Understanding cortical microcircuits requires thorough measurement of physiological properties of synaptic connections formed within and between diverse subclasses of neurons. Towards this goal, we combined spatially precise optogenetic stimulation with multicellular recording to deeply characterize intralaminar and translaminar monosynaptic connections to supragranular (L2/3) neurons in the mouse visual cortex. The reliability and specificity of multiphoton optogenetic stimulation were measured across multiple Cre lines and measurements of connectivity were verified by comparison to paired recordings and targeted patching of optically identified presynaptic cells. With a focus on translaminar pathways, excitatory and inhibitory synaptic connections from genetically defined presynaptic populations were characterized by their relative abundance, spatial profiles, strength, and short-term dynamics. Consistent with the canonical cortical microcircuit, layer 4 excitatory neurons and interneurons within L2/3 represented the most common sources of input to L2/3 pyramidal cells. More surprisingly, we also observed strong excitatory connections from layer 5 intratelencephalic neurons and potent translaminar inhibition from multiple interneuron subclasses. The hybrid approach revealed convergence to and divergence from excitatory and inhibitory neurons within and across cortical layers. Divergent excitatory connections often spanned hundreds of microns of horizontal space. In contrast, divergent inhibitory connections were more frequently measured from postsynaptic targets near each other.

The complexity of modern neurophysiology experiments requires specialized software to coordinate multiple acquisition devices and analyze the collected data. We have developed ACQ4, an open-source software platform for performing data acquisition and analysis in experimental neurophysiology. This software integrates the tasks of acquiring, managing, and analyzing experimental data. ACQ4 has been used primarily for standard patch-clamp electrophysiology, laser scanning photostimulation, multiphoton microscopy, intrinsic imaging, and calcium imaging. The system is highly modular, which facilitates the addition of new devices and functionality. The modules included with ACQ4 provide for rapid construction of acquisition protocols, live video display, and customizable analysis tools. Position-aware data collection allows automated construction of image mosaics and registration of images with 3-dimensional anatomical atlases. ACQ4 uses free and open-source tools including Python, NumPy/SciPy for numerical computation, PyQt for the user interface, and PyQtGraph for scientific graphics. Supported hardware includes cameras, patch clamp amplifiers, scanning mirrors, lasers, shutters, Pockels cells, motorized stages, and more. ACQ4 is available for download at http://www.acq4.org.

Layer 4 (L4) of primary auditory cortex (A1) receives a tonotopically organized projection from the medial geniculate nucleus of the thalamus. However, individual neurons in A1 respond to a wider range of sound frequencies than would be predicted by their thalamic input, which suggests the existence of cross-frequency intracortical networks. We used laser scanning photostimulation and uncaging of glutamate in brain slices of mouse A1 to characterize the spatial organization of intracortical inputs to L4 neurons. Slices were prepared to include the entire tonotopic extent of A1. We find that L4 neurons receive local vertically organized (columnar) excitation from layers 2 through 6 (L6) and horizontally organized excitation primarily from L4 and L6 neurons in regions centered ~300–500 μm caudal and/or rostral to the cell. Excitatory horizontal synaptic connections from layers 2 and 3 were sparse. The origins of horizontal projections from L4 and L6 correspond to regions in the tonotopic map that are approximately an octave away from the target cell location. Such spatially organized lateral connections may contribute to the detection and processing of auditory objects with specific spectral structures.

The most common synaptic connections between neurons in different cortical layers form the basis of the canonical cortical microcircuit. Understanding of cortical function will require further development and application of methods to efficiently characterize synaptic connections within and outside of the canonical pathway. Accordingly, we measured synaptic inputs onto superficial excitatory neurons in response to sequential two-photon optogenetic stimulation of neurons in deeper layers. Layer 4 excitatory neurons and somatostatin-neurons within layer 2/3 represented the most common sources of input. Although connections from excitatory and somatostatin-neurons in layer 5 were less common, the amplitudes of synaptic responses were equally strong. We examined synaptic strength across all connections, as well as the relationships between the strength of connections diverging from a common presynaptic neuron or converging to a single target. While the overall distribution indicates synaptic weight is concentrated to a few connections, strong excitatory connections are distributed across cells.