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Functional topography of single cortical cells: an intracellular approach combined with optical imaging
Brain Research Protocols 3 Ž1998. 199–208
Protocol
Functional topography of single cortical cells: an intracellular approach combined with optical imaging Peter ´ Buzas, ´ Ulf T. Eysel, Zoltan ´ F. Kisvarday ´
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Ruhr-UniÕersitat 150, ¨ Bochum, Medizinische Fakultat, ¨ Institut fur ¨ Physiologie, Abteilung fur ¨ Neurophysiologie, UniÕersitatsstrasse ¨ MA 4 r 149, 44801 Bochum, Germany Accepted 29 July 1998
Abstract Pyramidal cells mediating long-range corticocortical connections have been assumed to play an important role in visual perceptual mechanisms wC.D. Gilbert, Horizontal integration and cortical dynamics, Neuron 9 Ž1992. 1–13x. However, no information is available as yet on the specificity of individual pyramidal cells with respect to functional maps, e.g., orientation map. Here, we show a combination of techniques with which the functional topography of single pyramidal neurons can be explored in utmost detail. To this end, we used optical imaging of intrinsic signals followed by intracellular recording and staining with biocytin in vivo. The axonal and dendritic trees of the labelled neurons were reconstructed in three dimensions and aligned with corresponding functional orientation maps. The results indicate that, contrary to the sharp orientation tuning of neurons shown by the recorded spike activity, the efferent connections Žaxon terminal distribution. of the same pyramidal cells were found to terminate at a much broader range of orientations. q 1998 Elsevier Science B.V. All rights reserved. Themes: Sensory system Topics: Visual cortex, functional topography Keywords: Optical imaging; Intracellular recording and staining; Three-dimensional neuron reconstruction; Orientation selectivity
1. Type of research
Ø Analysis of the relationship between the physiological maps and anatomical reconstructions: a few days.
Functional topography of corticocortical connections.
2. Time required The protocol is subdivided into four major parts. Ø In vivo experiment: four days. Days 1–2: optical imaging of intrinsic signals. Days 2–4: electrophysiological recordings and tracer injections. Ø Tissue processing and histology: three days. Ø Three-dimensional reconstruction of the labelled neurons: 4–10 weeks or about 100–200 working hours per neuron depending on its complexity.
) Corresponding author. Fax: q49-234-709-4192; E-mail: [email protected]
3. Materials 3.1. Experimental animals Cats, 8–12 months old. 3.2. Special equipment Ø Electrophysiological laboratory equipped for acute in vivo experiments. Ø Amplifier for intracellular recording ŽAxoclamp-2A, Axon Instruments, Foster City, CA, USA.. Ø Electrode puller ŽP-87 FlamingrBrown micropipette puller, Sutter Instrument, Novato, CA, USA.. Ø Micropipette beveller ŽBV-10, Sutter Instrument..
1385-299Xr98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 5 - 2 9 9 X Ž 9 8 . 0 0 0 4 1 - 5
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Ø Optical imaging system Imager 2001 and data acquisitionranalysis software ŽOptical Imaging, Germantown, NY, USA.. Ø Visual stimulus generator for optical imaging and single cell electrophysiology ŽVision Research Graphics, Durham, NH, USA.. Ø Vibratome ŽModel 1000, Pelco International, Redding, CA, USA.. Ø Light microscope ŽLeica, Wetzlar, Germany. equipped with the three-dimensional neuron reconstruction system, Neurolucida ŽMicroBrightField, Colchester, VT, USA.. 3.3. Chemicals and reagents Ø Paraffin wax Žmelting point s 42–448C, Merck, Darmstadt, Germany.. Ø Dental cement ŽPaladur, Heraeus Kulzer, Wehrheim, Germany.. Ø Silicone oil Ž50 cSt, Aldrich Chemical, Milwaukee, WI, USA.. Ø Biocytin ŽSigma, Deisenhofen, Germany.. 4. Detailed procedure An overview of the entire experimental paradigm is shown in Fig. 1. 4.1. Preparation of the animal for in ÕiÕo optical imaging of intrinsic signals An anaesthetised, paralysed adult cat is prepared for acute optical imaging of intrinsic signals using a modified procedure of Blasdel and Salama w1x and Grinvald et al. w2x Žreviewed in Ref. w3x.. Briefly, initial surgical anaesthesia is introduced by a mixture of ketamin Ž7 mg kgy1 , Ketanest, Parke-Davis, Berlin, Germany. and xylazin Ž1 mg kgy1 , Rompun, Bayer Belgium, Sint-Truiden, Belgium. i.m. A catheter is inserted into the femoral artery to monitor blood pressure ŽBP-1 Pressure Monitor, World Precision Instruments, Sarasota, FL, USA. and to infuse a mixture of muscle relaxant Žalcuronium chloride, 0.15 mg kgy1 hy1 , Alloferin, Hoffman-La Roche, GrenzachWhylen, Germany. and glucose Ž24 mg kgy1 hy1, Glucosteril, Fresenius, Bad-Homburg, Germany. in Ringer solution ŽRingerlosung Fresenius, Fresenius, Bad-Homburg, ¨ Germany. continuously during the experiment. Artificial ventilation ŽCatrRabbit Ventilator, Ugo Basile, Comerio, Italy. and prolonged anaesthesia is provided through tracheal cannula using a 1:2 mixture of O 2 and N2 O supplied with 0.4–0.6% of halothane ŽHalothan Eurim, EurimPharm, Piding, Germany.. All pressure points are treated with the local anaesthetic, Xylocain gel ŽAstra Chemicals, WedelrHolstein, Germany.. The major physiological parameters Žend-tidal CO 2 : 3.0–4.0%, blood pressure: 100– 140 mmHg, heart rate: 160 miny1 , body temperature:
38–398C, and EEG. are continuously monitored and maintained. A bilateral pneumothorax Žsee also Section 4.5.1. is prepared to reduce artificial ventilation-related brain movement. For monitoring lung-function, a plastic tube connected to an elastic balloon Žsoft, powder-free latex exam gloves, Safeskin, Neufahrn, Germany. is inserted into the thorax on both sides of the chest. A 6 = 15 mm craniotomy is made between Horsley–Clarke coordinates AP y4 and q9 and LM q0.5 and q6.5 and a round, metal chamber is mounted over the exposed region using dental cement ŽPaladur. ŽFig. 2a.. Once the chamber is fixed, the dura mater is removed. At this step, great care is taken not to damage subdural blood vessels because optical imaging relies on light absorption differences between oxygenated and de-oxygenated haemoglobin, consequently, bleeding in the imaged region may cause artefacts. Another point that is important to mention is that removal of the dura mater inevitably causes bleeding of cut sinuses. There are several ways to stop dural bleeding, three are listed here. First, the cut edge of the dura is gently folded out so that the lumen of the damaged sinuses becomes squeezed onto the bone around the exposed region. Another method is to clamp the sinuses with a fine-tip forceps and hold for 1–2 min. If none of these methods helps in stopping the dural bleeding, heat coagulation of the sinuses with a fine-tip cauter device ŽFine Science Tools, Heidelberg, Germany. could be useful. The recording chamber has at least two outlets at its base, each connected to a stopcock via thin plastic tubes. These outlets serve to release or provide hydrostatic pressure when the chamber is closed with the cover-glass and when the cover-glass is removed. Finally, the chamber is filled with silicone oil Ž50 cSt, Sigma-Aldrich Chemie GmbH, Steinheim, Germany. and closed with the round cover-glass. It should be noted that during the experiment, the silicone oil gradually becomes milky probably due to an emulsification process with the watery based cerebrospinal fluid. In addition to this, blood could slowly diffuse into the oil that also causes deterioration of the optics and results in unwanted quality loss in the acquired optical images. Therefore, we recommend to clean the chamber and replace the silicone oil about every 8–10 h. 4.2. Optical imaging During optical imaging, the so-called vascular or green-image is first taken under green illumination Ž450 nm, Spindler and Hoyer, Gottingen, Germany. to show the ¨ pattern of surface blood vessels of the brain. It is essential to take a green-image every 2 h or before and after each recording session because of possible shifts in the focal depth, for example, due to mild cortical oedema or dehydration. Another important application of green-images is that they are used for aligning the optical images with the histological reconstructions Žsee Section 4.8.1.. For data acquisition, it is recommended to narrow down the image
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size to an area called ‘region of interest’ that saves a significant amount of computation time and disk space. It also improves the signal-to-noise ratio by eliminating potential noise sources from the images Že.g., from surrounding bone tissue and large blood vessels..
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Visual stimuli are generated with the Neurosequence program of the Vision Works stimulus generation system ŽVision Research Graphics, Durham, NH, USA.. Each stimulus presentation is triggered by the optical imaging system using a total of five output bits for coding the
Fig. 1. Scheme of the experimental paradigm, data acquisition, and analysis used. Numbers indicate the sequence of individual steps.
