Dissecting local circuits in vivo: Integrated optogenetic and electrophysiology approaches for exploring inhibitory regulation of cortical activity

Abstract

Local cortical circuit activity in vivo comprises a complex and flexible series of interactions between excitatory and inhibitory neurons. Our understanding of the functional interactions between these different neural populations has been limited by the difficulty of identifying and selectively manipulating the diverse and sparsely represented inhibitory interneuron classes in the intact brain. The integration of recently developed optical tools with traditional electrophysiological techniques provides a powerful window into the role of inhibition in regulating the activity of excitatory neurons. In particular, optogenetic targeting of specific cell classes reveals the distinct impacts of local inhibitory populations on other neurons in the surrounding local network. In addition to providing the ability to activate or suppress spiking in target cells, optogenetic activation identifies extracellularly recorded neurons by class, even when naturally occurring spike rates are extremely low. However, there are several important limitations on the use of these tools and the interpretation of resulting data. The purpose of this article is to outline the uses and limitations of optogenetic tools, along with current methods for achieving cell type-specific expression, and to highlight the advantages of an experimental approach combining optogenetics and electrophysiology to explore the role of inhibition in active networks. To illustrate the efficacy of these combined approaches, I present data comparing targeted manipulations of cortical fast-spiking, parvalbumin-expressing and low threshold-spiking, somatostatin-expressing interneurons in vivo.

Introduction

Neural circuits in the cortex are comprised of extensively interconnected populations of excitatory glutamatergic neurons and inhibitory GABAergic neurons. These two broad classes can be further divided into distinct subpopulations. This is particularly true of inhibitory interneurons, which can be grouped based on their morphology, molecular expression profiles, intrinsic firing properties, and synaptic connectivity (Ascoli et al., 2008, Markram et al., 2004, Moore et al., 2010). The availability of diverse inhibitory cell types provides a critical degree of functional flexibility to the network and allows for a wide range of computational operations within the local circuit. In an active network in vivo, each neuron receives a continuous barrage of excitatory and inhibitory synaptic inputs that result in a high-conductance state (Destexhe et al., 2003). In turn, that neuron contributes spike output to the surrounding local network. This continuous reciprocal exchange poses a considerable challenge to determining the roles of specific classes of neurons in contributing to information processing and generating ongoing patterns of cortical activity. The distributed and diverse nature of inhibitory populations increases this difficulty.

Considerable recent interest has focused on the roles of different interneuron populations in local cortical circuit operations. In particular, the relative impact of somatic and dendritic synaptic inhibition in regulating input integration and sensory processing remains unclear. In vitro evidence suggests that the intrinsic properties and synaptic positions of soma- and dendrite-targeting interneurons may cause them to be differentially recruited by excitatory inputs during sensory stimulation or other active conditions (Moore et al., 2010). In addition, previous work suggests that these two sources of inhibition may affect their postsynaptic targets on different time scales. However, these hypotheses have been difficult to test due to the difficulty of identifying and directly manipulating specific interneurons in vivo. The functional roles of some extremely sparse populations of inhibitory cells have likewise been difficult to elucidate in the intact brain. The development of technologies that allow targeted manipulation of specific cell types is thus critical to understanding interneuron participation in local circuit activity and ultimately in behavior and cognition.

Optogenetics, which provides tools for bidirectional optical control of neural activity by incorporation of artificially generated light-sensitive proteins into the cell membrane, represents a significant advance in our ability to dissect neural circuits in vivo. Optical manipulations via light-activated channels and pumps represent a highly reliable and physiologically appropriate method for activating and suppressing the firing of specific neural populations both in vitro (Adesnik and Scanziani, 2010, Boyden et al., 2005, Kuhlman and Huang, 2008, Petreanu et al., 2007, Sohal et al., 2009) and in vivo (Cardin et al., 2009, Gradinaru et al., 2009, Han et al., 2009, Huber et al., 2008, Tsai et al., 2009). Considerable recent work has highlighted the use of cell type-specific expression of the light-activated nonspecific cation channel Channelrhodopsin-2 (ChR2) and the light-activated chloride pump Halorhodopsin (eNpHR). The purpose of this review is to provide a detailed overview of the advantages and drawbacks of an experimental approach that integrates traditional electrophysiology and optogenetics and to present data illustrating the power of cell type-specificity in causally testing hypotheses about the functions of discrete populations of inhibitory neurons in vivo.

An integrative approach utilizing optical and electrophysiological methods provides two major advantages for in vivo exploration of network dynamics. Traditional extracellular recording techniques suffer from a lack of ability to identify the recorded neurons by class. Some cell types, such as fast-spiking interneurons, can be putatively identified by their characteristic waveform shape. However, this characterization has proven to be less than uniformly accurate. Most other types of inhibitory interneurons have broad spike waveforms and are indistinguishable from excitatory neurons in extracellular recordings. Furthermore, inhibitory cells are sparsely represented in the cortex and often have very low spontaneous firing rates, making them difficult to find. Inhibitory cells can be identified post hoc by recovering and staining intracellularly recorded neurons, but in vivo yields from these experiments are relatively low (Cardin et al., 2007, Hirsch et al., 2003). The reliability and precision of optically evoked firing, in combination with cell type-specific expression of ChR2, offers an unambiguous method for extracellular identification of cell type. Cells expressing ChR2 respond to brief light pulses with spikes at short latency and with a high degree of reliability, allowing the experimenter to rigorously identify the subpopulation of recorded neurons belonging to the targeted cell class (Cardin et al., 2009, Cardin et al., 2010, Lima et al., 2009). Multielectrode recordings can thus generate a higher yield of identified recordings. In addition, a major advantage for array recordings of large populations is the ability to artificially evoke many thousands of spikes from cells with normally low spontaneous firing rates, thereby enhancing spike waveform discrimination. A second, more obvious, advantage of the combined experimental approach is the ability to directly manipulate the level of activity of a specific class of cell in the context of observing ongoing network activity or behavior.

There are several inherent limitations on currently available optogenetic approaches and on their incorporation with traditional electrophysiological techniques. As discussed in Section 2.1, cell type specificity is limited by the choice of promoters, mouse lines, and vectors, making some cell types difficult to target. In addition, because ChR2 is distributed across the entire cell, light-evoked activation of ChR2 often causes conductance changes and depolarization simultaneously across entire targeted neurons, including the dendrites, cell body, and axon. It remains unclear whether calcium influx and synaptic vesicle release in response to this large, cell-wide depolarization is equivalent to that observed in response to naturally occurring spiking (Zhang et al., 2008). An additional concern is the relatively slow temporal kinetics of the onset and offset of ChR2, leading to light-evoked conductances and depolarization events that are significantly longer than naturally occurring events (Boyden et al., 2005, Gradinaru et al., 2010). Current optogenetic methods may thus be suboptimal for studies of synaptic transmission either in vitro or in vivo.

Because ChR2 activation generates robust current flow through ion channels, it can also produce misleading local field potential signals. For instance, activation of large layer 5 pyramidal neurons by light pulses at the cortical surface will preferentially depolarize the apical dendritic tufts of these cells. This localized current flow produces a superficial current sink, easily observed in the local field potential but unrelated to synaptic activity. Optoelectric artifacts, discussed below, can further obscure local field potential signals (Cardin et al., 2010, Han et al., 2009). An additional potential caveat to the use of optogenetic tools is the lack of any data on the cellular impact of long-term expression of these exogenous proteins. The potential for cell health to be compromised by high levels of expression of artificial rhodopsins over long periods remains unexplored.