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 primary motor cortex (M1), layer 5 projection neurons signal directly to distant motor structures to drive movement. Despite their pivotal position and acknowledged diversity these neurons are traditionally separated into broad commissural and corticofugal types, and until now no attempt has been made at resolving the basis for their diversity. We therefore probed the electrophysiological and morphological properties of retrogradely labeled M1 corticospinal (CSp), corticothalamic (CTh), and commissural projecting corticostriatal (CStr) and corticocortical (CC) neurons. An unsupervised cluster analysis established at least four phenotypes with additional differences between lumbar and cervical projecting CSp neurons. Distinguishing parameters included the action potential (AP) waveform, firing behavior, the hyperpolarisation-activated sag potential, sublayer position, and soma and dendrite size. CTh neurons differed from CSp neurons in showing spike frequency acceleration and a greater sag potential. CStr neurons had the lowest AP amplitude and maximum rise rate of all neurons. Temperature influenced spike train behavior in corticofugal neurons. At 26°C CTh neurons fired bursts of APs more often than CSp neurons, but at 36°C both groups fired regular APs. Our findings provide reliable phenotypic fingerprints to identify distinct M1 projection neuron classes as a tool to understand their unique contributions to motor function.

(1) and also ryanodine-sensitive release channels, activated by the pyridine nucleotide metabolite, cyclic adenosine 5Ј-diphosphate ribose (cADPR) (2). Recently, a third distinct Ca 2ϩ release mechanism has been identified which is potently and selectively activated by a novel pyridine nucleotide, nicotinic acid adenine dinucleotide phosphate (NAADP) (5, 6). NAADP releases Ca 2ϩ from egg homogenates and microsomal fractions in the presence of mitochondrial inhibitors and also when microinjected into the egg (6, 7). Neither heparin, an established InsP 3 receptor antagonist, nor the ryanodine receptor inhibitors, procaine and ruthenium red (8), or the selective cADPR antagonist 8-NH 2 -cADPR (9) block NAADP-induced Ca 2ϩ release (5, 6), indicating that NAADP acts at an alternative site. In addition, depletion of endoplasmic reticulum (ER) stores by pretreatment with thapsigargin (10 M) does not significantly affect NAADP Ca 2ϩ release, although it reduces release by both InsP 3 and cADPR (10). This result is in accordance with the suggestion that the NAADP-sensitive Ca 2ϩ store may reside in an intracellular compartment distinct from the InsP 3 -and cADPR-sensitive stores (6).We now report that non-releasing concentrations of NAADP fully and irreversibly inactivate the NAADP-sensitive Ca 2ϩ release mechanism both in intact sea urchin eggs and in homogenates and that this property is not shared by either InsP 3 or cADPR. Moreover, NAADP mobilizes Ca 2ϩ by activating a Ca 2ϩ release channel in the microsomal membrane, which is selectively blocked by classical inhibitors of L-type voltagesensitive Ca 2ϩ channels. These properties suggest that this release mechanism might contribute to complex patterns of Ca 2ϩ signals widely observed in intracellular signaling. MATERIALS AND METHODSCollection of Sea Urchin Eggs-Eggs were obtained by stimulating ovulation of female Lytechinus pictus (Marinus, Inc., Long Beach, CA) with a intracoelomic injection of KCl. These were then washed twice in artificial sea water (435 mM NaCl, 40 mM MgCl 2 , 15 mM MgSO 4 , 11 mM CaCl 2 , 10 mM KCl, 2.5 mM NaHCO 3 , 1.0 mM EDTA, pH 8.0), and jelly removed by filtration through 90-m nylon mesh.Ca 2ϩ Release Assays-Homogenates (2.5%) of unfertilized Lytechinus pictus eggs were prepared as described previously (11) and Ca 2ϩ loading was achieved by incubation at room temperature for 3 h in an intracellular medium consisting of 250 mM potassium gluconate, 250 mM Nmethylglucamine, 20 mM Hepes, pH 7.2,1 mM MgCl 2 , 0.5 mM ATP, 10 mM phosphocreatine, 10 units/ml creatine phosphokinase, 1 mg/ml oligomycin, 1 mg/ml antimycin, 1 mM sodium azide, 3 mM fluo-3. Free Ca 2ϩ concentration was measured by monitoring fluorescence intensity at excitation and emission wavelengths of 490 and 535 nm, respectively. Fluorimetry was performed at 17°C using 500 l of homogenate in a Perkin-Elmer LS-50B fluorimeter. Additions were made in a 5-l volume, and all chemicals were added in intracellular medium containing 10 M EGTA. Basal concentrations of Ca 2ϩ were typically...

1. The perforant path projection from layer III of the entorhinal cortex to CA1 of the hippocampus was studied within a hippocampal‐entorhinal combined slice preparation. We prevented contamination from the other main hippocampal pathways by removal of CA3 and the dentate gyrus. 2. Initially the projection was mapped using field potential recordings that suggested an excitatory sink in stratum lacunosum moleculare with an associated source in stratum pyramidale. 3. However, recording intracellularly from CA1 cells, stimulation of the perforant path produced prominent fast GABAA and slow GABAB IPSPs often preceded by small EPSPs. In a small number of cells we observed EPSPs only. 4. CNQX blocked excitatory and inhibitory responses. This indicated the presence of an intervening excitatory synapse between the inhibitory interneurone and the pyramidal cell. 5. Focal bicuculline applications revealed that the major site of GABAA inhibitory input was to stratum radiatum of CA1. 6. The inhibition activated by the perforant path was very effective at reducing simultaneously activated Schaffer collateral mediated EPSPs and suprathreshold‐stimulated action potentials. 7. Blockade of fast inhibition increased excitability and enhanced slow inhibition. Both increases relied upon the activation of NMDA receptors. 8. Perforant path inputs activated prominent and effective disynaptic inhibition of CA1 cells. This has significance for the output of hippocampal processing during normal behaviour and also under pathological conditions.