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stimulus type and one bit ŽGo-bit. for moving the stimulus during data acquisition. Orientation maps are acquired during the presentations of moving square-wave gratings at four orientations Ž08, 458, 908, and 1358. which move in one of the perpendicular directions and then in the opposite direction. The reversal in the direction of movement occurs at half of the data acquisition period. The stimulus gratings are shown on a 21Y computer monitor Ž120 Hz, non-interlaced mode, SONY, Pencoed, UK. 285 mm from the cat’s eyes. Spatial Ž0.1–0.2 cycle degy1 , 50% duty cycle. and temporal frequencies Ž1–2 Hz. are optimised in a few, short recordings for every new part of the cortex to be mapped. For obtaining orientation maps, individual stimuli are presented 25–50 times for 4.5 s followed by an interstimulus interval for 10 s when the animals view a blank screen. The cortex is illuminated with light at 605 " 5 nm ŽOmega Optical, Brattleboro, VT, USA. using a circular fibre optic slit lamp ŽSchott, Mainz, Germany.. Video images are recorded with a slow-scan Ž25 video frames sy1 . CCD camera ŽBischke CCD 6012P, 6 = 8 mm sensor. using a ‘tandem-lens’ of two objectives ŽSMC Pentax 1:1.2, 50 mm.. The images are collected and processed using the system Imager 2001 ŽOptical Imaging. and Matrox IM-640 imaging board controlled by the software VDAQ version 2.18k. 4.3. Analysis of the optical images For image analysis, each image acquired for a particular orientation is corrected for time related fluctuations in the signal using the ‘cocktail blank method’. Accordingly, each image is divided by the sum of images for all orientations resulting in so-called single condition maps using the MIX program of the system Imager 2001 ŽOptical Imaging.. The grey value distribution of each single condition map is ‘clipped’ by discarding the flanks outside "3 S.D. around mean to exclude possible artefacts and then scaled to the range between 0–255. Further evaluation of the images is done in two steps using a custom software
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written in IDL ŽResearch Systems, Boulder, CO, USA.. First, a high-pass filtering is applied using a 426-mm pixel-kernel followed by a low-pass filtering before the differential maps are vectorially summed. Orientation Žangle. maps are produced by summing the vector components pixel-by-pixel where the vector angle corresponds to the respective stimulus orientation and the vector length to the response intensity coded in grey values. The cortical area represented by a single image-pixel is measured by taking an image of a mesh-grid Ž10 mm grid.. After the optical imaging session, the chamber is carefully opened and the cover-glass and the gaskets are removed. The surface of the cortex is rinsed with several changes of Ringer solution or artificial cerebrospinal fluid until no silicone oil is visible. 4.4. Reference penetrations Reference penetrations are crucial landmarks in matching the functional Že.g., orientation maps. and morphological maps Že.g., the distribution of retrogradely labelled neurons.. For this purpose, long-tapered glass micropipettes with 10–15 mm external tip diameter were found most suitable because they only cause a very fine tissue scar w4,5x. The pipette is positioned perpendicular to the cortical surface, lowered into the cortex 1000 mm deep and the stereotactic position of its entry point is registered with 10 mm accuracy. In addition, the exact position of each penetration point is marked on an enlarged print of the green-image Žsurface vascular image.. In this way, reference penetrations with known antero-posterior and latero-medial distances and exact positions with respect to the surface blood vessels can be obtained. We recommend a minimum of five reference penetrations each separated by 500–1000 mm from its neighbours in the central 3r4 of the imaged regions. 4.5. Electrophysiology and tracer injection In this part of the experiment, an attempt is made to register the main electrophysiological parameters Žrecep-
Fig. 2. Panel Ža. shows the recording chamber used in optical imaging. The cover-glass and the silicone oil through which the imaging takes place are transparent. The CCD-camera and the illumination source are removed for clarity. In panel Žb., a schematic cross-section of the same chamber prepared for intracellular electrode application is seen. Once the recording electrode-tip establishes direct contact with the cortical surface, the chamber is filled-up with low-melting point paraffin wax. Panel Žc. shows the light microscopic image of the soma and part of the dendritic field of a layer III pyramidal cell. The pyramidal cell was intracellularly tested for its orientation preference and injected with biocytin. Due to the horizontal sectioning plane, the apical dendrite is visible only in more superficial sections Žc s capillary.. In panel Žd., a computer-assisted reconstruction of the same pyramidal cell is rotated into the parasagittal plane. In this view, the laminar distribution of the axon Žin red. and dendritic field Žin black. could be readily discerned. Comparison between the topography of the pyramidal cell shown above Žpanels e and f. and the optically imaged orientation map. In Že., the vascular image containing five reference penetrations Žstars. is overlaid with the density map of the axon terminals. Colour scheme to Že. indicates the number of terminals per image-pixel. In Žf., the orientation map is overlaid with the axon terminal distribution Žshown in black for clarity.. Notice that although the densest axon terminal clusters are found at similar orientations, the entire axon establishes contacts over a broad orientation range with respect to that of the soma location Žwhite asterisk.. Panels Žg. and Žh. provide a quantitative comparison between the orientation tuning measured electrophysiologically and the orientation distribution of efferent connections detected anatomically for the same neuron. The physiological orientation tuning of the cell Žpanel g. is based on the rate of the visually evoked spike response during the in vivo intracellular recording. The tuning curve of the boutons in panel Žh. shows the number of labelled boutons found in regions representing the various stimulus orientations. Clearly, the efferent connections represent a 1.5 times broader orientation tuning than that measured from the spike responses. Broken lines indicate the activity level between stimulus presentations in Žg. and the number of axon terminals if they were distributed evenly over all orientations in Žh..
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tive field characteristics. of single cortical neurons and subsequently stain the individual neurons intracellularly. It is advisable to search for neurons in the central region of the optically imaged functional map, since corticocortical connections often have a lateral extent up to 3–4 mm. For guiding the electrodes into functionally characterised locations, for example, into orientation centres, large prints of the surface vascular and functional maps could be useful.
4.5.1. Reducing brain moÕement for intracellular electrode applications Artificial ventilation as well as heartbeat result in movement of the brain hindering stable intracellular recording. The former could be substantially reduced by applying pneumothorax and setting the inhaled gas volume Ž20–25 ml for a 3-kg cat. so that the inflated lung does not reach the volume capacity of the thorax. It is also important to hang the chest by a hook inserted between the first thoracic spines. If necessary, further stabilisation could be achieved by puncturing the cisterna magna at the level of the first few cervical spines, thereby lowering the intracranial pressure Žsee Ref. w6x.. Another important factor in obtaining a ‘non-pulsating’ cortex is to maintain a slightly lower blood pressure Ž100–120 mmHg. using mild hyperventilation Ž3.0–3.8% CO 2 .. Finally, the electrode is positioned into its desired location under the control of an operating microscope and advanced until the pia mater is penetrated. At this stage, the impedance of the electrode is measured and the chamber is filled with a thick layer of low-melting point Ž42–448C. paraffin wax ŽFig. 2b..
4.5.2. Intracellular recording Choosing the right electrode might need some experimenting. In our hands, sharp borosilicate glass electrodes ŽGB150 F-8P, Science Products, Hofheim, Germany. pulled on a horizontal puller ŽP-87 FlamingrBrown micropipette puller, Sutter Instrument. worked best. The electrodes are filled with freshly prepared 0.5 M K-acetate containing 1–2% biocytin ŽSigma. and kept in a wet chamber for 1–2 h in a position such that the electrode shafts are pointing upright. During that time, the air-bubbles usually disappear from the electrode tip. To foster this process, vacuum can be applied using a 20-ml or largervolume syringe connected to the electrode shaft. It is advisable to use always fresh biocytin solution for two reasons. First, K-acetate decays with time resulting to conductivity loss. Second, biocytin might precipitate during long storage. The electrodes are bevelled on a BV-10 micropipette beveller ŽSutter Instrument. using an alumina-grinding plate Ž0.05 mm particle size, 22–278 bevelling angle, w7x.. As a result of the bevelling, the input resistance of the electrodes decreases Žincreased tip lumen. from an initial 100–120 M V to 40–105 M V final resistance and the tip becomes sharp. Hence, the electrode can
readily penetrate the cell membrane and pass sufficient current to drive biocytin into the intracellular space. Before use, the tip of each electrode is inspected under a light microscope Ž=100. for mechanical damage, surface contamination, and air-bubbles. Successful penetration of the cell membrane is indicated by a sudden 40–70-mV drop in the measured potential. After determining the position and extent of the receptive field using hand-held stimuli, orientation tuning curves are derived from recordings ŽSpike-2 software, Version 2.02, Cambridge Electronic Design, Cambridge, UK. in response to computer-generated moving light or dark bars at optimal length and velocity moving at each of eight equally spaced orientations presented in a pseudo-random manner on the 21Y computer monitor. The recorded potentials are digitised using the CED 1401 interface ŽCambridge Electronic Design Ltd., Cambridge, UK. and recorded on a personal computer. Orientation tuning is calculated as the average spike frequency during the presentation of the respective stimuli.
4.5.3. Intracellular deliÕery of biocytin Intracellular recording is followed by iontophoretic delivery of biocytin into the cell using positive 2–3.5 nA rectangular current pulses Ž200 ms ON 400 ms OFF duty cycles. for 1–5 min. The injection is briefly interrupted for a few times during which the orientation preference and receptive field location of the neuron are checked. After completion of the intracellular injection, the electrode is rapidly withdrawn from the cortex.
