Background

Within the visual cortex, orientation pinwheels (PCs) are singularity centers around which neuronal orientation preference systematically shifts. PCs have been the focus of many studies on how the brain encodes different aspects of object shape, such as linear oriented segments, curved segments, corners, and T junctions (Hubel and Wiesel, 1974; Das and Gilbert, 1999; Hashemi-Nezhad and Lyon, 2012). Single-unit recording has been the most common method for studying organization within orientation pinwheels (Hubel and Wiesel, 1974). However, due to limitations of blood vessel guided localization of microelectrodes and uncertainties of electrode perpendicularity (Nauhaus and Ringach, 2007), it is quite challenging to accurately target microelectrodes to pinwheel centers (PCs). Hashemi-Nezhad and Lyon (2012) used electrodes coated with Dil to nicely demonstrate that electrodes were truly perpendicular; however, the vasculature-based method still contained localization error. Nauhaus and Ringach (2007), carefully matched the electrode locations in a Utah array with locations in an optical imaged orientation map and achieved accurate PC localization, but such an approach offers little control over where the electrodes end up within orientation domains or pinwheel centers. Using 2-photon methods, Ohki et al. (2006) reported that the true PC is only 130 µm in diameter, underscoring the need for highly accurate electrode targeting (see also Nauhaus et al., 2008).

In this study, we developed a method for targeting PCs with high accuracy. This was achieved by: (1) Assuring perpendicular penetrations by imaging a large field of view with a narrow depth of field (~50 μm, front-to-front lens with f1.2 (Ratzlaff and Grinvald, 1991), and then ensuring the electrode paralleled exactly the axis of the optical imaging. Using this procedure, we have estimated that the deviation of electrodes from perpendicular is less than 2 deg (see Figure 3D). (2) Carefully targeting electrodes to PCs based on blood vessel guidance. (3) Further determining the precise PC center by assessing whether the local field potentials (LFP) exhibit ‘non-sine-like’ responses. We have previously shown (Li et al., 2019) using this methodology that penetrations with sine-like LFP response and small scatter in orientation preference are not true PC locations, while those with non-sine-like response and large scatter in orientation preference (> 60 deg, Maldonado et al., 1997) are the true PC locations. This additional non-sine-like criterion is essential for highly accurate determination of PC locations and has provided new understanding of orientation subdomain organization (Li et al., 2019). These detailed procedures are described below.