of these differences, it seems reasonable to suppose that cortical specializations in V1 and V2 suggest a dif-ferent emphasis of each area in processing the visual scene. Because retinal ganglion cell distribution does not change very much along the nasotemporal extension of the visual streak (Silveira et al., 1989), we suggest that intrinsic cortical circuitry, but also thalamic afferent projections (Towns et al., 1982; Harting and Huerta, 1983; Takahashi, 1985) may contribute to PIS differen-ces. In line with this interpretation, the visual receptive field size in V1 (Picanco-Diniz et al., 1991) is much smaller than in V2 and the cortical magnification factor along the visual streak representation in V1 and V2 also has remarkable differences (Picanc¸o Diniz et al., 1989).
Primary somatosensory area in the agouti
Our results confirmed previous work done by Pimentel-Souza and coworkers (1980) and provided additional evidence for the occurrence of a modular architecture in the agouti’s S1. In this species, area S1 contains a complete representation of the contralateral body surface, with the hindlimb represented more medi-ally, whereas the trunk, shoulder, forepaw, and the head are progressively arranged in a mediolateral dispo-sition, similar to other rodents (Campos and Welker, 1976; Sur et al., 1978; Krubitzer et al., 1986; Fabri and Burton, 1991; Slutsky et al., 2000; Remple et al., 2003; Campi et al., 2007; Rocha et al., 2007), primates (Kru-bitzer et al., 1995), marsupials (Catania et al., 2000), and monotremes (Krubitzer et al., 1995). In S1, neurons were strongly responsive to superficial stimulation of the skin and body hairs, with multiunit receptive fields characterized by sharp borders.
In S1, some body parts, such as the lips and the forepaw, are greatly magnified. This may be associated
with the agouti’s need for quick and precise food dis-crimination when foraging in tropical forests and shrub-lands. When feeding, the agouti sits on its hindpaws, leaving the forepaws free to manipulate food while keeping its eyes focused on the horizon, watching for predators (Fig. 1C). This close association between behavior and sensory representation has also been shown in the star-nosed mole cortex (Condylura cris-tata), where the enlarged representation of the tactile snout appendices reflects their relative behavioral importance rather than their innervation density (Cata-nia and Kaas, 1997). The capybara (Hydrochoerus hydrochoerus), the largest living rodent, uses its snout and lips actively during exploratory and feeding behav-ior, and these organs are selectively magnified in S1 (Campos and Welker, 1976). These results suggest that the magnified representation of behaviorally important body parts is common among mammals (Catania and Henry, 2006).
In this report, we provide the first description of the architecture of the agouti somatosensory cortex. Previ-ous work on this species focused either on electrophys-iological mapping of somatosensory representations (Pimentel-Souza et al., 1980) or on the analysis of the fine neuroanatomy of corticocortical connections origi-nating in this region (Rocha et al., 2007; Santiago et al., 2010). As is typical of S1 in other species (Kaas, 1983), area S1 in the agouti was characterized by the presence of a well-developed layer 4, more intense myelination, and higher CO-reactivity than adjacent regions. Immunoreactivity for SMI-32 in agouti S1 seems to agree with previous descriptions in other rodents, such as the rat (Kircaldie et al., 2002), ham-ster (Boire et al., 2005), and squirrel (Wong and Kaas, 2008). In all these rodent species S1 stands out as a region more intensely reactive for neurofilament pro-teins than neighboring areas, especially in its layers 3, 5, and 6. Additionally, layer 5 presented large SMI-32-reactive pyramidal neurons, contrasting with those of layer 3, which were also numerous but smaller. This pattern is also similar to what has been described for non-rodent species like the short-tailed opossum (Wong and Kaas, 2009a) and the tree shrew (Wong and Kaas, 2009b).