4.6. Tissue processing and histology After appropriate survival time Ž1–24 h., the animal receives an overdose of anaesthetics and is perfused transcardially with cold Ž48C., oxygenated Tyrode’s solution followed by a mixture of 2% paraformaldehyde and 0.5% glutaraldehyde in 0.1 M phosphate buffer ŽpH s 7.6.. Tissue blocks that are 2–3 mm larger on each side than the optically imaged region are dissected, the remaining parts of the dura mater and the arachnoidea are gently peeled-off with a fine-tip forceps and consecutive, 60–80-mm thick sections are cut using Vibratome. The cutting plane is set as parallel to the imaged cortical surface as possible. The sections are collected in 10 lots and rinsed in 0.1 M PB for 3 = 20 min to remove unbound fixative. Biocytin labelling is visualised in free-floating sections using the ABC method w8,9x with slight modifications and supplemented with cobalt intensification w10x. Briefly, the sections are incubated in 1:200 avidin–biotin-complexed horseradish peroxidase ŽABC, Vector Laboratories, Burlingame, CA, USA. in 0.05 M Tris-buffered saline ŽTBS, pH s 7.6. containing 0.05% Triton X-100 while
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gently agitated Ž48C, overnight.. The enzymatic reaction is revealed with 0.005% 3,3X-diaminobenzidine-4-HCl ŽDAB; Sigma. in TBS supplemented with CoCl 2 intensification Ž0.0025%. for 20 min. The latter is served to increase the contrast of the staining under the light microscope Žbluishblack precipitate. as well as improve the opacity of the reaction end-product for possible electron microscopy w10x. The reaction is completed in the presence of 0.001% H 2 O 2 for 1–5 min. Then, the sections are rinsed in TBS Ž2 = 15 min., PB Ž2 = 15 min., postfixed in 0.5% OsO4 Žin 0.1 M PB. for 10–20 min and dehydrated in ascending series of ethanol. At this phase of the procedure, there are two important factors to be taken into account. First, osmium tetroxide and dehydration make the tissue rigid and fragile. Secondly, large sections Žabout 10 = 5 mm2 . containing the grey and the white matter often become wrinkled due to uneven shrinkage. To prevent wrinkling, the sections are flat-embedded using a modified procedure of Somogyi and Freund w11x. Accordingly, the osmicated sections are kept between glass slides and coverslips during the entire dehydration process in ascending ethanol series. Then, they are transferred into propyleneoxide for 2 = 15 min and rapidly submerged into resin ŽDurcupan ACM, Fluka, Neu-Ulm, Germany. for 24 h at room temperature. Finally, the resin-embedded sections are mounted onto slides, coverslipped and cured at 568C for 24 h. 4.7. Three-dimensional reconstruction In order to obtain information on the spatial organisation of the labelled neurons with respect to the distribution of functional maps, the neuron reconstruction system, Neurolucida ŽMicroBrightField, Colchester, VT, USA. is used. The three-dimensional coordinates of the labelled soma, dendritic and axonal fields and axon terminals Žboutons. are reconstructed at =1000 magnification using a light microscope ŽLeica DMRB. fitted with a computer-controlled motorised stage ŽMarzhauser-Wetzlar, Wetzlar, ¨ Germany. and controller box ŽLUDL MC2000 XYZ, Ludl Electronic Products, Hawthorne, NY, USA.. 4.7.1. Finding the best match between adjoining sections A critical step in reconstructing axonal and dendritic fields that are sliced up during sectioning is how to find the best match between adjoining sections w12,4,5x. To this end, we use labelled fine processes, for example, axon collaterals and dendrites, which extend from one section to the other offering corresponding cut ends that can be matched with high precision. We recommend to choose several pairs of cut-ends in various parts of the estimated projection field of the labelled neuron. In our practice, cut-ends separated by 2–4 mm in the plane of the sections provide 5–10-mm matching accuracy. Once the labelled anatomical structures are compiled into a single structure, they are matched with the optical images as described in Section 4.8.1.
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4.7.2. Reference penetrations It has to be emphasised here that reference penetrations and tissue marks like surface blood vessels are crucial landmarks in obtaining the best match between the anatomical reconstructions and the optical images w4x. Therefore, attention is paid to find and reconstruct the exact location of the reference penetrations where they entered into the cortex. Unfortunately, the most superficial sections often contain blood vessels, large number of dark macrophages and connective tissue debris which obscure these locations. In this case, penetration marks from deeper sections are to be used and extrapolated to the surface sections. If visible, tracks of the recording electrodes with known entry points Žmarked on the green-image. could also be useful in the matching procedure. Finally, the reconstruction of the labelled neuronal processes Žaxonal and dendritic fields., the reference penetrations and other structures of interest Že.g., section contours. should be made in the same coordinate system. 4.8. Data analysis 4.8.1. Alignment of the reconstructed structures with the optical maps The aim of the aligning procedure is to transform the three-dimensionally digitised anatomical data into the two-dimensional coordinate system of the optical maps. In this procedure, only linear transformations are applied such as rotation, translation, and scaling. Accordingly, the anatomical coordinate system is defined by the X,Y,Z-axes of the motorised microscope stage, where the Z-axis represents the focus. This is then transformed into the ‘in vivo coordinate system’ where the XY plane corresponds to the plane of the optical image and the Z-axis represents the axis perpendicular to that plane. Because the optical image plane runs parallel with the cortical surface, the Z-axis corresponds to the cortical depth. The aligning procedure is carried out in four steps. First, the anatomical reconstruction is corrected for virtual shrinkage in the Z-axis caused by the optical density of the embedding medium, the epoxy resin, and the microscope immersion oil Žif used.. The microscopically measured Z-values are multiplied by the correction factor, f s n resinrn oil , where n resin s 1.549 is the experimentally determined index of refraction for the epoxy resin and n oil s 1.5180 ŽLeica. is the index of refraction for the immersion oil. In the second step, the data is tilted until the reconstructed structures are viewed from the plane of the optical images. In practice, small rotation about the X and Y axes is applied until the projection of each of the reference penetration tracks running perpendicular to the cortical surface is viewed as a spot. For all reference penetrations, the optimal rotation angles could be calculated using, for example, the least-square approximation algorithm. When the anatomical data have been rotated and projected into an image plane closely matching that of
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the optical image plane, the data are ready for the third step, correction for histology-related tissue shrinkage. The shrinkage factor in the XY plane is determined on the basis of reconstructed reference penetrations whose relative distances from each other have been measured in vivo w5x. Concerning Z-direction shrinkage, the shrinkage factor calculated for the XY plane is applied assuming that the tissue underwent equal shrinkage in each dimension. In the fourth step, the data are rotated around the Z-axis and translated until the corresponding reference penetration points of the anatomical reconstruction and of the optical image are matched using the least-square approximation algorithm.
imposed on the vascular image. The semi-coloured scheme on the right-hand side codes for the number of axon terminals per pixel Ž1–15.. The five reference penetrations marked by white stars serve to match the anatomical reconstruction with the corresponding vascular and orientation map images. In panel Žf., the same axon terminal density distribution is shown Žwithout colour-coding. in register with the functional orientation map. Interestingly, some axon clusters occupy regions Žyellow, ochre, and red. of similar orientations to that of the location of the parent soma and dendritic field Žwhite asterisk in f., whereas other clusters, although smaller and less dense, are found at dissimilar orientations.
4.8.2. QuantitatiÕe calculations In order to explore the functional topography of individual cells, the distribution of the labelled axon terminals is compared with the distribution of the functional orientation map w13,14x. The orientation map is composed of image pixels each covering a known area, whereas the axon terminals are represented by coordinate points. In order to carry out a quantitative analysis, the number of axon terminals is counted Žbinned. in every image pixel using a custom software written in IDL ŽResearch Systems.. The program generates a two-dimensional grid whose pixel size is identical to that of the optical image, overlays it onto the axon terminal distribution and counts how many axon terminals are found in every pixel Žbinning.. The resulting anatomical density map can now be directly compared with the orientation map on a pixel-by-pixel basis.
5.2. Comparison between the orientation tuning of spike actiÕity and the orientation topography of efferent connections of a layer III pyramidal cell
5. Results 5.1. Anatomy and functional topography of a layer III pyramidal cell in the cat Õisual cortex (area 18). In Fig. 2c,d,e,f, an example is provided for a biocytinlabelled layer III pyramidal whose efferent connections are compared with the distribution of orientation selectivity obtained with optical imaging of intrinsic signals. Here, we want to determine what orientations are encountered by the axon terminals of the layer III pyramidal cell. In Fig. 2c, the light microscopic image of the soma and spiny dendrites are viewed from the cortical surface Žc s capillary.. In panel Žd., the same three-dimensionally reconstructed pyramidal cell is seen from the parasagittal plane, so that its laminar distribution could be determined. Characteristically, the axon arborises in two main tiers of the cortex, in layers IIrIII and V. At about 1 mm lateral from the parent soma, clustering of the axon collaterals occurs, but other parts of the axonal field are not clustered at all. In panel Že., the density distribution of the reconstructed axon terminals is shown. Their distribution is determined on a pixel-by-pixel basis as described in Section 4.8 and super-
In panel Žg., the orientation tuning of the pyramidal cell is determined on the basis of visually evoked spike activity. When the distribution curve is fitted with a Gaussian curve Ždata are not shown., it provides a quantitative measure of the orientation tuning of the cell. The results reveal that this pyramidal neuron is selectiÕe Ž188, halfwidth at half-height. for a horizontal stimulus orientation. In panel Žh., the orientation distribution of the efferent connections Žaxon terminals. of the same cell is calculated as described in Section 4.8.2. Interestingly, the resulting ‘tuning’ curve for the axon terminals is 1.5 times broader than that seen for the spike activity Ž278, half-width at half-height., while their peak values differ only by 118 from each other. These findings suggest that the functional topography of long-range excitatory neurons is not strictly iso-specific because columns of dissimilar orientations are also linked together although to a lesser extent than those of similar orientations.
