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European Journal of Neuroscience, Vol. 11, pp. 4291±4308, 1999 ã European Neuroscience Association Orientation topography of layer 4 lateral networks revealed by optical imaging in cat visual cortex (area 18) Â va ToÂth and ZoltaÂn F. KisvaÂrday Tagrid Yousef, Tobias Bonhoeffer,1 Dae-Shik Kim,2 Ulf T. Eysel, E Abteilung fuÈr Neurophysiologie, Ruhr-UniversitaÈt Bochum, UniversitaÈtsstrasse 150, 44801, Bochum, Germany 1 Max-Planck Institute fuÈr Neurobiologie, Am Klopferspitz 18A, 82152 MuÈnchen-Martinsried, Germany 2 Center for Magnetic Resonance Research, University of Minnesota Medical School, 2021 6th Street, SE, Minneapolis, MN 55455, USA Keywords: anatomical tracing, lateral network, layer 4, optical imaging, orientation selectivity Abstract The functional speci®city of corticocortical connections with respect to the topography of orientation selectivity was studied by optical imaging of intrinsic signals and bulk injections of ¯uorescent latex beads (green and red) and biocytin into layer 4. The distributions of retrogradely labelled cells and anterogradely labelled axon terminals were histologically reconstructed from all cortical laminae, and the resulting anatomical maps compared with the optically imaged functional maps. Layer 4 injections produced extensive horizontal labelling up to 2±3 mm from the injection centres albeit without the clear patchy pattern described after layer 2/3 injections (Gilbert & Wiesel, 1989, J. Neurosci., 9, 2432±2442; KisvaÂrday et al., 1997, Cerebral Cortex, 7, 605±618). The functional (orientation) distribution of the labelled projections was analysed according to laminar location and lateral spread. With regard to the former, no major difference in the orientation topography between supragranular- (upper tier), granular- (middle tier) and infragranular (lower tier) layers was seen. Laterally, proximal and distal projections were distinguished and further dissected into three orientation categories, iso- (6 30°), oblique- (6 30±60°) and cross-orientations (6 60±90°) with respect to the orientation preference at the injection sites. The majority of distal connections (retrograde and anterograde) was equally distributed across orientations (35.4% iso-, 33.7% oblique-, and 30.9% cross-orientations) that are equivalent with a preponderance to dissimilar orientations (oblique- and cross-orientations, 64.6%). In one case, distal excitatory and inhibitory connections could be morphologically distinguished. For both categories, a marked bias to dissimilar orientations was found (excitatory, 63.7%; inhibitory, 86.6%). Taken together, these results suggest that the long-range layer 4 circuitry has a different functional role from that of the iso-orientation biased (52.9%, KisvaÂrday et al., 1997, Cerebral Cortex, 7, 605±618) layer 2/3 circuitry, and is perhaps involved in feature difference-based mechanisms, e.g. ®gure ground segregation. Introduction Recent electrophysiological and psychophysical experiments suggest that perceptual discrimination and grouping begin at the early level of visual cortical processing (Allman et al., 1985; von der Heydt & Peterhans, 1989; Nothdurft, 1993; Lamme, 1995; Kapadia et al., 1995; Baumann et al., 1997; Kastner et al., 1997). Although the underlying mechanisms of visual perception are not yet clear, the anatomical substrate of the above processing has been tentatively assigned to the long-range lateral network that could mediate direct effects between visuotopic locations where the receptive ®elds no longer overlap (Albus, 1975a; Tusa et al., 1979). On the basis of the diversity of the interactions seen in the electrophysiological and psychophysical experiments it is tempting to speculate that the longrange lateral networks should link cortical locations representing a broad spectrum of the cue elements, e.g. orientation. Indeed, our recent results obtained with focal tracer injections in layer 2/3 of the visual cortex of the cat showed only a moderate iso-orientation bias of long-range lateral connections implying that a considerable proportion of connections indeed link non-iso-orientations, including Correspondence: Dr Z. F. KisvaÂrday, as above. E-mail: [email protected] Received 24 May 1999, revised 19 August 1999, accepted 24 August 1999 cross-orientations (KisvaÂrday et al., 1997). Similar results were obtained in the primate and tree-shrew visual cortex (Malach et al., 1993, 1994; Bosking et al., 1997), casting doubt on former suggestions as to the strict speci®city of lateral connections to either iso- (Michalski et al., 1983; Nelson & Frost, 1985; Ts'o et al., 1986; Gilbert & Wiesel, 1989) or cross-orientations (Matsubara et al., 1985, 1987). Previous work on the functional speci®city of lateral networks has focused exclusively on layer 2/3. Because different cortical layers are composed of different neuron types, the question arises whether the layer 2/3 ®ndings could also be applied to deeper laminae. In the present study, we focused our interest on layer 4 connections. Electrophysiological studies showed the continuity of similar orientation preferences from layer 2/3 into layer 4 (Hubel & Wiesel, 1962, 1963; Albus, 1975b; Bauer, 1982; Murphy & Sillito, 1986). Anatomical studies have shown that there are spiny stellate (Martin & Whitteridge, 1984) and inhibitory basket cells (Somogyi et al., 1983) in layer 4 which send long, lateral axons over several mm similar to layer 2/3 pyramidal cells. Because layer 4 is the very ®rst stage in the cortical processing of the arriving retino-thalamic signals it would be important to know whether the lateral connections here are distributed in a functionally, e.g. orientation, speci®c manner. To this end, the retrograde tracer, ¯uorescent latex beads, and the 4292 T. Yousef et al. anterograde tracer, biocytin, were injected into layer 4 and the distributions of the labelled connections were reconstructed and analysed with respect to functional orientation maps obtained with the optical imaging technique based on intrinsic signal detection (Bonhoeffer & Grinvald, 1991, 1993). The results show that the longrange component of the layer 4 lateral network establishes an overall non-iso-orientation-biased topography suggesting a different role from its layer 2/3 counterpart. Materials and methods Surgical procedures Normal adult cats were acutely prepared for surgery using standard procedures in accordance with institutional and Federal Guidelines for the Care and Use of Laboratory Animals; initial surgical anaesthesia was induced with intramuscular injection of a mixture of ketamin, 7 mg/kg (Ketanest, Parke-Davis, Berlin, Germany) and xylazin, 1 mg/kg (Rompun, Bayer Belgium, SintTruiden, Belgium). All wounds and pressure points were treated with the local anaesthetic, xylocain gel (Astra Chemicals, Wedel/ Holstein, Germany) in order to minimize pain and discomfort to the animal. During cranial surgery and throughout the entire optical imaging experiments, the animals were lightly anaesthetized (N2O : O2, 70 : 30% plus Na-pentothal, 2 mg/kg/h) and paralysed (alcuronium chloride, 0.06 mg/kg/h, i.a.), and their physiological conditions (blood pressure, 95±120 mmHg; heart rate; body temperature, 38.5 °C; expiratory CO2, 3.5±4%; and EEG) continuously monitored. From the beginning of the experiments, the corneas were protected from drying out using neutral contact lenses and eye drops of 1.5% saline. Thirty minutes prior to optical imaging the nictitating membranes were retracted and the pupils dilated with 5% phenylephrinhydrochloride (Neosynephrin-POS, Ursapharm, SaarbruÈcken, Germany) and 1% atropinsulphate (Atropin-POS, Ursapharm, SaarbruÈcken, Germany), respectively. At this phase of the experiment, correction lenses were chosen on the basis of tapetal re¯ection. A craniotomy (14 3 7 mm2) was made on the dorsal surface of the skull centred to Horsley±Clarke co-ordinates A1 and L4 to expose the quasi-central visual representation of areas 17 and 18 (Fig. 1). A metal chamber of 30 mm inner diameter was cemented onto the skull using dental cement (Paladur, Heraeus Kulzer GmbH, Wehrheim, Germany). The dura mater was cut along the wall of the craniotomy and removed. Damaged dura vessels were either cauterized (Fine