SUMMARY Modern genetic approaches are powerful in providing access to diverse cell types in the brain and facilitating the study of their function. Here we report a large set of driver and reporter transgenic mouse lines, including 23 new driver lines targeting a variety of cortical and subcortical cell populations and 26 new reporter lines expressing an array of molecular tools. In particular, we describe the TIGRE2.0 transgenic platform and introduce Cre-dependent reporter lines that enable optical physiology, optogenetics, and sparse labeling of genetically-defined cell populations. TIGRE2.0 reporters broke the barrier in transgene expression level of single-copy targeted-insertion transgenesis in a wide range of neuronal types, along with additional advantage of a simplified breeding strategy compared to our first-generation TIGRE lines. These novel transgenic lines greatly expand the repertoire of high-precision genetic tools available to effectively identify, monitor, and manipulate distinct cell types in the mouse brain.

We present a unique, extensive, and open synaptic physiology analysis platform and dataset. Through its application, we reveal principles that relate cell type to synaptic properties and intralaminar circuit organization in the mouse and human cortex. The dynamics of excitatory synapses align with the postsynaptic cell subclass, whereas inhibitory synapse dynamics partly align with presynaptic cell subclass but with considerable overlap. Synaptic properties are heterogeneous in most subclass-to-subclass connections. The two main axes of heterogeneity are strength and variability. Cell subclasses divide along the variability axis, whereas the strength axis accounts for substantial heterogeneity within the subclass. In the human cortex, excitatory-to-excitatory synaptic dynamics are distinct from those in the mouse cortex and vary with depth across layers 2 and 3.

Generating a comprehensive description of cortical networks requires a large-scale, systematic approach. To that end, we have begun a pipeline project using multipatch electrophysiology, supplemented with two-photon optogenetics, to characterize connectivity and synaptic signaling between classes of neurons in adult mouse primary visual cortex (V1) and human cortex. We focus on producing results detailed enough for the generation of computational models and enabling comparison with future studies. Here, we report our examination of intralaminar connectivity within each of several classes of excitatory neurons. We find that connections are sparse but present among all excitatory cell classes and layers we sampled, and that most mouse synapses exhibited short-term depression with similar dynamics. Synaptic signaling between a subset of layer 2/3 neurons, however, exhibited facilitation. These results contribute to a body of evidence describing recurrent excitatory connectivity as a conserved feature of cortical microcircuits.

One of the largest components of the delayed outward current active during normal physiology in many mammalian neurons such as medium spiny neurons of the striatum and tufted–mitral cells of the olfactory bulb, has gone unnoticed and is due to a Na+-activated-K+-current. Previous studies of K+ currents in mammalian neurons may have overlooked this large outward component because the sodium channel blocker tetrodotoxin (TTX) is typically used in such studies; we find that TTX also eliminates this delayed outward component as a secondary consequence. Unexpectedly we found that the activity of a persistent inward sodium current (persistent INa) is highly effective in activating this large Na+-dependent (TTX-sensitive) delayed outward current. Using siRNA techniques we identified SLO2.2 (Slack) channels as carriers of this delayed outward current. These findings have far reaching implications for many aspects of cellular and systems neuroscience, as well as clinical neurology and pharmacology.

We report a novel coupled system of sodium-activated potassium currents (IKNa) and persistent sodium currents (INaP), the components of which are widely distributed throughout the brain. Its existence and importance has not been previously recognized. Although IKNa was known to exist in many cell types, the source of Na+ which activates IKNa remained a mystery. We now show in single membrane patches generated from the somas of rat neurons that sodium influx through INaP is sufficient for activation of KNa channels, without substantial contribution from the transient sodium current or bulk [Na+]i. INaP was found to be active at cell membrane resting potentials, a finding that may explain why IKNa can be evoked from negative holding potentials. These results show an unanticipated role for INaP in activating a negative feedback system countering the excitable effects INaP; the interrelatedness of INaP and IKNa suggests new ways neurons can tune their excitability.

Principal neurons of the medial superior olive (MSO) convey azimuthal sound localization cues through modulation of their rate of action potential firing. Previous intracellular studies in vitro have shown that action potentials appear highly attenuated at the soma of MSO neurons, potentially reflecting specialized action potential initiation and/or a physically distant site of generation. To examine this more directly, we made dual patch-clamp recordings from MSO principal neurons in gerbil brainstem slices. Using somatic and dendritic whole-cell recordings, we show that graded action potentials at the soma are highly sensitive to the rate of rise of excitation and undergo strong attenuation in their backpropagation into the dendrites (length constant, 76 μm), particularly during strong dendritic excitation. Using paired somatic whole-cell and axonal loose-patch recordings, we show that action potentials recorded in the axon at distances > 25 μm are all-or-none, and uniform in amplitude even when action potentials appear graded at the soma. This proximal zone corresponded to the start of myelination in the axon, as assessed with immunocytochemical staining for myelin basic protein in single-labelled neurons. Finally, the axon was capable of sustaining remarkably high firing rates, with perfect entrainment occurring at frequencies of up to 1 kHz. Together, our findings show that action potential signalling in MSO principal neurons is highly secure, but shows a restricted invasion of the somatodendritic compartment of the cell. This restriction may be important for minimizing distortions in synaptic integration during the high frequencies of synaptic input encountered in the MSO.

SUMMARYModern genetic approaches are powerful in providing access to diverse types of neurons within the mammalian brain and greatly facilitating the study of their function. We here report a large set of driver and reporter transgenic mouse lines, including 23 new driver lines targeting a variety of cortical and subcortical cell populations and 26 new reporter lines expressing an array of molecular tools. In particular, we describe the TIGRE2.0 transgenic platform and introduce Cre-dependent reporter lines that enable optical physiology, optogenetics, and sparse labeling of genetically-defined cell populations. TIGRE2.0 reporters broke the barrier in transgene expression level of single-copy targeted-insertion transgenesis in a wide range of neuronal types, along with additional advantage of a simplified breeding strategy compared to our firstgeneration TIGRE lines. These novel transgenic lines greatly expand the repertoire of highprecision genetic tools available to effectively identify, monitor, and manipulate distinct cell types in the mouse brain.

Understanding cortical function will require a detailed and comprehensive knowledge of local circuit properties. The Allen Institute for Brain Science is beginning a large-scale project using multipatch electrophysiology, supplemented with 2-photon optogenetics, to characterize local connectivity and synaptic signaling between major classes of neurons in the adult mouse primary visual cortex and neurosurgical samples from human frontal and temporal cortex. We focus on generating results that are detailed enough for the generation of computational models and enable rigorous comparison with future studies. Here we report our examination of the intralaminar connectivity within each of several classes of excitatory neurons. We find that connections are sparse but present among all excitatory cell types and layers we sampled, with the most sparse connections in layers 5 and 6. Almost all synapses in mouse exhibited short-term depression with similar dynamics. Synaptic signaling between a subset of layer 2/3 neurons, however, exhibited facilitation. These results contribute to a body of evidence describing recurrent excitatory connectivity as a conserved feature of cortical microcircuits.