6. Discussion We have compared the orientation tuning characteristics of a layer III pyramidal cell with the orientation distribution of its efferent connections using optical imaging of intrinsic signals in combination with intracellular recording and staining with biocytin. Of the many possible pitfalls concerning in vivo optical imaging and subsequent single cell analysis, there are two important technical issues which deserve further consideration. 6.1. Stability of the cortex during in ÕiÕo intracellular recording and staining It is well-known that respiration and heartbeat generate rhythmic intracranial pulsation rendering intracellular electrophysiology difficult. This is particularly true for optical
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imaging preparations where large cortical regions are exposed. Therefore, every effort has to be made to reduce brain movement. Bilateral pneumothorax is very helpful in reducing ventilation-related brain pulsation. Inhaled gas-volume should also be carefully set to a value that ensures sufficient ventilation on the one hand, but creates minimal changes in the intrathoracic pressure on the other hand. The other basic principle in preventing brain pulsation during electrophysiological recording is to restore the intracranial pressure over the exposed region. For this, a commonly used method is to mount a chamber onto the skull and fill it up with agar–agar Ž3–4%. and wax on top. Unfortunately, at the contact site with the brain, the agar could readily dissolve into the cerebrospinal fluid, while on the outer surface, unless protected by a film of oil or wax, it dries out and shrinks. The resulting effect is that the agar provides no reliable seal against the intracranial pressure. These problems turned our attention to low-melting point paraffin wax that has the following advantages over agar application. Ø First, paraffin wax does not dissolve in the cerebrospinal fluid nor does it dry out. Ø Second, being an inert substance to brain tissue, it does not provoke an inflammatory reaction. We routinely use wax for covering exposed cortical surfaces where the dura had been removed for several days. In none of the cases was the cortex affected by the wax. Ø Third, paraffin wax has low-heat capacity unlike the watery based agar solution. Thus, a direct application of 43–458C wax onto the cortex does not cause heat-coagulation in the tissue probably due to rapid convection by surface blood vessels. Our histological sections showed no heat-related damage even after repeated wax applications. Conversely, when 43–458C agar solution was used, many neurons in layers I and II contained vacuoles and some of them showed signs of early degeneration. Ø Fourth, paraffin wax has high ohmic resistance that is also advantageous for monitoring intracellular electrical activity because the capacitance of the electrodes is lower than when using agar. Ø Fifth, when the electrode is withdrawn, the wax-plug could be taken out at once, whereas agar often breaks into small fragments. Finally, we found that the higher the wax level is in the chamber, the better stability is provided. Prior to wax application, the inner wall of the chamber and the exposed bone surface should be cleared of silicone oil and washing solution ŽRinger solution or artificial cerebrospinal fluid, ACSF. to provide a good seal. During intracellular recording, strong membrane potential fluctuations associated with heartbeat andror respiration could indicate leakage in the chamber. In this case, when droplets are often visible on the surface of the chamber, it is recommended to remove the wax-plug and rinse and drain the chamber thoroughly before applying new wax.
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After completion of the electrophysiological recordings, the animal is perfused without removing the chamber and the wax-plug that helps to preserve the three-dimensional structure of the exposed cortical region. 6.2. Morphological analysis An important step in analysing the functional topography of the labelled neurons is to find the best match between the coordinate systems of the anatomical reconstruction and functional maps acquired with the optical imaging technique. For that, it is essential to preserve the three-dimensional geometry of the cortical tissue during all phases of the experiment. Particularly, care should be taken to avoid development of cortical oedema because it radically distorts tissue geometry by pushing the cortex outwards through the craniotomy. Therefore, attention should be paid to proper settings of artificial ventilation and to apply sufficient level of anaesthesia, each of which could lead to brain oedema when inadequately used. Another point that is also important in preserving tissue geometry concerns histological treatments. Drying the sections onto slides causes dramatic shrinkage in the Z-axis; therefore, it is not the choice to be used. Instead, the sections should be postfixed in OsO4 , dehydrated and embedded in resin as described in detail in Section 4.6. During this procedure, a strong, heavy metal-based scaffold is provided to the membranes and the soluble tissue compounds are replaced with resin. In this manner, the entire tissue structure and its in vivo geometry are largely conserved. Further details of the procedure can be found in histological protocols described by Somogyi and Freund w11x. During the matching process between the anatomical and the optical imaging data, only linear transformations are used assuming little or no tissue distortion. Severe, non-linear distortions are very cumbersome to correct and they are not considered here. Concerning the precision of the matching procedure, there are two major factors to be mentioned. Pixel resolution Žusually in the range of a few tens of microns. of the optical images is one that is based on the optics, the camera resolution and the applied binning factor of camera-pixels. The other factor concerns the accuracy of marking the location of the reference penetrations onto the vascular map. Critically viewed, uncertainty is inherent to this procedure due to the relatively low magnification of the operating microscope Ž=25.. Taken together, we estimate that the lateral mismatch between corresponding reference penetration points marked on the vascular image and found in the tissue sections is less than 40 mm. 6.3. AlternatiÕe and support protocols A critical issue of the present study is to align the anatomical reconstructions with the optically imaged functional maps. For that, we use reference penetrations at
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known locations of the imaged cortex Žsee Section 4.4.. Previous studies have used a different alignment approach w14–16x. Commonly, the vascular image of the cortical surface is aligned with the drawing of blood vessel contours and radial vessel profiles Žwhich ‘dive’ into the cortex. taken from the first few tissue sections. In this process, global scaling, translation, and rotation are applied. Data from deeper sections are overlaid using blood vessel profiles that course perpendicular to the cortical surface. It is estimated that the above aligning procedure provides 50–100 mm error w14x, whereas ours is less than 40 mm.
7. Quick procedure Ø Anaesthetised paralysed adult cat is prepared for optical imaging of intrinsic signals. Orientation preference map is recorded in the selected region. Ø Reference penetrations are made right after the optical imaging using empty glass-micropipettes. Ø Intracellular recording and staining with biocytin in the optically imaged region. Ø Tissue fixation using transcardial perfusion, sectioning tissue blocks on Vibratome, biocytin histochemistry and resin-embedding. Ø Three-dimensional reconstruction of intracellularly stained cells, reference penetrations and section contours. Ø Data analysis: aligning the anatomical reconstructions with the optical image maps, quantitative calculations.
8. Essential literature references References w3–5,8,11x.
Acknowledgements
´ Toth Authors thank Ms. Eva ´ and Ms. Christa Schlauß for their excellent technical assistance, and Mr. Robert ´ Magyar and Dr. Martin Rausch for their computer expertise. Special thanks to Dr. Maxim Volgushev for helping us with the electrophysiology set-up and Dr. Trichur Vidyasagar for his comments on the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft ŽEy8r17-1; Ey8r21 and SFB509, TPA6 to ZFK. and the European Communities ŽSC10329-C..
References w1x G.G. Blasdel, G. Salama, Voltage-sensitive dyes reveal modular organization in monkey striate cortex, Nature 321 Ž1986. 579–585. w2x A. Grinvald, E. Lieke, R.D. Frostig, C.D. Gilbert, T.N. Wiesel, Functional architecture of cortex revealed by optical imaging of intrinsic signals, Nature 324 Ž1986. 361–364. w3x T. Bonhoeffer, A. Grinvald, Optical imaging based on intrinsic signals, The methodology, in: A.W. Toga, J.C. Mazziotta ŽEds.., Brain Mapping: The Methods, Academic Press, San Diego, 1996, pp. 55–97. w4x Z.F. Kisvarday, D.-S. Kim, U.T. Eysel, T. Bonhoeffer, Relationship ´ between lateral inhibitory connections and the topography of the orientation map in cat visual cortex, Eur. J. Neurosci. 6 Ž1994. 1619–1632. w5x Z.F. Kisvarday, E. Toth, ´ ´ M. Rausch, U.T. Eysel, Orientation-specific relationship between populations of excitatory and inhibitory lateral connections in the visual cortex of the cat, Cereb. Cortex 7 Ž1997. 605–618. w6x C.M. Gray, D.A. McCormick, Chattering cells: superficial pyramidal neurons contributing to the generation of synchronous oscillations in the visual cortex, Science 274 Ž1996. 109–113. w7x K.T. Brown, D.G. Flaming, Instrumentation and technique for beveling fine micropipette electrodes, Brain Res. 86 Ž1975. 172–180. w8x K. Horikawa, W.E. Armstrong, A versatile means of intracellular labeling: injection of biocytin and its detection with avidin conjugates, J. Neurosci. Methods 25 Ž1988. 1–11. w9x M.A. King, P.M. Louis, B.E. Hunter, O.W. Walker, Biocytin: a versatile anterograde neroanatomical tract-tracing alternative, Brain Res. 497 Ž1989. 361–367. w10x J.C. Adams, Heavy metal intensification of DAB-based HRP reaction product, J. Histochem. Cytochem. 29 Ž1981. 775. w11x P. Somogyi, T. Freund, Immunocytochemistry and synaptic relationships of physiologically characterized HRP-filled neurons, in: L. Heimer, L. Zaborszky ŽEds.., Neuronal Tract-tracing Methods 2, Plenum, New York, 1989, pp. 239–264. w12x Z.F. Kisvarday, U.T.T. Eysel, Functional and structural topography ´ of horizontal inhibitory connections in cat visual cortex, Eur. J. Neurosci. 5 Ž1993. 1558–1572. w13x Z.F. Kisvarday, T. Bonhoeffer, D.-S. Kim, U.T. Eysel, Functional ´ topography of horizontal neuronal networks in cat visual cortex ŽArea 18., in: A. Aertsen, V. Braitenberg ŽEds.., Brain Theory: Biological Basis and Computational Principles, Elsevier, Amsterdam, 1996, pp. 97–122. w14x W.H. Bosking, Y. Zhang, B. Schofield, D. Fitzpatrick, Orientation selectivity and the arrangement of horizontal connections in tree shrew striate cortex, J. Neurosci. 17 Ž1997. 2112–2127. w15x R. Malach, R.B. Tootell, D. Malonek, Relationship between orientation domains, cytochrome oxidase stripes, and intrinsic horizontal connections in squirrel monkey area V2, Cereb. Cortex 4 Ž1994. 151–165. w16x T. Yoshioka, G.G. Blasdel, J.B. Levitt, J.S. Lund, Relation between patterns of intrinsic lateral connectivity, ocular dominance, and cytochrome oxidase-reactive regions in macaque monkey striate cortex, Cereb. Cortex 6 Ž1996. 297–310.