Science Tools GmbH, Heidelberg, Germany) or squeezed with a ®netip forceps until bleeding entirely stopped. The surface of the exposed cortex was cleaned with arti®cial cerebrospinal ¯uid or Ringer solution, and all tissue debris was carefully removed. The chamber was ®lled with silicone oil (50 cSt viscosity, Aldrich, Milwaukee, WI, USA) and sealed with a round glass-cover (Fig. 1A). Visual stimuli and optical recording Full-®eld visual stimuli of sinusoidal drifting (10±15 °/s) square-wave gratings (0.1±0.4 cycles per degree) of four different orientations were presented binocularly using a video screen (SONY) in 120 Hz non-interlaced mode and the Neuroseq software (Vision Research Graphics, Durham, NH, USA). For obtaining orientation maps the stimulus grating was moved in opposite directions at each presentation. Individual stimuli were presented 25±50 times for 4.5 s followed by an inter-stimulus interval for 10 s when the animals viewed a blank screen. The cortex was illuminated with light at 605 6 5 nm using a circular ®bre optic slit lamp (Schott, Mainz, Germany). Video images were recorded with a slow-scan CCD camera (Bischke CCD 6012P, FIG. 1. Photographs of the exposed cortical region subjected to the present study. (A) Part of area 18 of the left hemisphere is seen through the glass window of the imaging chamber. (B) A block of the same cortical region is shown after histological ®xation. Asterisk indicates the location of an injection site where green ¯uorescent latex beads were delivered by pressure. Dots denote, approximately, the border between areas 17 and 18 (Tusa et al., 1979) and the arrow points to the mm scale at the zero position of the ear-bar. Same scale applies in A. 6 3 8 mm sensor) attached to a `tandem-lens' of two objectives (SMC Pentax 1 : 1.2, 50 mm) resulting in a picture resolution of 21.28 mm per camera pixel. The images were collected and processed using the system Imager 2001 (Optical Imaging, Germantown, NY, USA) and Matrox IM-640 imaging board controlled by the software VDAQ218k. Image analysis Typically 10 blocks were collected and combined, each containing ®ve stimulus trials of individual stimuli. The images were analysed in several steps. To correct for uneven illumination each image acquired for a particular orientation and direction was divided by the sum of images for all orientations and directions resulting in single condition maps using the TVMIX program of the system Imager 2001 (Optical Imaging). Differential maps were generated by dividing single condition maps of opposite polarity (i.e. single condition maps obtained with gratings of perpendicular orientations). Further evaluation of the images was done in two steps using a custom software written in IDL (Research Systems, Boulder, CO, USA). A band-pass ®ltering was used to reduce noise (DC shift in the images and small blood vessels) but retaining the dominant structure of the orientation maps. Before the differential maps were vectorially summed, high-pass ®ltering with a pixel-kernel equivalent of 1064 mm had been applied followed by a low-pass ®ltering. The high-pass ®ltering parameter was chosen on the basis of the expected periodicity of the orientation domains (~ 1.25 mm, Swindale et al., 1987) and the low pass-®ltering parameter on the basis of the smallest orientation domains, in the range of 200±300 mm in diameter. Orientation (angle) maps were produced by summing the vector components of differential maps on a pixel-by-pixel basis and displayed with colour coding. Ó 1999 European Neuroscience Association, European Journal of Neuroscience, 11, 4291±4308 Functional topography of orientation selectivity in layer 4 4293 FIG. 2. Columnar arrangement of orientation selectivity in part of an orientation map revealed by electrophysiological recordings. (A) Zoomed-in orientation angle map coded by the right-hand scheme in which the white spot indicates the locations of a recording site. Black bars represent averaged orientation vectors at each location. (B) Polar-plots show orientation and direction responses of single sites at known distances from the cortical surface. (C) The average orientation preference (thick bar) along the recording track was determined from the orientation preferences calculated by Fourier analysis (WoÈrgoÈtter & Eysel, 1987) of the individual responses (thin bars). There is a clear match between the orientation preference shown by optical imaging and electrophysiological recordings including layer 4. Scale bar, 100 mm. Electrophysiological recordings from orientation columns It was important to verify that the optically imaged orientation preferences re¯ect the orientation preferences of the underlying cortical tissue at any given image pixel position. Therefore, recordings of multiunit, or occasionally single unit, activity were made from several locations penetrating through several layers (Fig. 2). All penetrations were made perpendicular to the cortical surface. Their locations were chosen on the basis of single condition and angle maps and, eventually, determined with the help of vascular images. Electrical responses of single cells and multiunit clusters were recorded with glass pipettes (inner tip diameter 5±10 mm) ®lled with 0.5 M Na-acetate. For visual stimulation, computer-controlled moving light bars (Cambridge Electronic Design, CED-1401 laboratory interface) were used generated with a `Picasso' image synthesizer (Innisfree, Cambridge, MA, USA) and presented on a Tektronix 608 monitor 28.5 cm in front of the animal's eye. The stimulus orientation was varied in pseudo-random sequence by multiples of 22.5° and drifted with optimal velocity along the orthogonal axis of the orientation across the receptive ®eld area. Peristimulus time histograms (not shown) and polar-plots (Fig. 2B) were generated from ®ve stimulus sweeps in opposite directions at each orientation. The preferred orientation at each recording site was calculated using Fourier analysis of the raw data (SDO-analysis, WoÈrgoÈtter & Eysel, 1987). Tracer injections and histology The surface vascular pattern of the imaged regions served as reference for guiding the injection pipette into particular locations of the orientation preference maps. Extracellular pressure injections of ¯uorescent latex beads (10±15 mm tip diameter, 10±36 nL, Molecular Probes, Eugene, OR, USA) or iontophoretic delivery of 2% biocytin dissolved in 0.5 M Na-acetate (6±8 mm tip diameter, positive 0.6± 0.7 mA, 1-s ON/1-s OFF duty cycle for 20 min; Sigma, Deisenhofen, Germany) were made into the middle cortical layer. In order to align the histological images with optical images four to eight additional reference penetrations were made around each injection using empty Ó 1999 European Neuroscience Association, European Journal of Neuroscience, 11, 4291±4308 4294 T. Yousef et al. FIG. 3. Matching between optical images and histological sections. (A and B) Superimposed images of surface vascular maps and reconstructions of sections contours. Cross-hairs mark the location of reference penetrations made by empty glass electrodes in the vascular maps which caused small, recognisable damage to the tissue along their courses. The light microscopic image of one of these such penetrations is seen at the upper (C) and lower surface (D) of an osmicated section. Reference penetrations served to match the optical images with the histological reconstructions (see Materials and methods). (E and F) Zoomed-in vascular images of (A and B), respectively, showing the goodness of matching of reference penetration positions as viewed in the in vivo optical image (cross-hairs) and as found in the ®xed tissue (small dots). Notice that none of the corresponding locations differs by more than ~ 40 mm. Asterisks mark tracer injection sites. Scale bars, 1 mm (A and B); 50 mm (C and D); 1 mm (E and F). glass-pipettes (10±15 mm tip diameter). Their precise positions in the vascular maps were marked on enlarged photographic images as well as their stereotaxic locations documented (Fig. 3). After 30±45 h the animals received an overdose of anaesthetics and were perfused transcardially with a mixture of 2±4% paraformaldehyde and 0.