Modular architecture of the agouti S1
The modular architecture of S1 can be revealed with the help of a variety of enzymatic histochemical techni-ques, such as CO (Land and Simons, 1985; Catania and Kaas, 1997; Campi et al., 2007), succinate dehy-drogenase (Wallace, 1987), and NADPH-diaphorase (Franca and Volchan, 1995; Pereira et al., 2000; Nogueira-Campos et al., 2012). In the agouti, we used I.A. Dias et al.
both histochemical and immunohistochemical methods to provide evidence for modularity in S1 sections. SMI-32 wasn’t able to reveal barrel-like modules in the agouti, similar to other rodents such as the rat (Kirkcal-die et al., 2002) and the squirrel (Wong and Kaas, 2008). However, it was evident that SMI-32 immunore-activity in area S1 was not homogeneous, especially when different regions along the medial to lateral extent of area S1 were compared. A similar pattern can also be observed in the hamster (fig. 3 of Boire et al., 2005) and reflects cytoarchitectural heterogeneity (Kir-caldie et al., 2002).
In tangential sections and similar to other rodents ((Wong-Riley and Welt, 1980; Krubitzer et al., 1986; Fabri and Burton, 1991; Campi et al., 2007; Rocha et al., 2007), and even primates (Weller, 1993; Wallace et al., 2000) and marsupials (Weller, 1993; Catania et al., 2000), S1 in the agouti was readily distinct from adjacent cortex because of intense myelination and strong CO activity. Additionally, these techniques revealed cortical isomorphs in S1 that were separated by weakly stained septal regions. For instance, a clear septum separated the forepaw representation from the face representation (Figs. 7, 8). Another septum sepa-rated the lower lip from the upper lip representation (Figs. 7, 8). The existence of septa separating body iso-morphs were also described in S1 of other rodent spe-cies (Krubitzer et al., 1986; Remple et al., 2003), primates (Krubitzer and Kaas, 1990; Jain et al., 1998, 2001), insectivores (Cusick et al., 1985), chiroptera (Krubitzer and Calford, 1992), and carnivores (Johnson, 1990). In small rodents and primates septa demarcate the limits of functional modules (Krubitzer et al., 1986; Remple et al., 2003). Generally, these septa represent regions of rich callosal and corticocortical connections (Qi and Kaas, 2004).
In the agouti, both CO and myelin staining also revealed an intrinsic modular architecture in S1’s face area, sug-gesting the existence of isomorphs associated with indi-vidual facial whiskers, similar to the posteromedial barrel subfield of smaller rodents (Woolsey et al., 1975). How-ever, in the agouti, these modules are less evident than both in rats and mice (Woolsey and Van der Loos, 1970; Welker and Woolsey, 1974). The agouti has diurnal habits, with greater visual acuity than rats and mice (Picanco-Diniz et al., 1989, 1991), and does not seem to depend much on their whiskers for active exploration. This behav-ior is similar to that of the gray squirrel (Sciurus carolinen-sis), a diurnal rodent with a well-developed visual cortex, but also lacking a well-defined barrel field (see Woolsey et al., 1975; Krubitzer et al., 1986).
Our results also demonstrate the existence of a small area located lateral to S1, with a rostrocaudal
disposi-tion in the parietal cortex, that corresponds to the sec-ondary somatosensory area (S2) found in a wide range of other mammals, both metatherians (e.g., opossum: Beck et al., 1996) and eutherians (e.g., rat: Walker and Sinha, 1972; gray squirrel: Krubitzer et al., 1986). Because S2 is not found in prototherians, such as the platypus (Ornithorhynchus anatinus), it is considered a cortical area phylogenetically more recent than S1 (Kru-bitzer et al., 1995).
ACKNOWLEDGMENTS
This study is part of the requirements for the Ph.D. degree of Ivanira Amaral Dias.
CONFLICT OF INTEREST
The authors declare no identified conflict of interest.
ROLE OF AUTHORS
All authors had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study con-cept and design: Pereira A, Houzel JC, Picanc¸o-Diniz CW, Lent R. Acquisition of data: Dias IA, Bahia CP, Mayer AO, Santiago LF, Silveira LCL, Picanc¸o-Diniz CW, Pereira A. Analysis and interpretation of data: Dias IA, Bahia CP, Mayer AO, Picanc¸o-Diniz CW, Pereira A. Draft-ing of the article: Dias IA, Bahia CP, Picanc¸o-Diniz CW, Pereira A. Critical revision of the article for important intellectual content: Lent R, Franca JG, Houzel JC. Sta-tistical analysis: Dias IA, Picanc¸o-DIniz CW. Obtained funding: Lent R, Franca JG, Bahia CP, Pereira A. Study supervision: Pereira A.
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