Protocol
Functional topography of single cortical cells: an intracellular approach combined with optical imaging Peter ´ Buzas, ´ Ulf T. Eysel, Zoltan ´ F. Kisvarday ´
)
Ruhr-UniÕersitat 150, ¨ Bochum, Medizinische Fakultat, ¨ Institut fur ¨ Physiologie, Abteilung fur ¨ Neurophysiologie, UniÕersitatsstrasse ¨ MA 4 r 149, 44801 Bochum, Germany Accepted 29 July 1998
Abstract Pyramidal cells mediating long-range corticocortical connections have been assumed to play an important role in visual perceptual mechanisms wC.D. Gilbert, Horizontal integration and cortical dynamics, Neuron 9 Ž1992. 1–13x. However, no information is available as yet on the specificity of individual pyramidal cells with respect to functional maps, e.g., orientation map. Here, we show a combination of techniques with which the functional topography of single pyramidal neurons can be explored in utmost detail. To this end, we used optical imaging of intrinsic signals followed by intracellular recording and staining with biocytin in vivo. The axonal and dendritic trees of the labelled neurons were reconstructed in three dimensions and aligned with corresponding functional orientation maps. The results indicate that, contrary to the sharp orientation tuning of neurons shown by the recorded spike activity, the efferent connections Žaxon terminal distribution. of the same pyramidal cells were found to terminate at a much broader range of orientations. q 1998 Elsevier Science B.V. All rights reserved. Themes: Sensory system Topics: Visual cortex, functional topography Keywords: Optical imaging; Intracellular recording and staining; Three-dimensional neuron reconstruction; Orientation selectivity
1. Type of research
Ø Analysis of the relationship between the physiological maps and anatomical reconstructions: a few days.
Functional topography of corticocortical connections.
2. Time required The protocol is subdivided into four major parts. Ø In vivo experiment: four days. Days 1–2: optical imaging of intrinsic signals. Days 2–4: electrophysiological recordings and tracer injections. Ø Tissue processing and histology: three days. Ø Three-dimensional reconstruction of the labelled neurons: 4–10 weeks or about 100–200 working hours per neuron depending on its complexity.
) Corresponding author. Fax: q49-234-709-4192; E-mail: [email protected]
3. Materials 3.1. Experimental animals Cats, 8–12 months old. 3.2. Special equipment Ø Electrophysiological laboratory equipped for acute in vivo experiments. Ø Amplifier for intracellular recording ŽAxoclamp-2A, Axon Instruments, Foster City, CA, USA.. Ø Electrode puller ŽP-87 FlamingrBrown micropipette puller, Sutter Instrument, Novato, CA, USA.. Ø Micropipette beveller ŽBV-10, Sutter Instrument..
1385-299Xr98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 1 3 8 5 - 2 9 9 X Ž 9 8 . 0 0 0 4 1 - 5
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Ø Optical imaging system Imager 2001 and data acquisitionranalysis software ŽOptical Imaging, Germantown, NY, USA.. Ø Visual stimulus generator for optical imaging and single cell electrophysiology ŽVision Research Graphics, Durham, NH, USA.. Ø Vibratome ŽModel 1000, Pelco International, Redding, CA, USA.. Ø Light microscope ŽLeica, Wetzlar, Germany. equipped with the three-dimensional neuron reconstruction system, Neurolucida ŽMicroBrightField, Colchester, VT, USA.. 3.3. Chemicals and reagents Ø Paraffin wax Žmelting point s 42–448C, Merck, Darmstadt, Germany.. Ø Dental cement ŽPaladur, Heraeus Kulzer, Wehrheim, Germany.. Ø Silicone oil Ž50 cSt, Aldrich Chemical, Milwaukee, WI, USA.. Ø Biocytin ŽSigma, Deisenhofen, Germany.. 4. Detailed procedure An overview of the entire experimental paradigm is shown in Fig. 1. 4.1. Preparation of the animal for in ÕiÕo optical imaging of intrinsic signals An anaesthetised, paralysed adult cat is prepared for acute optical imaging of intrinsic signals using a modified procedure of Blasdel and Salama w1x and Grinvald et al. w2x Žreviewed in Ref. w3x.. Briefly, initial surgical anaesthesia is introduced by a mixture of ketamin Ž7 mg kgy1 , Ketanest, Parke-Davis, Berlin, Germany. and xylazin Ž1 mg kgy1 , Rompun, Bayer Belgium, Sint-Truiden, Belgium. i.m. A catheter is inserted into the femoral artery to monitor blood pressure ŽBP-1 Pressure Monitor, World Precision Instruments, Sarasota, FL, USA. and to infuse a mixture of muscle relaxant Žalcuronium chloride, 0.15 mg kgy1 hy1 , Alloferin, Hoffman-La Roche, GrenzachWhylen, Germany. and glucose Ž24 mg kgy1 hy1, Glucosteril, Fresenius, Bad-Homburg, Germany. in Ringer solution ŽRingerlosung Fresenius, Fresenius, Bad-Homburg, ¨ Germany. continuously during the experiment. Artificial ventilation ŽCatrRabbit Ventilator, Ugo Basile, Comerio, Italy. and prolonged anaesthesia is provided through tracheal cannula using a 1:2 mixture of O 2 and N2 O supplied with 0.4–0.6% of halothane ŽHalothan Eurim, EurimPharm, Piding, Germany.. All pressure points are treated with the local anaesthetic, Xylocain gel ŽAstra Chemicals, WedelrHolstein, Germany.. The major physiological parameters Žend-tidal CO 2 : 3.0–4.0%, blood pressure: 100– 140 mmHg, heart rate: 160 miny1 , body temperature:
38–398C, and EEG. are continuously monitored and maintained. A bilateral pneumothorax Žsee also Section 4.5.1. is prepared to reduce artificial ventilation-related brain movement. For monitoring lung-function, a plastic tube connected to an elastic balloon Žsoft, powder-free latex exam gloves, Safeskin, Neufahrn, Germany. is inserted into the thorax on both sides of the chest. A 6 = 15 mm craniotomy is made between Horsley–Clarke coordinates AP y4 and q9 and LM q0.5 and q6.5 and a round, metal chamber is mounted over the exposed region using dental cement ŽPaladur. ŽFig. 2a.. Once the chamber is fixed, the dura mater is removed. At this step, great care is taken not to damage subdural blood vessels because optical imaging relies on light absorption differences between oxygenated and de-oxygenated haemoglobin, consequently, bleeding in the imaged region may cause artefacts. Another point that is important to mention is that removal of the dura mater inevitably causes bleeding of cut sinuses. There are several ways to stop dural bleeding, three are listed here. First, the cut edge of the dura is gently folded out so that the lumen of the damaged sinuses becomes squeezed onto the bone around the exposed region. Another method is to clamp the sinuses with a fine-tip forceps and hold for 1–2 min. If none of these methods helps in stopping the dural bleeding, heat coagulation of the sinuses with a fine-tip cauter device ŽFine Science Tools, Heidelberg, Germany. could be useful. The recording chamber has at least two outlets at its base, each connected to a stopcock via thin plastic tubes. These outlets serve to release or provide hydrostatic pressure when the chamber is closed with the cover-glass and when the cover-glass is removed. Finally, the chamber is filled with silicone oil Ž50 cSt, Sigma-Aldrich Chemie GmbH, Steinheim, Germany. and closed with the round cover-glass. It should be noted that during the experiment, the silicone oil gradually becomes milky probably due to an emulsification process with the watery based cerebrospinal fluid. In addition to this, blood could slowly diffuse into the oil that also causes deterioration of the optics and results in unwanted quality loss in the acquired optical images. Therefore, we recommend to clean the chamber and replace the silicone oil about every 8–10 h. 4.2. Optical imaging During optical imaging, the so-called vascular or green-image is first taken under green illumination Ž450 nm, Spindler and Hoyer, Gottingen, Germany. to show the ¨ pattern of surface blood vessels of the brain. It is essential to take a green-image every 2 h or before and after each recording session because of possible shifts in the focal depth, for example, due to mild cortical oedema or dehydration. Another important application of green-images is that they are used for aligning the optical images with the histological reconstructions Žsee Section 4.8.1.. For data acquisition, it is recommended to narrow down the image
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size to an area called ‘region of interest’ that saves a significant amount of computation time and disk space. It also improves the signal-to-noise ratio by eliminating potential noise sources from the images Že.g., from surrounding bone tissue and large blood vessels..
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Visual stimuli are generated with the Neurosequence program of the Vision Works stimulus generation system ŽVision Research Graphics, Durham, NH, USA.. Each stimulus presentation is triggered by the optical imaging system using a total of five output bits for coding the
Fig. 1. Scheme of the experimental paradigm, data acquisition, and analysis used. Numbers indicate the sequence of individual steps.