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.6). For the bead- Ó 1999 European Neuroscience Association, European Journal of Neuroscience, 11, 4291±4308 Functional topography of orientation selectivity in layer 4 4295 injected animals only paraformaldehyde was used. Large blocks of area of interest were dissected, and frozen sections (30 mm) from the bead-injected, and vibratome sections from the biocytin-injected tissue were cut parallel to the cortical surface. In the case of bead injections, alternate series of sections were collected. One series was mounted onto chrome-gelatine-coated slides in a 1 : 1 mixture of toluol and DePeX (SERVA Finebiochemica GmbH KG, Heidelberg, Germany) for reconstructing the distribution of labelled cells. Onethird (OI17RHr) or one-quarter (OI20LHg and OI20LHr) of the section series was stained for Nissl substance for determining laminar boundaries, and another third (OI17RHr) or quarter (OI20LHg and OI20LHr) of the series underwent osmication (1% OsO4 for 20 min, dehydration and embedding in resin, Durcupan ACM, Fluka Chemie AG, Buchs, Switzerland). The latter series served to recover the location of reference penetrations. In the case of biocytin injections, all sections were treated with osmium followed by embedding in resin. Reconstruction of the labelling Fluorescent labelling is known to fade with time and under intense illumination ¯uorescence rapidly bleaches. Therefore, great care was taken to preserve the ¯uorescent signal partly by storing the sections at 4 °C and partly by illuminating only the inspected region during reconstruction. No such precaution with the resin-embedded, biocytin-labelled specimens was necessary. Labelled neuronal somata and axon terminals were reconstructed from the entire depth of the cortex using 3 100 objective and the reconstruction procedure reported earlier (KisvaÂrday et al., 1997). Brie¯y, the position of each labelled structure was registered according to x- and y-coordinates with the help of a personal computer and the reconstruction system Neurolucida (MicroBrightField, Colchester, VT, USA). Reference penetrations were determined from osmicated sections (Fig. 3C and D) and their exact positions reconstructed in a similar manner to labelled structures. Data of single sections were assigned a z-value according to their distances from the cortical surface. Finally, all individual sections of the same tissue block were aligned. In this procedure, small blood vessels (< 20 mm diameter) that run from one section into the adjoining section (bead-injected cases) or labelled ®bres with corresponding cut ends (biocytin-injected cases) served to ®nd the best match. In this manner, the data of many individual sections were compiled into a single data ®le resulting in a semi-three-dimensional reconstruction of the labelling. Concerning the bead injections it needs to be added that the Nissl-stained and osmium-treated sections could not be used for cell mapping. Therefore, the distributions shown for the bead injections represent only part of the entire labelling. FIG. 4. Light microscopy of ¯uorescent bead labelling in 30-mm horizontal sections. (A) The injection site is shown at the level of upper layer IV at a depth of 630 mm from the pial surface. The core region of the injection site is characterized by the intense ¯uorescence of the tracer (diameter, < 320 mm) around which labelled neurons are readily visible. The labelling density rapidly falls off over 100±150 mm from the injection centre indicating an uptake zone con®ned mainly to the core region. The framed area is enlarged in B, showing bead-labelled cells in the halo zone (arrows). At distal locations the labelling intensity of neurons was somewhat lower than for cells at the injection site. Nonetheless strongly labelled cells either alone (C) or in clusters (D) could be found up to several mm from the injection site. In the present analysis, only cells with unequivocal labelling were taken into account. Scale bars, 100 mm (A); 10 mm (B,C); 50 mm (D). Ó 1999 European Neuroscience Association, European Journal of Neuroscience, 11, 4291±4308 4296 T. Yousef et al. FIG. 5. Light microscopy of a layer 4 biocytin injection. (A) The injection site and the resulting proximal axon terminal and soma labelling are shown. In remote regions, two types of labelled axons could be distinguished. (B and C) Inhibitory axons of the basket cell type which characteristically formed en passant terminals and often contacted perikarya (asterisks). (D and E) Putative excitatory axons gave off a mixture of en passant terminals (arrows in D) and terminals with short stalks (arrowheads in D). Frequently these axon segments could be traced back to main, myelinated axons radiating out from the injection site. The arrow in E indicates the direction of a thick, myelinated axon that branched into a cluster of thin collaterals. Scale bars, 100 mm (A); 10 mm (B and C); 20 mm (D and E). FIG. 6. Orientation topography of layer 4 projections revealed by a combination of optical imaging and biocytin injection. (A±D) Single condition maps each recorded during visual stimulation by moving gratings of one of four orientations (insets). Dark zones represent highly active regions whereas white zones are less active. (E) The orientation angle map was generated by vector summation of the four single condition maps shown in (A±D). The colour-coded scheme indicates which region prefers which orientation. (F±K) Topography of biocytin-labelled connections in the same, optically imaged region. The axon terminal (F) and soma (G) labelling are displayed in a density matrix form where every pixel corresponds to a particular orientation image pixel. The colour-coded logarithmic scale indicates the number of labelled structures per pixel. For a better comparison of the anatomical and optical images, a cross-hair is provided in each panel centred onto the injection site. Accordingly, the injected zone is found in a yellow-orange zone of the angle map (E) representing close to horizontal stimulus orientation. Notice that the axon terminal labelling shows signs of clustering from ~ 800 mm (circle) from the injection centre. No clear clustering in the soma labelling is visible. (H) The reconstructed labelled terminals (red dots) and somata (white dots) are viewed in a frontal plane. The injection centre corresponds to the highest density of labelling in the middle of layer 4. From this plane the labelling is smoothly distributed around the injection site. The bouton cluster that occurs in the medial bank of the gyrus, probably corresponding to area 17, lays outside the optically imaged region (broken line) and, therefore, is not part of the analysis. (I±K) Horizontal views of the labelling in the three main tiers reveal no marked differences in the distribution patterns. The outlined areas refer to the region of the optical images. At each level, clustering is present, however, the clusters strongly differ in size, form and density from one another. Most of the somata were found in regions of high bouton density indicating reciprocity in the projections. L, lateral; V, ventral. Scale bars, 1 mm (A±D); 1 mm (E±G); 1 mm (H±K). Ó 1999 European Neuroscience Association, European Journal of Neuroscience, 11, 4291±4308 Functional topography of orientation selectivity in layer 4 4297 Ó 1999 European Neuroscience Association, European Journal of Neuroscience, 11, 4291±4308 4298 T. Yousef et al. Aligning procedure The anatomical reconstructions were carefully aligned with the optical images using reference penetrations (see Fig. 3). First, the anatomical data were corrected for shrinkage caused by the ®xative and subsequent histological treatment. In the following, all ®gures have been corrected for tissue shrinkage. Then the same data were tilted and rotated into the plane of the optical images. In practice, this plane was found when the reconstructed reference penetrations running perpendicular to that plane were viewed as spots. At this stage, the anatomical reconstructions can be superimposed onto the optical images using the corresponding reference penetration marks (Fig. 3A and B). As shown in Fig. 3E and F, our aligning method had an estimated error of < 50 mm. Quantitative calculations Optical maps are composed of pixels each covering a known area, whereas the anatomical data represent coordinate points. In