Summary An increasingly powerful approach for studying brain circuits relies on targeting genetically encoded sensors and effectors to specific cell types. However, current approaches for this are still limited in functionality and specificity. Here we utilize several intersectional strategies to generate multiple transgenic mouse lines expressing high levels of novel genetic tools with high specificity. We developed driver and double reporter mouse lines and viral vectors using the Cre/Flp and Cre/Dre double recombinase systems, and established a new, retargetable genomic locus, TIGRE, which allowed the generation of a large set of Cre/tTA dependent reporter lines expressing fluorescent proteins, genetically encoded calcium, voltage, or glutamate indicators, and optogenetic effectors, all at substantially higher levels than before. High functionality was shown in example mouse lines for GCaMP6, YCX2.60, VSFP Butterfly 1.2, and Jaws. These novel transgenic lines greatly expand the ability to monitor and manipulate neuronal activities with increased specificity.

In the rodent primary somatosensory cortex, the configuration of whiskers and sinus hairs on the snout and of receptor-dense zones on the paws is topographically represented as discrete modules of layer IV granule cells (barrels) and thalamocortical afferent terminals 1,2 . The role of neural activity, particularly activity mediated by NMDARs (N-methyl-D-aspartate receptors), in patterning of the somatosensory cortex has been a subject of debate [3][4][5][6] . We have generated mice in which deletion of the NMDAR1 (NR1) gene is restricted to excitatory cortical neurons, and here we show that sensory periphery-related patterns develop normally in the brainstem and thalamic somatosensory relay stations of these mice. In the somatosensory cortex, thalamocortical afferents corresponding to large whiskers form patterns and display critical period plasticity, but their patterning is not as distinct as that seen in the cortex of normal mice. Other thalamocortical patterns corresponding to sinus hairs and digits are mostly absent. The cellular aggregates known as barrels and barrel boundaries do not develop even at sites where thalamocortical afferents cluster. Our findings indicate that cortical NMDARs are essential for the aggregation of layer IV cells into barrels and for development of the full complement of thalamocortical patterns.The rodent somatosensory system is an excellent model to study molecular mechanisms underlying the establishment of patterned topographic connections between the sensory periphery and the brain. Trigeminal and dorsal column pathways convey a somatosensory map from the periphery to the neocortex by relay stations in the brainstem and the ventrobasal thalamus. At each level, pre-synaptic afferents and their target cells establish a patterned array of modules corresponding to the arrangement of whiskers and sinus hairs on the snout (trigeminal pathway), and receptor-dense zones of the paws (dorsal column pathway) 2,7,8 . These patterns are consolidated during the first postnatal week, and are subject to alterations if the sensory periphery is perturbed by nerve or whisker lesions during a critical period 2,7,8 . Thus, early neural activity might be essential in the development and plasticity of central patterns much like that of the vertebrate visual system 9,10 . Within this context, NMDAR activity has attracted much attention 9,11 . Initial studies using pharmacological blockade of NMDARs in postnatal rat somatosensory cortex did not detect effects on thalamocortical patterning, but revealed functional impairment of thalamocortical connectivity and whisker-specific responsiveness of barrel neurons 4,5 . In contrast, genetic manipulations of NMDARs showed an absence of periphery-related patterns in the somatosensory cortex 6 . This result could not be ascribed to direct effects on the neocortex, however, as it could have been a consequence of impaired patterns at the brainstem level 6,12,13 . To delineate the specific role(s) of cortical NMDARs in patterning, here we disrupted the ...

Population signals from neuronal ensembles in cortex during behavior are commonly measured with EEG, local field potential (LFP), and voltage-sensitive dyes. A genetically encoded voltage indicator would be useful for detection of such signals in specific cell types. Here we describe how this goal can be achieved with Butterfly, a voltage-sensitive fluorescent protein (VSFP) with a subthreshold detection range and enhancements designed for voltage imaging from single neurons to brain in vivo. VSFP-Butterfly showed reliable membrane targeting, maximum response gain around standard neuronal resting membrane potential, fast kinetics for single-cell synaptic responses, and a high signal-to-noise ratio. Butterfly reports excitatory postsynaptic potentials (EPSPs) in cortical neurons, whisker-evoked responses in barrel cortex, 25-Hz gamma oscillations in hippocampal slices, and 2- to 12-Hz slow waves during brain state modulation in vivo. Our findings demonstrate that cell class-specific voltage imaging is practical with VSFP-Butterfly, and expand the genetic toolbox for the detection of neuronal population dynamics.

The deep cerebellar nuclei (DCN) integrate inputs from the brain stem, the inferior olive, and the spinal cord with Purkinje cell output from cerebellar cortex and provide the major output of the cerebellum. Despite their crucial function in motor control and learning, the various populations of neurons in the DCN are poorly defined and characterized. Importantly, differences in electrophysiological properties between glutamatergic and GABAergic cells of the DCN have been largely elusive. Here, we used glutamate decarboxylase (GAD) 67-green fluorescent protein (GFP) knock-in mice to unambiguously identify GABAergic (GAD-positive) and non-GABAergic (GAD-negative, most likely glutamatergic) neurons of the DCN. Morphological analysis of DCN neurons patch-clamped with biocytin-containing electrodes revealed a significant overlap in the distributions of the soma sizes of GAD-positive and GAD-negative cells. Compared with GAD-negative DCN neurons, GAD-positive DCN neurons fire broader action potentials, display stronger frequency accommodation, and do not reach as high firing frequencies during depolarizing current injections. Furthermore, GAD-positive cells display slower spontaneous firing rates and have a more shallow frequency-to-current relationship than the GAD-negative cells but exhibit a longer-lasting rebound depolarization and associated spiking after a transient hyperpolarization. In contrast to the rather homogeneous population of GAD-positive cells, the GAD-negative cells were found to consist of two distinct populations as defined by cell size and electrophysiological features. We conclude that GABAergic DCN neurons are specialized to convey phasic spike rate information, whereas tonic spike rate is more faithfully relayed by the large non-GABAergic cells.

BackgroundFluorescent proteins have been used to generate a variety of biosensors to optically monitor biological phenomena in living cells. Among this class of genetically encoded biosensors, reporters for membrane potential have been a particular challenge. The use of presently known voltage sensor proteins is limited by incorrect subcellular localization and small or absent voltage responses in mammalian cells.ResultsHere we report on a fluorescent protein voltage sensor with superior targeting to the mammalian plasma membrane and high responsiveness to membrane potential signaling in excitable cells.Conclusions and SignificanceThis biosensor, which we termed VSFP2.1, is likely to lead to new methods of monitoring electrically active cells with cell type specificity, non-invasively and in large numbers, simultaneously.