Optical imaging combined with targeted electrical recordings, microstimulation, or tracer injections
Introduction
In the cerebral cortex, neurons with similar receptive fields and response properties are often clustered in columns running from the pia to the white matter (Mountcastle, 1957, Hubel and Wiesel, 1962, Hubel and Wiesel, 1963, Hubel and Wiesel, 1965, for review see Mountcastle, 1997). On the other hand, the tangential organization of the cortex is such that response properties undergo a gradual change, usually in a systematic manner, forming representations of the environment on the cortical surface. How the morphology and the response properties of single neurons are related to the overall functional architecture of cortical columns remains a fundamental question in neurobiology.
One effective way to study the cortex is by recording its electrical activity with microelectrodes. It is more than 40 years since the activity of a single unit in an awake behaving animal was first recorded (Hubel, 1959, Jasper et al., 1960). In these experiments the dura can be left intact, allowing the animal's brain to be maintained in good condition for periods of up to several years. A complementary technique, used to study the cortex at the level of cell populations, is optical imaging. This method has long been used to study the principles underlying cortical development, organization and function, and to process sensory information in vivo. More than 100 groups are currently using this technique (partial list: Grinvald et al., 1984, Grinvald et al., 1986, Grinvald et al., 1991, Orbach et al., 1985, Blasdel and Salama, 1986, Ts'o et al., 1990, Bonhoeffer and Grinvald, 1991, Bartfeld and Grinvald, 1992, Das and Gilbert, 1995, Godecke and Bonhoeffer, 1996, Sheth et al., 1996, Wang et al., 1996, Weliky et al., 1996, Crair et al., 1997, Rubin and Katz, 1999, Shtoyerman et al., 2000, Womelsdorf et al., 2001). In contrast to single-unit recording, optical imaging based on intrinsic signals or on voltage-sensitive dyes requires removal of the dura, so that the cortex is exposed. Recently we developed a transparent silicone dural substitute, enabling optical imaging for periods longer than a year (Shtoyerman et al., 1995, Shtoyerman et al., 2000, Grinvald et al., 1999, Slovin et al., 1999, Slovin et al., 2000, Arieli et al., 2002). Use of this device made it possible to combine optical imaging and the classical techniques based on microelectrodes.
The integration of optical imaging with several powerful techniques based on microelectrodes is described in this report. This integration has greatly extended the ability to tackle problems that cannot be explored by any single approach. The combined approach has enabled us, for example: (1) to confirm functional maps obtained by optical imaging; (2) to study the relationship of the morphology of the axonal and dendritic arborization of a single neuron to the cortical functional architecture; (3) to show that the network dynamics are constrained by the cortical functional architecture; (4) to examine how the dynamic state of the cortical network influences the response to a stimulus; and (5) to determine how the dynamic activity of neuronal populations over a large cortical area affects the spontaneous spike trains at the single neuron level. These and other issues can now also be addressed in relation to the behavior and the conscious state of the animal (Seidemann et al., 2000, Seidemann et al., 2002, Shtoyerman et al., 2000, Slovin et al., 2000). It is important to note that many of the techniques described here are also useful for research based on physiological recording without optical imaging. For example, they could completely change the approach to chronic electrophysiological recording in behaving monkeys, since the electrodes can be targeted into a selected cortical site under full visual control.
We describe here the details of the ‘sliding-top cranial window’ and the ‘electrode positioner microdrive’, allowing the technique to be replicated by others. We also give examples of results obtained while combining optical imaging with the various electrode-based techniques in the multiple studies mentioned above (Shmuel and Grinvald, 1996, Shoham et al., 1997, Sterkin et al., 1999, Tsodyks et al., 1999). More detailed information is available upon request. A brief report of an earlier version of the micromanipulator was published in a review (Grinvald et al., 1999).
Section snippets
Sliding-top cranial window
Upon exposure of the brain, required for high-resolution optical imaging experiments, two major problems are encountered: (1) movement of the brain due to heartbeat pulsation and respiration, and (2) danger of brain infection caused by contamination. In principle, these problems can be eliminated by the use of a sealed cranial window. To allow the use of microelectrodes under visual control, an electrode should be inserted through the transparent window in a way that enables it to be
Results
The sliding-top cranial window with the electrode positioner microdrive for combined optical and electrophysiological recordings has been used for a number of studies in our laboratory (Shmuel and Grinvald, 1996, Shoham et al., 1997, Sterkin et al., 1999, Tsodyks et al., 1999, Shoham and Grinvald, 2001). The aim of the experiments was to record V1/V2 neurons from the visual cortex and to relate the electrical activity of single neurons to the functional anatomy and the population dynamics
Conclusions
The ‘sliding-top cranial window’ and its removable ‘electrode positioner microdrive’ described here have made it possible to integrate optical imaging with several powerful techniques based on microelectrodes. The advantage of the sliding-top cranial window is that it has a completely transparent cover, which can be moved with a drawer-like action over the chamber's outer lips, so that it can be easily and completely filled with liquid, and the cover can then be slid over it so that the chamber
Acknowledgements
We thank the Instrument Design Unit of the Weizmann Institute, and especially Jehoshua Wolowelsky, Nurit Gideon, Benjamin Pasmantirer, and Lilia Goffer, for their major contribution in planning the cranial window and the microdrive. Special thanks are due to Abraham Inhoren and Dado Binyamin from the Instrument Workshop of the Weizmann Institute. We thank Rina Hildesgeim, Tal Kenet, Amir Shmuel, and Doron Shoham for participating in the experiments. Many thanks to Shirley Smith for the
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