order to obtain a quantitative view of the orientation distribution of the labelled connections the aligned anatomical data set was converted into a density distribution map using a pixel resolution (21.28 mm grid constant) identical to that of the orientation maps. Orientation tuning curves were calculated by counting the number of labelled structures in each image pixel and the results were binned at every 22.5°. For a direct comparison with previous quantitative results on the orientation distribution of lateral projections (KisvaÂrday et al., 1997; Bosking et al., 1997) the labelling was grouped according to iso(6 30°), oblique- (6 30±60°) and cross-orientation (6 60±90°) categories with reference to orientation preferences of the injection sites (Table 2). Furthermore, the anatomical data were dissected into proximal and distal regions by drawing a circle around the injection core in the centre. Instead of using a ®xed value, we applied a qualitative de®nition whereby the radius of the circle varied from case to case. Because the clustering phenomenon is generally associated with the long-range system, projections shorter than the ®rst occurring clusters were considered proximal and projections longer than that distal. Accordingly, the radii dividing proximal and distal projections ranged between 460 and 850 mm, providing an average of 740 mm for the seven cases. Results Electrophysiological ®ndings We tested whether the average orientation preferences detected by the optical imaging technique differed from the orientation preferences recorded with electrodes. To this end the orientation tuning of cortical cells was determined at 19 domain locations of four animals. In all cases, the optically imaged orientation preferences matched the electrophysiologically determined preferred orientations. Secondly, we carried out a laminar analysis for orientation selectivity to ®nd out whether optical imaging could be applied for determining orientation preferences below layer 3. Therefore, at 12 of the 19 recording sites, orientation tunings were determined from several layers including layer 4. An example for the results of such penetrations and the orientation map of the same region is shown in Fig. 2. The recording site (white spot in A) was in an orientation domain and had a preference for near-horizontal orientations (orientation coding is in grey-scale). Individual recordings at 12 different cortical depths of the same site revealed similar orientation preferences as shown by the generated polar plots (Fig. 2B). In a more condensed form, the same set of data is represented by the bar-cluster which showed a scatter < 20° around the average orientation preference of this cortical column (Fig. 2C). Importantly, at all other penetrations a similarly small orientation scatter was found when layers 2±4 were considered. Anatomical ®ndings Injection sites In order to explore the functional speci®city of lateral connections of layer 4, small injections of ¯uorescent latex beads (red and green) and biocytin were made into regions of orientation domains. We selected these locations for our injections because here the orientation preferences between neighbouring columns change in small steps, hence our injections could be restricted to a reasonably narrow range of orientations showing an average standard deviation of 8.5° for the seven injection sites. The locations of the injections were chosen to be in the middle of the images so that much of the resulting labelling could be con®ned to a large part of the imaged regions. In some cases, however, the injections could only be made 50±200 mm lateral from their desired locations in order to avoid damage to surface blood vessels. The overall lateral extent of the injection sites was 150± 320 mm in diameter as marked by the dense tracer deposit and the surrounding halo region. In Figs 4A and B, and 5A, light micrographs show representative examples of the core and halo regions of the injection sites taken at a low magni®cation. The laminar position of the injections and the surrounding labelling was determined in horizontal Nissl-stained and osmicated sections using qualitative criteria, e.g. differences in cell and ®bre density, soma size and the presence of large neurons, for example large pyramidal cells in the border region of layers 3/4 and in layer 5. In addition to these landmarks, rotation of the three-dimensional reconstructions into a plane perpendicular to the cortical surface was also helpful (Fig. 6H). We used a simpli®ed scheme of the sixlayered cortex whereby the super®cial layers (1±3) represented the upper tier, layer 4 the middle tier and the deep layers (5±6) the lower tier of the cortex. In the case of OI25LH, the border region between the middle and lower tiers could not be determined unequivocally. Therefore, the labelling was analysed by pooling the data of these two cortical tiers together. All seven injections were con®ned primarily to layer 4. Nonetheless, in two bead-injected cases the involvement of other layers cannot be excluded. Case OI20LHr comprised the lowermost division of layer 3 (inset to Fig. 10C) and case OI20LHg overlapped partly with the uppermost division of layer 5 (inset to Fig. 10E). In the core regions, not all labelled cells and axon terminals could be traced due to the strong tracer deposit. Around the core region, however, both types of tracer injection had a halo zone where the labelled structures could already be distinguished. In case of the bead injections directly above the core, labelled cells were numerous probably as a result of back¯ow of the tracer and, below the core, labelled cells could take up the tracer via their ascending dendrites (see insets of Fig. 10). The latter phenomenon was also observed in the biocytin-injected cases (Fig. 6H). Here, many layer 6 cells below the layer 4 injection were labelled probably by taking up the tracer via their profusely branching apical dendrites. General topography of the labelling Fluorescent latex beads are known to be transported retrogradely. We used this tracer to reveal neuronal somata with projection to the injection site. Conversely, biocytin, commonly used as anterograde tracer, was selected to determine the termination of axons of cells located at the injection sites. While biocytin is indeed a potent anterograde tracer, to some degree, it can also be transported retrogradely as indicated by labelled somata at remote locations from Ó 1999 European Neuroscience Association, European Journal of Neuroscience, 11, 4291±4308 Functional topography of orientation selectivity in layer 4 4299 TABLE 1. Summary of the major experimental parameters Injection site Case Tracer Core radius (mm) Laminar extent Preferred orientation (°) OI20LHr OI20LHg OI17RHr OI25LH Red latex beads (10 nL) Green latex beads (10 nL) Red latex beads (36 nL) Biocytin 75 120 100 95 Upper 4 Mid 4 Mid 4 Upper 4 OI26RH Biocytin 106 F081292 F261093 Biocytin Biocytin 160 92 Sections (n) (mm) 101 116 82 67 23* 23* 15* 21 30 30 30 70 Upper 4 122 17 70 Entire 4 Mid 4 76 87 28 21 70 70 Labelled somata (S) and boutons (B) (n) 7184(S) 26519(S) 4488(S) 650(S) 38005(B) 141(S) 35739(B) 260525(B) 551(S) 59778(B) Maximum lateral extent of labelling (mm) 3.93 3 3.05² 5.87 3 3.54² 3.77 3 3.90² 4.40 3 3.48 3.74 3 3.66² 3.05 3 2.76² 5.26 3 3.55² 4.94 3 3.90² *Numbers represent alternate sections used in the reconstructions. ²Refers to anterior±posterior and medial±lateral directions (A±P 3 M±L). the injections. We utilized this dual characteristic of biocytin for simultaneous detection of reciprocal projections. Labelled somata and axon terminals were found up to several mm from the injection centres. In Fig. 4C, a light microscopic example is shown for a cell labelled retrogradely with red-¯uorescent beads. Such neurons were occasionally found in clusters up to several mm from the injections (Fig. 4D). It is readily seen that the intensity of the ¯uorescent label of these remote cells is similar to the labelling intensity of those lying proximal to the injection site (Fig. 4B). Interestingly, biocytin-labelled somata were always less numerous than bead-labelled somata although the injections were similar in size. This indicates that the axon terminals have a lower af®nity in taking up