Brain networks include a large diversity of functionally distinct neuronal elements each with unique properties enabling computational capacities and supporting brain functions. validate this strategy by recordings of identified subtypes of GABAergic and glutamatergic neurons in deep cortical layers subcortical cholinergic neurons and neurons in the thalamic reticular nucleus in anesthetized and awake mice. We propose this method as an important complement to existing technologies to relate specific cell type activity to brain circuitry function and behavior. Introduction Most brain structures consist of a large diversity of neuronal subtypes differing in their precise anatomical location morphology connectivity molecular composition biophysical properties and activity patterns. To understand network function and the neural basis of behavior it is necessary to measure the activity of precisely identified cell types in the intact brain. However studying the activity of specific cell types has been limited especially when optical access is restricted or when the cells of interest do not represent the majority within a brain structure. Here we report the development and validation of a high-yield method for the targeted recording and labeling of genetically identified neurons throughout the brain which provides their activity and precise anatomical location morphological and molecular properties. Several approaches have been employed to record the activity of different cell types in the brain. Extracellular blind unit recordings can take advantage of spike waveform properties to separate putative excitatory and inhibitory cells (Csicsvari et al. 1999 However since both populations contain cell types exhibiting overlapping spectra of spike shapes assignment of recorded units to particular cell types remains difficult (Fuentealba et al. 2008 Vigneswaran et al. 2011 The use of glass electrodes and blind patch technology enable dye loading of recorded neurons solving this ambiguity (Pinault and Deschenes 1998 In principle the full morphology and anatomical position can be recovered and the expression of molecular markers determined through SJ 172550 immunohistochemistry (Klausberger et al. 2003 Despite the high degree of precision of such an approach its low yield makes its use impractical when studying infrequent cell types. The recent development of optical and genetic technologies has dramatically facilitated the targeting and recording of the activity of identified cell populations. Using fluorescent protein expression in specific cell types in transgenic animals two-photon targeted patching (TPTP) enables the visualization of genetically defined neurons in order to direct the recording electrode to the cell type of interest for extracellular recording of spiking activity or intracellular membrane potential measurements (Margrie et al. 2003 However light scattering through brain tissue has largely restricted the use of two-photon guided methodologies to superficial brain structures such as upper cortical layers. The expression of channelrhodopsin-2 (ChR2) under specific promoters in combination with tetrodes or silicon probes coupled to a light source overcomes this limitation (Lima et al. 2009 Anikeeva et al. 2012 Roux et al. 2014 Through this strategy genetically defined cells that conditionally express ChR2 can be identified through SJ 172550 their tight temporal responsiveness to light stimulation allowing the ‘tagging’ of extracellularly recorded SJ 172550 units and monitoring of their activity regardless of recording depth. This method has been successfully applied to record the activity of genetically tagged neuronal populations in freely behaving animals (Kvitsiani PKACa et al. 2013 Stark et al. 2013 However unlike TPTP this technique cannot offer information on the morphology precise anatomical position and membrane potential dynamics of the recorded cells and SJ 172550 as such relies largely on the genetic identification provided by the conditional expression of ChR2 and spike waveform isolation. Strict dependence on genetic identification is not sufficient for the unambiguous identification of cell types. Many widely used transgenic mouse lines do not target single neuronal subtypes but rather neuronal subpopulations that include substantial and physiologically relevant heterogeneity (Huang 2014 For instance cortical GABAergic interneurons expressing parvalbumin (PV) can exhibit basket and chandelier morphologies (Rudy SJ 172550 et al. 2011 with potentially opposite postsynaptic impacts.