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stimulus type and one bit ŽGo-bit. for moving the stimulus during data acquisition. Orientation maps are acquired during the presentations of moving square-wave gratings at four orientations Ž08, 458, 908, and 1358. which move in one of the perpendicular directions and then in the opposite direction. The reversal in the direction of movement occurs at half of the data acquisition period. The stimulus gratings are shown on a 21Y computer monitor Ž120 Hz, non-interlaced mode, SONY, Pencoed, UK. 285 mm from the cat’s eyes. Spatial Ž0.1–0.2 cycle degy1 , 50% duty cycle. and temporal frequencies Ž1–2 Hz. are optimised in a few, short recordings for every new part of the cortex to be mapped. For obtaining orientation maps, individual stimuli are presented 25–50 times for 4.5 s followed by an interstimulus interval for 10 s when the animals view a blank screen. The cortex is illuminated with light at 605 " 5 nm ŽOmega Optical, Brattleboro, VT, USA. using a circular fibre optic slit lamp ŽSchott, Mainz, Germany.. Video images are recorded with a slow-scan Ž25 video frames sy1 . CCD camera ŽBischke CCD 6012P, 6 = 8 mm sensor. using a ‘tandem-lens’ of two objectives ŽSMC Pentax 1:1.2, 50 mm.. The images are collected and processed using the system Imager 2001 ŽOptical Imaging. and Matrox IM-640 imaging board controlled by the software VDAQ version 2.18k. 4.3. Analysis of the optical images For image analysis, each image acquired for a particular orientation is corrected for time related fluctuations in the signal using the ‘cocktail blank method’. Accordingly, each image is divided by the sum of images for all orientations resulting in so-called single condition maps using the MIX program of the system Imager 2001 ŽOptical Imaging.. The grey value distribution of each single condition map is ‘clipped’ by discarding the flanks outside "3 S.D. around mean to exclude possible artefacts and then scaled to the range between 0–255. Further evaluation of the images is done in two steps using a custom software
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written in IDL ŽResearch Systems, Boulder, CO, USA.. First, a high-pass filtering is applied using a 426-mm pixel-kernel followed by a low-pass filtering before the differential maps are vectorially summed. Orientation Žangle. maps are produced by summing the vector components pixel-by-pixel where the vector angle corresponds to the respective stimulus orientation and the vector length to the response intensity coded in grey values. The cortical area represented by a single image-pixel is measured by taking an image of a mesh-grid Ž10 mm grid.. After the optical imaging session, the chamber is carefully opened and the cover-glass and the gaskets are removed. The surface of the cortex is rinsed with several changes of Ringer solution or artificial cerebrospinal fluid until no silicone oil is visible. 4.4. Reference penetrations Reference penetrations are crucial landmarks in matching the functional Že.g., orientation maps. and morphological maps Že.g., the distribution of retrogradely labelled neurons.. For this purpose, long-tapered glass micropipettes with 10–15 mm external tip diameter were found most suitable because they only cause a very fine tissue scar w4,5x. The pipette is positioned perpendicular to the cortical surface, lowered into the cortex 1000 mm deep and the stereotactic position of its entry point is registered with 10 mm accuracy. In addition, the exact position of each penetration point is marked on an enlarged print of the green-image Žsurface vascular image.. In this way, reference penetrations with known antero-posterior and latero-medial distances and exact positions with respect to the surface blood vessels can be obtained. We recommend a minimum of five reference penetrations each separated by 500–1000 mm from its neighbours in the central 3r4 of the imaged regions. 4.5. Electrophysiology and tracer injection In this part of the experiment, an attempt is made to register the main electrophysiological parameters Žrecep-
Fig. 2. Panel Ža. shows the recording chamber used in optical imaging. The cover-glass and the silicone oil through which the imaging takes place are transparent. The CCD-camera and the illumination source are removed for clarity. In panel Žb., a schematic cross-section of the same chamber prepared for intracellular electrode application is seen. Once the recording electrode-tip establishes direct contact with the cortical surface, the chamber is filled-up with low-melting point paraffin wax. Panel Žc. shows the light microscopic image of the soma and part of the dendritic field of a layer III pyramidal cell. The pyramidal cell was intracellularly tested for its orientation preference and injected with biocytin. Due to the horizontal sectioning plane, the apical dendrite is visible only in more superficial sections Žc s capillary.. In panel Žd., a computer-assisted reconstruction of the same pyramidal cell is rotated into the parasagittal plane. In this view, the laminar distribution of the axon Žin red. and dendritic field Žin black. could be readily discerned. Comparison between the topography of the pyramidal cell shown above Žpanels e and f. and the optically imaged orientation map. In Že., the vascular image containing five reference penetrations Žstars. is overlaid with the density map of the axon terminals. Colour scheme to Že. indicates the number of terminals per image-pixel. In Žf., the orientation map is overlaid with the axon terminal distribution Žshown in black for clarity.. Notice that although the densest axon terminal clusters are found at similar orientations, the entire axon establishes contacts over a broad orientation range with respect to that of the soma location Žwhite asterisk.. Panels Žg. and Žh. provide a quantitative comparison between the orientation tuning measured electrophysiologically and the orientation distribution of efferent connections detected anatomically for the same neuron. The physiological orientation tuning of the cell Žpanel g. is based on the rate of the visually evoked spike response during the in vivo intracellular recording. The tuning curve of the boutons in panel Žh. shows the number of labelled boutons found in regions representing the various stimulus orientations. Clearly, the efferent connections represent a 1.5 times broader orientation tuning than that measured from the spike responses. Broken lines indicate the activity level between stimulus presentations in Žg. and the number of axon terminals if they were distributed evenly over all orientations in Žh..
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tive field characteristics. of single cortical neurons and subsequently stain the individual neurons intracellularly. It is advisable to search for neurons in the central region of the optically imaged functional map, since corticocortical connections often have a lateral extent up to 3–4 mm. For guiding the electrodes into functionally characterised locations, for example, into orientation centres, large prints of the surface vascular and functional maps could be useful.
4.5.1. Reducing brain moÕement for intracellular electrode applications Artificial ventilation as well as heartbeat result in movement of the brain hindering stable intracellular recording. The former could be substantially reduced by applying pneumothorax and setting the inhaled gas volume Ž20–25 ml for a 3-kg cat. so that the inflated lung does not reach the volume capacity of the thorax. It is also important to hang the chest by a hook inserted between the first thoracic spines. If necessary, further stabilisation could be achieved by puncturing the cisterna magna at the level of the first few cervical spines, thereby lowering the intracranial pressure Žsee Ref. w6x.. Another important factor in obtaining a ‘non-pulsating’ cortex is to maintain a slightly lower blood pressure Ž100–120 mmHg. using mild hyperventilation Ž3.0–3.8% CO 2 .. Finally, the electrode is positioned into its desired location under the control of an operating microscope and advanced until the pia mater is penetrated. At this stage, the impedance of the electrode is measured and the chamber is filled with a thick layer of low-melting point Ž42–448C. paraffin wax ŽFig. 2b..
4.5.2. Intracellular recording Choosing the right electrode might need some experimenting. In our hands, sharp borosilicate glass electrodes ŽGB150 F-8P, Science Products, Hofheim, Germany. pulled on a horizontal puller ŽP-87 FlamingrBrown micropipette puller, Sutter Instrument. worked best. The electrodes are filled with freshly prepared 0.5 M K-acetate containing 1–2% biocytin ŽSigma. and kept in a wet chamber for 1–2 h in a position such that the electrode shafts are pointing upright. During that time, the air-bubbles usually disappear from the electrode tip. To foster this process, vacuum can be applied using a 20-ml or largervolume syringe connected to the electrode shaft. It is advisable to use always fresh biocytin solution for two reasons. First, K-acetate decays with time resulting to conductivity loss. Second, biocytin might precipitate during long storage. The electrodes are bevelled on a BV-10 micropipette beveller ŽSutter Instrument. using an alumina-grinding plate Ž0.05 mm particle size, 22–278 bevelling angle, w7x.. As a result of the bevelling, the input resistance of the electrodes decreases Žincreased tip lumen. from an initial 100–120 M V to 40–105 M V final resistance and the tip becomes sharp. Hence, the electrode can
readily penetrate the cell membrane and pass sufficient current to drive biocytin into the intracellular space. Before use, the tip of each electrode is inspected under a light microscope Ž=100. for mechanical damage, surface contamination, and air-bubbles. Successful penetration of the cell membrane is indicated by a sudden 40–70-mV drop in the measured potential. After determining the position and extent of the receptive field using hand-held stimuli, orientation tuning curves are derived from recordings ŽSpike-2 software, Version 2.02, Cambridge Electronic Design, Cambridge, UK. in response to computer-generated moving light or dark bars at optimal length and velocity moving at each of eight equally spaced orientations presented in a pseudo-random manner on the 21Y computer monitor. The recorded potentials are digitised using the CED 1401 interface ŽCambridge Electronic Design Ltd., Cambridge, UK. and recorded on a personal computer. Orientation tuning is calculated as the average spike frequency during the presentation of the respective stimuli.
4.5.3. Intracellular deliÕery of biocytin Intracellular recording is followed by iontophoretic delivery of biocytin into the cell using positive 2–3.5 nA rectangular current pulses Ž200 ms ON 400 ms OFF duty cycles. for 1–5 min. The injection is briefly interrupted for a few times during which the orientation preference and receptive field location of the neuron are checked. After completion of the intracellular injection, the electrode is rapidly withdrawn from the cortex.