biocytin compared with latex beads, whereas the opposite applies for somatic and dendritic uptake when the two tracers are compared. Distinction between excitatory and inhibitory cells It is known that biocytin reveals the ®ne morphological details of cells in a Golgi-stain manner, a characteristic that could be utilized for distinguishing between excitatory and inhibitory components of the labelled structures. We reported previously (KisvaÂrday et al., 1997) that in addition to spiny neurons, biocytin stains basket cells of the GABAergic cell population, some of which provide long-range lateral inhibitory connections. In one of the four biocytin cases (OI26RH), the quality of the staining allowed us to distinguish between presumed excitatory and inhibitory elements on the basis of morphological characteristics. In Fig. 5, examples for basket (Fig. 5B and C) and presumed excitatory (Fig. 5D and E) axon collaterals are illustrated. Typically, the basket axons gave off large en passant boutons which often terminated on somata. Excitatory axons emitted en passant and club-like boutons of various sizes. The global branching pattern also differed between the two types of axon. Basket axons rami®ed more frequently and did not form distinct patches although small clustering could occur. Long-range excitatory axons emitted thin collaterals some of which could form patches. The few labelled basket somata found proximal to the injection site (< 300 mm, Fig. 8E) gave off smooth, beaded dendrites. Contrary to this, spiny neurons were quite numerous and could be found up to 3 mm (e.g. in Fig. 6G). Three-dimensional analysis of the labelling After registering the three-dimensional position of each labelled cell body and axon terminal, their laminar position was determined using histological information of Nissl-stained sections and anatomical landmarks visible in the osmicated sections. The reconstructed distributions were divided into three main tiers using computergenerated planes running quasi-parallel to the cortical surface and approximating laminar divisions. It needs to be added here that area 18 is not an entirely ¯at structure, hence some large sections contained more than one lamina. By the same token, the planes used for dividing cortical tiers could not exactly follow the depth ¯uctuation of the laminar boundaries in the tissue. Thus, a small proportion of the labelling was probably assigned to neighbouring tiers. In general, all layer 4 injections resulted in labelling that spanned virtually the entire cortical depth. There was no major difference in the pattern of labelling between different laminae, proximal to the column of injection the labelling was diffuse while more distally clustering occurred. Labelled somata and axon terminals showed a tendency to be in close neighbourhood indicating that the projections are highly reciprocal. An example is provided in Fig. 6H±K for a biocytin injection showing a similar, quasi-clustered labelling pattern of somata (white dots) and axon terminals (red dots) in each of the three main tiers. When the entire labelling is viewed from the same plane in a form of density distribution, it is obvious that the overall pattern strongly resembles the pattern of the single tiers indicating a columnar-like arrangement for the most part of the densely labelled locations (Fig. 6F and G). A similar trend was found for all other injections including the bead injections illustrated in Fig. 10. Another feature that is worth note concerns the clustering pattern of the layer 4 lateral projections. The examples illustrated in Figs 6, 8 and 10 revealed a different clustering pattern from those reported for the labelling after layer 2/3 injections (Gilbert & Wiesel, 1989; KisvaÂrday et al., 1997). The layer 4 injections resulted in only a modest number of clusters possessing irregular size and shape (Figs 6F, 8A, and 10A, C and E) and, in general, were less obvious than in the case of the layer 2/3 injections mentioned above. Furthermore, the average lateral extent of layer 4 projections measured here was 4.3 mm (Table 1) compared with the 6.5±8 mm of the layer 2/3 circuitry (Gilbert & Wiesel, 1989; KisvaÂrday et al., 1997). There was, however, an interesting common characteristic between the layer 2/3 results and the seven layer 4 cases shown here. Namely, the cell and axon terminal density values between the labelled clusters seldom showed zero values. This observation indicates that for the layer 4 projections, too, there is a kind of `baseline' between the injection site and many locations especially at distances shorter than 2 mm. All layer 4 injections gave rise to labelling up to 2±3 mm from the injection site mainly in the same layer. The only exception was found in the case of OI20LHg in which the injection site included part of upper layer 5. Here the lateral extent of the Ó 1999 European Neuroscience Association, European Journal of Neuroscience, 11, 4291±4308 4300 T. Yousef et al. FIG. 7. Quantitative orientation distribution of biocytin-labelled axon terminals and somata shown in Fig. 6. (A) The orientation preference of the injection site was determined by counting the number of orientation pixels corresponding to the core and immediate halo region. (B and C) The results, respectively, for the entire bouton and soma labelling. (D±I) The same data with respect to different tiers. Proximal (broken lines) and distal (solid lines) projections differ in their distributions. The former occupy mainly similar orientations and the latter display a strong tendency to link many different orientations. labelling was up to 4 mm mainly in the lower cortical tier, suggesting that the most distal projections of this case are the product of layer 5 pyramidal neurons rather than projections of cells in layers 2/3 and 4 (Gilbert & Wiesel, 1983; Martin & Whitteridge, 1984; Gabbott et al., 1987). comparing anatomical density maps and orientation maps are shown in Figs 6A±G and 8A±E for biocytin injections, and Fig. 10A±F for bead injections. In order to foster a better comparison of corresponding parts of the maps, a cross-hair centred on the injection site was overlaid in each ®gure panel. Comparison between the distribution of layer 4 lateral projections and orientation maps Quantitative results We were interested to know the speci®city of layer 4 lateral projections with respect to functional orientation preference maps. To this end, the number of labelled structures (axon terminals and somata, see Table 2) were counted in each image pixel and displayed as density distribution matrices. This format of the anatomical data was used to carry out a quantitative analysis of their orientation speci®city at the spatial resolution of a single image pixel (see Materials and methods for details). Examples for A quantitative measure on the orientation speci®city of lateral connections in layer 4 was obtained on entire cell distributions rather than taking only individual clusters into account. As a ®rst step, the orientation preferences of the injection sites were determined in a circle representing the core and halo regions. The results of two biocytin cases are shown, respectively, in Figs 7A and 8F, and three bead injection cases in Fig. 10G±I. Interestingly, three of the seven injection sites showed skewed orientation distributions over a relatively broad range (e.g. Fig. Ó 1999 European Neuroscience Association, European Journal of Neuroscience, 11, 4291±4308 Functional topography of orientation selectivity in layer 4 4301 FIG. 8. Topography of labelled excitatory and inhibitory connections in superposition with the vascular image of the part of area 18 under study. (A±D) Density distributions of excitatory (A) and inhibitory (B) axon terminals and somata possessing spiny (C) and smooth (D) dendrites. Excitatory connections (A and C) show larger lateral extent than inhibitory connections (B and D). Distal excitatory boutons display weak clustering while distal inhibitory boutons provided only two small clumps. No clustering in the soma labelling was seen. (F) The orientation distribution of the injection site is skewed because it was not in the middle of an orientation domain, some 250 mm away from the nearest orientation centre as shown in E. Scale bars, 1 mm (A±E). 8F), while the others were symmetric to a narrow orientation range (e.g. Fig. 10I). With a close look at the orientation maps, one sees that the injections of the former category were not in the middle of an orientation domain, hence they occupied a few other orientations in addition to the dominating one. A laminar analysis of the projections was carried out using the categories upper, middle and lower tiers instead of individual laminae. Examples of this analysis are shown in Figs 7 and 9. The orientation distribution curves differed only in minor details and the main tendencies remained the same throughout the different tiers. Therefore, for the sake of simplicity, the data of individual tiers were pulled together in each case and used for analysing the orientation speci®city of the projections as a function of lateral distance from the injection site. In this analysis, proximal and distal projections were separated on the basis of qualitative criteria (see Materials and methods). The results showed that proximal projections always had a major peak at similar orientation preferences with respect to that of the injection sites (broken lines in graphs of Figs 7, 9 and 10). In addition to this, in the cases where one or two nearby orientation centres ¯anked the injection site, other high peaks could also occur at different orientations (Fig. 10K and L). An interesting feature was that none of the distribution curves including those of distal projections was symmetric to the average orientation preference of the injection site. Most of the distributions were skewed even in the cases where the injection site had a narrow, symmetrically distributed orientation range (e.g. Fig. 10G). This ®nding suggests that in¯uences Ó 1999 European Neuroscience Association, European Journal of Neuroscience, 11, 4291±4308 4302 T. Yousef et al. FIG. 9. Orientation speci®city of lateral excitatory and inhibitory connections according to the main tiers of cortical lamination. There is no major difference between excitatory and inhibitory components in either tier. Proximal projections prefer similar orientations and distal projections have the tendency to link dissimilar orientations. In the upper tier, the nine labelled somata at the injection site were found at iso-orientations (not shown). Same conventions apply as in Fig. 7. other than solely orientation selectivity could also play a role in the organization of the projections. Concerning the orientation preference of distal projections, a different picture emerged from that seen for the proximal projections. They commonly represented the entire orientation range in a manner that resulted in an overall preference to non-iso-orientations. When the curves of the distal projections are viewed in detail it is clear that the peaks can occur up to 90° around the orientation preference of the injection sites. This trend was found in all other instances in addition to those shown in Figs 7, 9 and 10. In one case, the excitatory and inhibitory structures could be analysed separately. Figure 9 shows the quantitative results for the three tiers of this case. When the middle tier projections are viewed, there is no clear difference between the excitatory (Fig. 9C) and inhibitory (Fig. 9D) distribution curves. Concerning the upper tier, however, there was a marked difference between the distal excitatory and inhibitory curves (Fig. 9B). While this situation could be typical for upper tier projections, the overall distribution, which was clearly dominated by the middle-tier projections, showed no marked differences between excitatory and inhibitory connections. A similar picture emerged when the labelled excitatory (P-type) somata were taken into account (Fig. 9E). Proximally, they chie¯y occupied similar orientations and distally they preferred a broad range of orientations. Inhibitory (B-type) somata were found only proximal to the injection site at similar orientations (Fig. 9E). Comparison between the labelling methods In order to summarize the orientation distributions according to the applied methods, the following three labelling categories were established; ¯uorescent bead-labelled somata (Fig. 11A and B), biocytin-labelled somata [including excitatory and inhibitory types (Fig. 11C and D) representing retrograde projections] and biocytin-labelled axon terminals [(Fig. 11E and F) representing anterograde projections]. The average `orientation tuning' curve in each category is indicated by a thick line and the data of individual cases by thin grey lines. With regard to proximal projections (Fig. 11A, C and E), there are some notable differences between the different methods. In the case of bead-labelled cells (Fig. 11A) and anterogradely labelled terminals (Fig. 11E), strong non-iso-orientation components were present around the dominant iso-orientation peak that were not seen for the biocytin-labelled somata (Fig. 11C). Concerning distal projections (Fig. 11B, D and F), a common feature of all labelling categories was that the distribution curves were more ¯at than their proximal counterparts and showed no clear-cut preference to any particular orientation range. Interestingly, in the case of biocytin-labelled somata (Fig. 11D) where a strong ¯uctuation Ó 1999 European Neuroscience Association, European Journal of Neuroscience, 11, 4291±4308 Functional topography of orientation selectivity in layer 4 4303 in the curve was present the lowest intensity was found around similar orientations. In order to provide an overall view of the quantitative results, the data presented in Fig. 11 are shown in a cumulative form in Fig. 12A where all cases (anterograde and retrograde) are pulled together. The main message of the resulting graphs is that distal connections show a preference towards dissimilar orientations whereas proximal projections are engaged with similar orientations. For a direct comparison of the present results with the known orientation preference of the layer 2/3 long-range circuitry (KisvaÂrday et al., 1997), the distribution values were expressed in a simple form as shown in Fig. 12B. We used a previously established method for dissecting the orientation map into categories of iso(6 30°), oblique- (6 30±60°) and cross-orientations (6 60±90°) with respect to the orientation preferences at the injection sites, and analysed the pooled data of the layer 4 connections accordingly. There are two important facts to note. First, there is no appreciable difference between the three orientation categories within any of the labelling groups corresponding to anterograde and retrograde labelling (Fig. 12B). Secondly, each group provided very similar distributions that indicate their strong reciprocal relationship. Taken together, the above quantitative data for the distal connections show that all orientation categories are rather equally represented. As a consequence of such an orientation topography, non-iso-orientation connections of oblique- and cross-orientations exceed iso-orientation connections by a ratio of two to one. Discussion The present study was undertaken to explore the orientation selectivity of lateral connections with respect to layer 4. The main ®nding is that lateral connections link all orientations in a rather balanced manner. Because by de®nition the iso-orientation category represents only one-third of the entire orientation range, the ®ndings are equivalent with a bias of the layer 4 lateral connections to noniso-orientations. These data together with previous ®ndings on the iso-orientation bias of the layer 2/3 circuitry (Gilbert & Wiesel, 1989; Malach et al., 1993; Bosking et al., 1997; KisvaÂrday et al., 1997) extend our view as to the heterogeneity of lateral connection to different layers, in this case to layer 4. Technical considerations The major conclusion of the present study on the orientation speci®city of layer 4 projections relies largely on data where no distinction between inhibitory and excitatory components of the labelling could be made. In a previous study, using a combination of electrophysiological mapping of orientation selectivity and anatomical tracing of layer 2/3 neuronal connections, we showed that inhibitory projections tend to surround the termination sites of the excitatory projections (KisvaÂrday