4.6. Tissue processing and histology After appropriate survival time Ž1–24 h., the animal receives an overdose of anaesthetics and is perfused transcardially with cold Ž48C., oxygenated Tyrode’s solution followed by a mixture of 2% paraformaldehyde and 0.5% glutaraldehyde in 0.1 M phosphate buffer ŽpH s 7.6.. Tissue blocks that are 2–3 mm larger on each side than the optically imaged region are dissected, the remaining parts of the dura mater and the arachnoidea are gently peeled-off with a fine-tip forceps and consecutive, 60–80-mm thick sections are cut using Vibratome. The cutting plane is set as parallel to the imaged cortical surface as possible. The sections are collected in 10 lots and rinsed in 0.1 M PB for 3 = 20 min to remove unbound fixative. Biocytin labelling is visualised in free-floating sections using the ABC method w8,9x with slight modifications and supplemented with cobalt intensification w10x. Briefly, the sections are incubated in 1:200 avidin–biotin-complexed horseradish peroxidase ŽABC, Vector Laboratories, Burlingame, CA, USA. in 0.05 M Tris-buffered saline ŽTBS, pH s 7.6. containing 0.05% Triton X-100 while
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gently agitated Ž48C, overnight.. The enzymatic reaction is revealed with 0.005% 3,3X-diaminobenzidine-4-HCl ŽDAB; Sigma. in TBS supplemented with CoCl 2 intensification Ž0.0025%. for 20 min. The latter is served to increase the contrast of the staining under the light microscope Žbluishblack precipitate. as well as improve the opacity of the reaction end-product for possible electron microscopy w10x. The reaction is completed in the presence of 0.001% H 2 O 2 for 1–5 min. Then, the sections are rinsed in TBS Ž2 = 15 min., PB Ž2 = 15 min., postfixed in 0.5% OsO4 Žin 0.1 M PB. for 10–20 min and dehydrated in ascending series of ethanol. At this phase of the procedure, there are two important factors to be taken into account. First, osmium tetroxide and dehydration make the tissue rigid and fragile. Secondly, large sections Žabout 10 = 5 mm2 . containing the grey and the white matter often become wrinkled due to uneven shrinkage. To prevent wrinkling, the sections are flat-embedded using a modified procedure of Somogyi and Freund w11x. Accordingly, the osmicated sections are kept between glass slides and coverslips during the entire dehydration process in ascending ethanol series. Then, they are transferred into propyleneoxide for 2 = 15 min and rapidly submerged into resin ŽDurcupan ACM, Fluka, Neu-Ulm, Germany. for 24 h at room temperature. Finally, the resin-embedded sections are mounted onto slides, coverslipped and cured at 568C for 24 h. 4.7. Three-dimensional reconstruction In order to obtain information on the spatial organisation of the labelled neurons with respect to the distribution of functional maps, the neuron reconstruction system, Neurolucida ŽMicroBrightField, Colchester, VT, USA. is used. The three-dimensional coordinates of the labelled soma, dendritic and axonal fields and axon terminals Žboutons. are reconstructed at =1000 magnification using a light microscope ŽLeica DMRB. fitted with a computer-controlled motorised stage ŽMarzhauser-Wetzlar, Wetzlar, ¨ Germany. and controller box ŽLUDL MC2000 XYZ, Ludl Electronic Products, Hawthorne, NY, USA.. 4.7.1. Finding the best match between adjoining sections A critical step in reconstructing axonal and dendritic fields that are sliced up during sectioning is how to find the best match between adjoining sections w12,4,5x. To this end, we use labelled fine processes, for example, axon collaterals and dendrites, which extend from one section to the other offering corresponding cut ends that can be matched with high precision. We recommend to choose several pairs of cut-ends in various parts of the estimated projection field of the labelled neuron. In our practice, cut-ends separated by 2–4 mm in the plane of the sections provide 5–10-mm matching accuracy. Once the labelled anatomical structures are compiled into a single structure, they are matched with the optical images as described in Section 4.8.1.
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4.7.2. Reference penetrations It has to be emphasised here that reference penetrations and tissue marks like surface blood vessels are crucial landmarks in obtaining the best match between the anatomical reconstructions and the optical images w4x. Therefore, attention is paid to find and reconstruct the exact location of the reference penetrations where they entered into the cortex. Unfortunately, the most superficial sections often contain blood vessels, large number of dark macrophages and connective tissue debris which obscure these locations. In this case, penetration marks from deeper sections are to be used and extrapolated to the surface sections. If visible, tracks of the recording electrodes with known entry points Žmarked on the green-image. could also be useful in the matching procedure. Finally, the reconstruction of the labelled neuronal processes Žaxonal and dendritic fields., the reference penetrations and other structures of interest Že.g., section contours. should be made in the same coordinate system. 4.8. Data analysis 4.8.1. Alignment of the reconstructed structures with the optical maps The aim of the aligning procedure is to transform the three-dimensionally digitised anatomical data into the two-dimensional coordinate system of the optical maps. In this procedure, only linear transformations are applied such as rotation, translation, and scaling. Accordingly, the anatomical coordinate system is defined by the X,Y,Z-axes of the motorised microscope stage, where the Z-axis represents the focus. This is then transformed into the ‘in vivo coordinate system’ where the XY plane corresponds to the plane of the optical image and the Z-axis represents the axis perpendicular to that plane. Because the optical image plane runs parallel with the cortical surface, the Z-axis corresponds to the cortical depth. The aligning procedure is carried out in four steps. First, the anatomical reconstruction is corrected for virtual shrinkage in the Z-axis caused by the optical density of the embedding medium, the epoxy resin, and the microscope immersion oil Žif used.. The microscopically measured Z-values are multiplied by the correction factor, f s n resinrn oil , where n resin s 1.549 is the experimentally determined index of refraction for the epoxy resin and n oil s 1.5180 ŽLeica. is the index of refraction for the immersion oil. In the second step, the data is tilted until the reconstructed structures are viewed from the plane of the optical images. In practice, small rotation about the X and Y axes is applied until the projection of each of the reference penetration tracks running perpendicular to the cortical surface is viewed as a spot. For all reference penetrations, the optimal rotation angles could be calculated using, for example, the least-square approximation algorithm. When the anatomical data have been rotated and projected into an image plane closely matching that of
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the optical image plane, the data are ready for the third step, correction for histology-related tissue shrinkage. The shrinkage factor in the XY plane is determined on the basis of reconstructed reference penetrations whose relative distances from each other have been measured in vivo w5x. Concerning Z-direction shrinkage, the shrinkage factor calculated for the XY plane is applied assuming that the tissue underwent equal shrinkage in each dimension. In the fourth step, the data are rotated around the Z-axis and translated until the corresponding reference penetration points of the anatomical reconstruction and of the optical image are matched using the least-square approximation algorithm.
imposed on the vascular image. The semi-coloured scheme on the right-hand side codes for the number of axon terminals per pixel Ž1–15.. The five reference penetrations marked by white stars serve to match the anatomical reconstruction with the corresponding vascular and orientation map images. In panel Žf., the same axon terminal density distribution is shown Žwithout colour-coding. in register with the functional orientation map. Interestingly, some axon clusters occupy regions Žyellow, ochre, and red. of similar orientations to that of the location of the parent soma and dendritic field Žwhite asterisk in f., whereas other clusters, although smaller and less dense, are found at dissimilar orientations.
4.8.2. QuantitatiÕe calculations In order to explore the functional topography of individual cells, the distribution of the labelled axon terminals is compared with the distribution of the functional orientation map w13,14x. The orientation map is composed of image pixels each covering a known area, whereas the axon terminals are represented by coordinate points. In order to carry out a quantitative analysis, the number of axon terminals is counted Žbinned. in every image pixel using a custom software written in IDL ŽResearch Systems.. The program generates a two-dimensional grid whose pixel size is identical to that of the optical image, overlays it onto the axon terminal distribution and counts how many axon terminals are found in every pixel Žbinning.. The resulting anatomical density map can now be directly compared with the orientation map on a pixel-by-pixel basis.
5.2. Comparison between the orientation tuning of spike actiÕity and the orientation topography of efferent connections of a layer III pyramidal cell
5. Results 5.1. Anatomy and functional topography of a layer III pyramidal cell in the cat Õisual cortex (area 18). In Fig. 2c,d,e,f, an example is provided for a biocytinlabelled layer III pyramidal whose efferent connections are compared with the distribution of orientation selectivity obtained with optical imaging of intrinsic signals. Here, we want to determine what orientations are encountered by the axon terminals of the layer III pyramidal cell. In Fig. 2c, the light microscopic image of the soma and spiny dendrites are viewed from the cortical surface Žc s capillary.. In panel Žd., the same three-dimensionally reconstructed pyramidal cell is seen from the parasagittal plane, so that its laminar distribution could be determined. Characteristically, the axon arborises in two main tiers of the cortex, in layers IIrIII and V. At about 1 mm lateral from the parent soma, clustering of the axon collaterals occurs, but other parts of the axonal field are not clustered at all. In panel Že., the density distribution of the reconstructed axon terminals is shown. Their distribution is determined on a pixel-by-pixel basis as described in Section 4.8 and super-
In panel Žg., the orientation tuning of the pyramidal cell is determined on the basis of visually evoked spike activity. When the distribution curve is fitted with a Gaussian curve Ždata are not shown., it provides a quantitative measure of the orientation tuning of the cell. The results reveal that this pyramidal neuron is selectiÕe Ž188, halfwidth at half-height. for a horizontal stimulus orientation. In panel Žh., the orientation distribution of the efferent connections Žaxon terminals. of the same cell is calculated as described in Section 4.8.2. Interestingly, the resulting ‘tuning’ curve for the axon terminals is 1.5 times broader than that seen for the spike activity Ž278, half-width at half-height., while their peak values differ only by 118 from each other. These findings suggest that the functional topography of long-range excitatory neurons is not strictly iso-specific because columns of dissimilar orientations are also linked together although to a lesser extent than those of similar orientations.