et al., 1997). As a result of such an arrangement, the inhibitory elements provided a smaller contribution to iso-orientation locations than the iso-orientation biased excitatory ones. On the basis of these ®ndings a logical assumption is that the percentage values provided here for the oblique- and cross-orientation categories (in Fig. 12B) re¯ect largely inhibitory rather than excitatory connections. We think that this is not the case, because in the single case, where inhibitory and excitatory components could be distinguished, strong non-iso-orientation components were provided both by the excitatory and inhibitory projections at distal locations (Figs 8 and 9). Moreover, the known limited lateral extent of inhibitory projections (1±1.5 mm, for review see KisvaÂrday, 1992) indicates that the most distal projections must represent purely excitatory links. Another point that should be mentioned regards the orientation distribution curves in Fig. 11. As is always the case when different tracers are used, the present study showed some noticeable differences between the soma labelling by latex beads and biocytin (Fig. 11A±D). Although we do not know exactly the underlying factors for the observed differences, we think that the different uptake mechanisms involved and the mode of tracer delivery, pressure injection for the beads and iontophoresis for biocytin played an important role. A similar reasoning may apply when comparing the different data in the literature (Matsubara et al., 1987; Gilbert & Wiesel, 1989). A third point that needs to be taken into account is whether the ®ltering properties of the optical images can signi®cantly affect the orientation distribution of the analysed connections. In order to know this we carried out calculations for three cases (OI20LHr, OI20LHg, OI17RHr). In each case, the optical data were processed with nine different combinations of high- and low-pass ®lters (Fig. 13A), and then the resulting images used for calculating the orientation distribution of the anatomical connections. As one can see in Fig. 13B and C, the orientation distributions of the connections were nearly identical, there was no statistically signi®cant difference (nonparametric test, Pearson correlation) between the curves. Therefore, we are sure that changing the ®ltering parameters of the images does not affect the statistics of the connections. Finally, it is well known that orientation selectivity in the same cortical columns varies slightly from cell to cell (present study of area 18, < 20°; area 17, 5±15° in Albus, 1975b; and 9±18° in Murphy & Sillito, 1986). This scatter of orientation selectivity has to be taken into account for estimating the speci®city of connections between different locations. Using the above values the exactness of our calculations, between any two cortical points, is estimated to be in the range of 6 30±40°, corresponding to a maximum error of 30±45%. Although such a potential error seems to be quite high, it is very much in line with the broad orientation tuning width of individual cells in area 18 (70°, see ®gure 7/6B in Orban, 1984). Orientation speci®city of layer 4 lateral connections Previous studies on the topographic relationship between functional maps and anatomical connections have chie¯y been restricted to the super®cial layers. One of the reasons for this stems from the notion that optical imaging signals derive largely from super®cial layers and upper layer 4 (Bonhoeffer & Grinvald, 1995). In this regard, our optical imaging camera was focused 750 mm below the cortical surface, instead of the commonly used 600 mm, to lower the focal plane well into layer 4. When consecutive images were taken at 750 and 600 mm from the surface there was no appreciable difference between the resulting orientation maps. There are two possibilities in this regard. First, our camera cannot view structures deeper than layer 3, and the images simply re¯ect activity changes from the super®cial layers. Alternatively, our camera can detect deep layer signals, and the correspondence between upper and lower layer images shows that orientation selectivity is organized in columns. At this stage we do not know whether our camera did indeed image layer 4. The second reason that most studies concentrate on super®cial layer connections is due to the well-known presence of pyramidal cells which provide extensive patchy projections (Rockland & Lund, 1982; Gilbert & Wiesel, 1983; KisvaÂrday & Eysel, 1992). However, there is anatomical evidence that other layers could also play an important role in horizontal interactions. Indeed, some layer 4 spiny stellate cells give rise to clustered axons in layer 3 (Martin & Whitteridge, 1984), and some layer 5 pyramidal cells also have clustered projections in their own layer and in overlying layers 1±3 Ó 1999 European Neuroscience Association, European Journal of Neuroscience, 11, 4291±4308 4304 T. Yousef et al. Ó 1999 European Neuroscience Association, European Journal of Neuroscience, 11, 4291±4308 Functional topography of orientation selectivity in layer 4 4305 (Gilbert & Wiesel, 1983; Martin & Whitteridge, 1984; Gabbott et al., 1987). These anatomical ®ndings strongly suggest that lateral connections originating in the supragranular layers represent only part of the horizontal system in the visual cortex. Then the question arises whether the orientation preferences of layer 4 lateral connections differ from those of the more super®cial and deep layers. Our quantitative results clearly demonstrated that layer 4 distal projections possess an almost equal preference to iso-, obliqueand cross-orientations (see Fig. 12) that is not the case for the layer 2/ 3 projections of area 18 (KisvaÂrday et al., 1997). Concerning the orientation topography of the deep layers, there are no available data yet. FIG. 11. Quantitative evaluation of the orientation speci®city of layer 4 lateral projections as distinguished by different labelling methods: (A and B) with ¯uorescent latex beads; (C and D) retrograde biocytin labelling; (E and F) anterograde biocytin labelling. Left-hand panels show graphs of proximal, right-hand panels distal projections. Individual cases are drawn by grey lines and averages shown by black lines. Generally, proximal projections are engaged with similar orientations although other orientations are also represented for the bead labelling. A different distribution is seen for distal projections, they are distributed to many orientations with a bias to dissimilar orientations. FIG. 10. Orientation topography of ¯uorescent bead-labelled cells after layer 4 injections. (A, C and E) The three cases of bead labelling in superimposition with vascular maps. (B, D and F) Their corresponding orientation angle maps. Insets on the left-hand side indicate the laminar location of the injection cores (in red), the halo regions (in black) and sites of possible back¯ow (in grey) of the tracer along the injection track. In all three cases, the main body of the injection was con®ned to layer 4 (see insets), although for case OI20LHg with some additional contribution to upper layer 5. The labelling was densest at proximal locations (within the circles). More distally, only a few and less densely labelled clusters emerged providing an overall pattern that resembles the biocytin cases. The orientation preferences at the injection sites shown in (G±I) were calculated from respective angle maps shown in (B, D and F). Of the three injections, case OI20LHr was made closest to the middle of an orientation domain; this resulted in the narrowest orientation range, a single orientation bin in (G). Concerning the labelled projections (J±L), in none of the cases could a clear segregation according to a narrow orientation range be observed. Importantly, distal projections (solid lines) show an overall preference to dissimilar orientations. The same conventions apply as in Fig. 7. Scale bar, 1 mm (A±F). Ó 1999 European Neuroscience Association, European Journal of Neuroscience, 11, 4291±4308 4306 T. Yousef et al. FIG. 12. (A) Cumulative orientation distribution of layer 4 projections as revealed by the present study, including retrograde and anterograde labelling. Proximal projections, grey line; distal projections, black line. Broken line indicates the percentage of distal projections if they were evenly distributed to orientations. (B) Bar graphs show the cumulative results for anterograde (biocytin-labelled boutons), retrograde (bead + biocytin-labelled somata) and all (anterograde + retrograde) cases according to categories of iso- (6 30°), oblique- (6 30±60°) and cross-orientations (6 60±90°) with respect to the orientation preferences calculated for the injections. Non-iso-orientation (oblique- and cross-orientations) connections account for 64% of all distal projections. TABLE 2. Orientation distribution of proximal and distal projections Case Label OI20LHr Somata OI20LHg Somata OI17RHr Somata OI25LH Boutons Somata OI26RH Boutons Somata F081292 Boutons F261093 Boutons Somata Average Distance from initial segment Orientation preference Iso Proximal Distal Proximal Distal Proximal Distal Proximal Distal Proximal Distal Proximal Distal Proximal Distal Proximal Distal Proximal Distal Proximal Distal 48.9 41.3 45.9 36.0 33.8 30.7 60.8 41.2 89.7 37.0 66.4 32.6 73.6 40 29.2 38.9 68.9 30.3 82.7 26.0 Proximal Distal 60.0 35.4 (2531) (829) (7931) (3336) (1147) (338) (18325) (3245) (559) (10) (17950) (2840) (89) (8) (38547) (54842) (23719) (7685) (354) (32) Oblique Cross 28.0 19.4 16.0 25.3 32.0 28.5 15.8 35.1 4.0 37 24.4 42.8 19.0 50 32.7 35.5 30.9 35.5 17.3 27.6 23.1 39.3 38.1 38.7 34.2 40.8 23.4 23.7 6.3 26.0 9.2 24.6 7.4 10 38.1 25.6 0.2 34.2 0.0 46.4 22.0 33.7 (1449) (389) (2759) (2341) (1083) (313) (4763) (2763) (25) (10) (6581) (3724) (23) (10) (43156) (41592) (10640) (8995) (74) (34) Total (100%) (1199) (787) (6571) (3581) (1158) (449) (7038) (1871) (39) (7) (2498) (2146) (9) (2) (50337) (32051) (92) (8647) (0) (57) (5179) (2005) (17261) (9258) (3388) (1100) (30126) (7879) (623) (27) (27029) (8710) (121) (20) (132040) (128485) (34451) (25327) (428) (123) 18.0 30.9 Data are given as a percentages, with absolute values in parentheses. Previous estimates on the orientation speci®city of lateral connections For the aforementioned reasons, virtually all available information on the speci®city of lateral projections with respect to orientation are concerned with layer 2/3. Brie¯y, the results obtained in cat could be divided into two major groups as to whether they support the connectivity concepts of linking similar (Michalski et al., 1983; Nelson & Frost, 1985; Ts'o et al., 1986; Gilbert & Wiesel, 1989) or dissimilar orientations (Matsubara et al., 1985, 1987). Interestingly, recent cross-correlation data indicate that lateral connections are neither highly speci®c nor random for orientation (Hata et al., 1991; Tamura et al., 1996). Functional±anatomical results strongly support such a view. In the study of KisvaÂrday et al. (1997), 53±59% of excitatory and 46±48% of inhibitory connections were shown to prefer similar orientations, whereas a considerable proportion of both functional types connected to dissimilar orientations including crossorientations. Concerning other species, the data on the orientation speci®city of lateral connections have also been restricted to the super®cial layers. In Ó 1999 European Neuroscience Association, European Journal of Neuroscience, 11, 4291±4308 Functional topography of orientation selectivity in layer 4 4307 their measurements did not include projections outside the patches, where non-iso-orientations are likely to be encountered, the actual numerical ®gure given in their report probably represents an underestimate of the total proportion of non-iso-orientation connections. In V1 of the tree shrew, the orientation analysis of corticocortical connections was carried out by taking into account patch as well as interpatch regions (Bosking et al., 1997). The result obtained for distances longer than 500 mm from the injection centres showed that an average of 57.6% of the connections occupied sites with orientation preferences within 6 35° of the average preferred orientations of the injection sites. This ®gure is in concordance with our recent calculation obtained in layer 2/3 of the cat visual cortex (average, 52.9%, > 650 mm using 6 30° con®dence range in KisvaÂrday et al., 1997) but it differs from the present layer 4 data. Functional implications FIG. 13. An example of testing whether different ®ltering parameters can signi®cantly change the distribution of orientation domains and, therefore, the orientation statistics of lateral connections. (A) The nine combinations of highand low-pass ®ltering values applied in the test. (B and C) The resulting orientation distribution graphs are shown for the proximal and distal connections of case OI20LHr, respectively. Clearly, in both panels, the nine graphs have an almost identical course indicating that ®ltering of optical images plays little if any role in the orientation statistics of the connections studied. the primate (Malach et al., 1993, 1994) and the tree shrew visual cortex (Bosking et al., 1997), optical imaging of orientation selectivity was combined with anterograde tracing of biocytin. In the study of Malach et al. (1993), approximately half of the patchy projections were found at orientations which were not present at the injection sites. Because The present ®ndings show that the layer 4 horizontal network has a complex relationship with orientation maps. We believe that such a circuitry could account for electrophysiological and psychophysical results that are dif®cult to interpret by simple network rules, e.g. `like connects to like'. Layer 4, being the main recipient layer of the primary afferents, is assumed to be involved in the generation of novel receptive ®eld features like orientation selectivity. In this regard, recent data from our laboratory using the remote inactivation paradigm indicate that in layer 4 lateral cross-orientation inhibitory interactions have an in¯uence on the sharpening of orientation selectivity (Crook et al., 1998). The present study provides anatomical support for the observed effects. Furthermore, our results are compatible with the view in which the above inhibitory effects could be mediated either directly using cross-orientation inhibition or indirectly, via crossoriented excitatory connections terminating on inhibitory cells. At this stage, we can only anticipate that a similar circuitry to the one shown here underlies a range of other electrophysiological observations. For example, in primate V1 some neurons prefer complex visual stimuli consisting of radically different orientation components, respectively, within and outside of their `classical' receptive ®elds (Sillito et al., 1995). In cat visual cortex, there are socalled double orientation tuned neurons in most layers including layer 4 which are best driven by cross-like ®gures (Shevelev et al., 1994, 1995). Conceivably, the `input±output' sites of these neurons ought to be engaged with many different orientations as in the network presented here. Finally, a potential implication of lateral connections associated with a broad range of functional speci®city is that the individual neuron is subjected to a host of modulatory effects (Gilbert & Wiesel, 1990; Kapadia et al., 1995). Following this line of thought onto large networks, such interactions might be crucial in accounting for some psychophysical effects. For example, ®gureground segmentation tasks and perceptual grouping of distributed feature elements probably involve mechanisms based on feature dissimilarity (Nothdurft, 1992; Lamme, 1995; Kastner et al., 1997; Baumann et al., 1997) or feature similarity (for review see Singer & Gray, 1995), respectively. The present study suggests that for orientation cues such interactions could be mediated at a ®rst stage already by the layer 4 circuitry. Acknowledgements The authors would like to thank Ms D. Strehler and Ms P. Hentrich for photography, Mr RoÂbert Magyar for his computer expertize, and Drs PeÂter BuzaÂs, Maxim Volgushev and Bashir Ahmed for helpful comments on earlier version of the manuscript. This work was supported by the Deutsche Ó 1999 European Neuroscience Association, European Journal of Neuroscience, 11, 4291±4308 4308 T. Yousef et al. Forschungsgemeinschaft (SFB509, TPA6 to Z.F.K. and Ey 8/23) and the European Communities (SC1 0329-C). References Albus, K. 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