6. Discussion We have compared the orientation tuning characteristics of a layer III pyramidal cell with the orientation distribution of its efferent connections using optical imaging of intrinsic signals in combination with intracellular recording and staining with biocytin. Of the many possible pitfalls concerning in vivo optical imaging and subsequent single cell analysis, there are two important technical issues which deserve further consideration. 6.1. Stability of the cortex during in ÕiÕo intracellular recording and staining It is well-known that respiration and heartbeat generate rhythmic intracranial pulsation rendering intracellular electrophysiology difficult. This is particularly true for optical
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imaging preparations where large cortical regions are exposed. Therefore, every effort has to be made to reduce brain movement. Bilateral pneumothorax is very helpful in reducing ventilation-related brain pulsation. Inhaled gas-volume should also be carefully set to a value that ensures sufficient ventilation on the one hand, but creates minimal changes in the intrathoracic pressure on the other hand. The other basic principle in preventing brain pulsation during electrophysiological recording is to restore the intracranial pressure over the exposed region. For this, a commonly used method is to mount a chamber onto the skull and fill it up with agar–agar Ž3–4%. and wax on top. Unfortunately, at the contact site with the brain, the agar could readily dissolve into the cerebrospinal fluid, while on the outer surface, unless protected by a film of oil or wax, it dries out and shrinks. The resulting effect is that the agar provides no reliable seal against the intracranial pressure. These problems turned our attention to low-melting point paraffin wax that has the following advantages over agar application. Ø First, paraffin wax does not dissolve in the cerebrospinal fluid nor does it dry out. Ø Second, being an inert substance to brain tissue, it does not provoke an inflammatory reaction. We routinely use wax for covering exposed cortical surfaces where the dura had been removed for several days. In none of the cases was the cortex affected by the wax. Ø Third, paraffin wax has low-heat capacity unlike the watery based agar solution. Thus, a direct application of 43–458C wax onto the cortex does not cause heat-coagulation in the tissue probably due to rapid convection by surface blood vessels. Our histological sections showed no heat-related damage even after repeated wax applications. Conversely, when 43–458C agar solution was used, many neurons in layers I and II contained vacuoles and some of them showed signs of early degeneration. Ø Fourth, paraffin wax has high ohmic resistance that is also advantageous for monitoring intracellular electrical activity because the capacitance of the electrodes is lower than when using agar. Ø Fifth, when the electrode is withdrawn, the wax-plug could be taken out at once, whereas agar often breaks into small fragments. Finally, we found that the higher the wax level is in the chamber, the better stability is provided. Prior to wax application, the inner wall of the chamber and the exposed bone surface should be cleared of silicone oil and washing solution ŽRinger solution or artificial cerebrospinal fluid, ACSF. to provide a good seal. During intracellular recording, strong membrane potential fluctuations associated with heartbeat andror respiration could indicate leakage in the chamber. In this case, when droplets are often visible on the surface of the chamber, it is recommended to remove the wax-plug and rinse and drain the chamber thoroughly before applying new wax.
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After completion of the electrophysiological recordings, the animal is perfused without removing the chamber and the wax-plug that helps to preserve the three-dimensional structure of the exposed cortical region. 6.2. Morphological analysis An important step in analysing the functional topography of the labelled neurons is to find the best match between the coordinate systems of the anatomical reconstruction and functional maps acquired with the optical imaging technique. For that, it is essential to preserve the three-dimensional geometry of the cortical tissue during all phases of the experiment. Particularly, care should be taken to avoid development of cortical oedema because it radically distorts tissue geometry by pushing the cortex outwards through the craniotomy. Therefore, attention should be paid to proper settings of artificial ventilation and to apply sufficient level of anaesthesia, each of which could lead to brain oedema when inadequately used. Another point that is also important in preserving tissue geometry concerns histological treatments. Drying the sections onto slides causes dramatic shrinkage in the Z-axis; therefore, it is not the choice to be used. Instead, the sections should be postfixed in OsO4 , dehydrated and embedded in resin as described in detail in Section 4.6. During this procedure, a strong, heavy metal-based scaffold is provided to the membranes and the soluble tissue compounds are replaced with resin. In this manner, the entire tissue structure and its in vivo geometry are largely conserved. Further details of the procedure can be found in histological protocols described by Somogyi and Freund w11x. During the matching process between the anatomical and the optical imaging data, only linear transformations are used assuming little or no tissue distortion. Severe, non-linear distortions are very cumbersome to correct and they are not considered here. Concerning the precision of the matching procedure, there are two major factors to be mentioned. Pixel resolution Žusually in the range of a few tens of microns. of the optical images is one that is based on the optics, the camera resolution and the applied binning factor of camera-pixels. The other factor concerns the accuracy of marking the location of the reference penetrations onto the vascular map. Critically viewed, uncertainty is inherent to this procedure due to the relatively low magnification of the operating microscope Ž=25.. Taken together, we estimate that the lateral mismatch between corresponding reference penetration points marked on the vascular image and found in the tissue sections is less than 40 mm. 6.3. AlternatiÕe and support protocols A critical issue of the present study is to align the anatomical reconstructions with the optically imaged functional maps. For that, we use reference penetrations at
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known locations of the imaged cortex Žsee Section 4.4.. Previous studies have used a different alignment approach w14–16x. Commonly, the vascular image of the cortical surface is aligned with the drawing of blood vessel contours and radial vessel profiles Žwhich ‘dive’ into the cortex. taken from the first few tissue sections. In this process, global scaling, translation, and rotation are applied. Data from deeper sections are overlaid using blood vessel profiles that course perpendicular to the cortical surface. It is estimated that the above aligning procedure provides 50–100 mm error w14x, whereas ours is less than 40 mm.
7. Quick procedure Ø Anaesthetised paralysed adult cat is prepared for optical imaging of intrinsic signals. Orientation preference map is recorded in the selected region. Ø Reference penetrations are made right after the optical imaging using empty glass-micropipettes. Ø Intracellular recording and staining with biocytin in the optically imaged region. Ø Tissue fixation using transcardial perfusion, sectioning tissue blocks on Vibratome, biocytin histochemistry and resin-embedding. Ø Three-dimensional reconstruction of intracellularly stained cells, reference penetrations and section contours. Ø Data analysis: aligning the anatomical reconstructions with the optical image maps, quantitative calculations.
8. Essential literature references References w3–5,8,11x.
Acknowledgements
´ Toth Authors thank Ms. Eva ´ and Ms. Christa Schlauß for their excellent technical assistance, and Mr. Robert ´ Magyar and Dr. Martin Rausch for their computer expertise. Special thanks to Dr. Maxim Volgushev for helping us with the electrophysiology set-up and Dr. Trichur Vidyasagar for his comments on the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft ŽEy8r17-1; Ey8r21 and SFB509, TPA6 to ZFK. and the European Communities ŽSC10329-C..
References w1x G.G. Blasdel, G. Salama, Voltage-sensitive dyes reveal modular organization in monkey striate cortex, Nature 321 Ž1986. 579–585. w2x A. Grinvald, E. Lieke, R.D. Frostig, C.D. Gilbert, T.N. Wiesel, Functional architecture of cortex revealed by optical imaging of intrinsic signals, Nature 324 Ž1986. 361–364. w3x T. Bonhoeffer, A. Grinvald, Optical imaging based on intrinsic signals, The methodology, in: A.W. Toga, J.C. Mazziotta ŽEds.., Brain Mapping: The Methods, Academic Press, San Diego, 1996, pp. 55–97. w4x Z.F. Kisvarday, D.-S. Kim, U.T. Eysel, T. Bonhoeffer, Relationship ´ between lateral inhibitory connections and the topography of the orientation map in cat visual cortex, Eur. J. Neurosci. 6 Ž1994. 1619–1632. w5x Z.F. Kisvarday, E. Toth, ´ ´ M. Rausch, U.T. Eysel, Orientation-specific relationship between populations of excitatory and inhibitory lateral connections in the visual cortex of the cat, Cereb. Cortex 7 Ž1997. 605–618. w6x C.M. Gray, D.A. McCormick, Chattering cells: superficial pyramidal neurons contributing to the generation of synchronous oscillations in the visual cortex, Science 274 Ž1996. 109–113. w7x K.T. Brown, D.G. Flaming, Instrumentation and technique for beveling fine micropipette electrodes, Brain Res. 86 Ž1975. 172–180. w8x K. Horikawa, W.E. Armstrong, A versatile means of intracellular labeling: injection of biocytin and its detection with avidin conjugates, J. Neurosci. Methods 25 Ž1988. 1–11. w9x M.A. King, P.M. Louis, B.E. Hunter, O.W. Walker, Biocytin: a versatile anterograde neroanatomical tract-tracing alternative, Brain Res. 497 Ž1989. 361–367. w10x J.C. Adams, Heavy metal intensification of DAB-based HRP reaction product, J. Histochem. Cytochem. 29 Ž1981. 775. w11x P. Somogyi, T. Freund, Immunocytochemistry and synaptic relationships of physiologically characterized HRP-filled neurons, in: L. Heimer, L. Zaborszky ŽEds.., Neuronal Tract-tracing Methods 2, Plenum, New York, 1989, pp. 239–264. w12x Z.F. Kisvarday, U.T.T. Eysel, Functional and structural topography ´ of horizontal inhibitory connections in cat visual cortex, Eur. J. Neurosci. 5 Ž1993. 1558–1572. w13x Z.F. Kisvarday, T. Bonhoeffer, D.-S. Kim, U.T. Eysel, Functional ´ topography of horizontal neuronal networks in cat visual cortex ŽArea 18., in: A. Aertsen, V. Braitenberg ŽEds.., Brain Theory: Biological Basis and Computational Principles, Elsevier, Amsterdam, 1996, pp. 97–122. w14x W.H. Bosking, Y. Zhang, B. Schofield, D. Fitzpatrick, Orientation selectivity and the arrangement of horizontal connections in tree shrew striate cortex, J. Neurosci. 17 Ž1997. 2112–2127. w15x R. Malach, R.B. Tootell, D. Malonek, Relationship between orientation domains, cytochrome oxidase stripes, and intrinsic horizontal connections in squirrel monkey area V2, Cereb. Cortex 4 Ž1994. 151–165. w16x T. Yoshioka, G.G. Blasdel, J.B. Levitt, J.S. Lund, Relation between patterns of intrinsic lateral connectivity, ocular dominance, and cytochrome oxidase-reactive regions in macaque monkey striate cortex, Cereb. Cortex 6 Ž1996. 297–310.