Molecular tools for recording and intervention of neuronal activity.

Observing the activity of neural networks is critical for the identification of learning and memory processes, as well as abnormal activities of neural circuits in disease, particularly for the purpose of tracking disease progression. Methodologies for describing the activity history of neural networks using molecular biology techniques first utilized genes expressed by active neurons, followed by the application of recently developed techniques including optogenetics and incorporation of insights garnered from other disciplines, including chemistry and physics. In this review, we will discuss ways in which molecular biological techniques used to describe the activity of neural networks have evolved along with the potential for future development.

Serotonin in the orbitofrontal cortex enhances cognitive flexibility.

Cognitive flexibility is a brain's ability to switch between different rules or action plans depending on the context. However, cellular level understanding of cognitive flexibility have been largely unexplored. We probed a specific serotonergic pathway from dorsal raphe nuclei (DRN) to the orbitofrontal cortex (OFC) while animals are performing reversal learning task. We found that serotonin release from DRN to the OFC promotes reversal learning. A long-range connection between these two brain regions was confirmed anatomically and functionally. We further show that spatiotemporally precise serotonergic action directly enhances the excitability of OFC neurons and offers enhanced spike probability of OFC network. Serotonergic action facilitated the induction of synaptic plasticity by enhancing Ca2+ influx at dendritic spines in the OFC. Thus, our findings suggest that a key signature of flexibility is the formation of choice specific ensembles via serotonin-dependent synaptic plasticity.

Impacts of the invasive Spartina anglica on C-S-Hg cycles and Hg(II) methylating microbial communities revealed by hgcA gene analysis in intertidal sediment of the Han River estuary, Yellow Sea.

We investigated the impact of invasive vegetation on mercury cycles, and identified microorganisms directly related to Hg(II) methylation using hgcA gene in vegetated mud flats (VMF) inhabited by native Suaeda japonica (SJ) and invasive Spartina anglica (SA), and unvegetated mud flats (UMF) in Ganghwa intertidal sediments. Sulfate reduction rate (SRR) and rate constants of Hg(II) methylation (Km) and methyl-Hg demethylation (Kd) were consistently greater in VMF than in UMF, specifically 1.5, 2 and 11.7 times higher, respectively, for SA. Both Km and Kd were significantly correlated with SRR and the abundance of sulfate-reducing bacteria. These results indicate that the rhizosphere of invasive SA provides a hotspot for Hg dynamics coupled with sulfate reduction. HgcA gene analysis revealed that Hg(II)-methylators were dominated by Deltaproteobacteria, Chloroflexi and Euryarchaeota, comprising 37.9%, 35.8%, and 6.5% of total hgcA gene sequences, respectively, which implies that coastal sediments harbor diverse Hg(II)-methylating microorganisms that previously underrepresented.

Tagging active neurons by soma-targeted Cal-Light.

Verifying causal effects of neural circuits is essential for proving a direct circuit-behavior relationship. However, techniques for tagging only active neurons with high spatiotemporal precision remain at the beginning stages. Here we develop the soma-targeted Cal-Light (ST-Cal-Light) which selectively converts somatic calcium rise triggered by action potentials into gene expression. Such modification simultaneously increases the signal-to-noise ratio of reporter gene expression and reduces the light requirement for successful labeling. Because of the enhanced efficacy, the ST-Cal-Light enables the tagging of functionally engaged neurons in various forms of behaviors, including context-dependent fear conditioning, lever-pressing choice behavior, and social interaction behaviors. We also target kainic acid-sensitive neuronal populations in the hippocampus which subsequently suppress seizure symptoms, suggesting ST-Cal-Light's applicability in controlling disease-related neurons. Furthermore, the generation of a conditional ST-Cal-Light knock-in mouse provides an opportunity to tag active neurons in a region- or cell-type specific manner via crossing with other Cre-driver lines. Thus, the versatile ST-Cal-Light system links somatic action potentials to behaviors with high temporal precision, and ultimately allows functional circuit dissection at a single cell resolution.

Sulfur and methane oxidation by a single microorganism.

Natural and anthropogenic wetlands are major sources of the atmospheric greenhouse gas methane. Methane emissions from wetlands are mitigated by methanotrophic bacteria at the oxic-anoxic interface, a zone of intense redox cycling of carbon, sulfur, and nitrogen compounds. Here, we report on the isolation of an aerobic methanotrophic bacterium, 'Methylovirgula thiovorans' strain HY1, which possesses metabolic capabilities never before found in any methanotroph. Most notably, strain HY1 is the first bacterium shown to aerobically oxidize both methane and reduced sulfur compounds for growth. Genomic and proteomic analyses showed that soluble methane monooxygenase and XoxF-type alcohol dehydrogenases are responsible for methane and methanol oxidation, respectively. Various pathways for respiratory sulfur oxidation were present, including the Sox-rDsr pathway and the S4I system. Strain HY1 employed the Calvin-Benson-Bassham cycle for CO2 fixation during chemolithoautotrophic growth on reduced sulfur compounds. Proteomic and microrespirometry analyses showed that the metabolic pathways for methane and thiosulfate oxidation were induced in the presence of the respective substrates. Methane and thiosulfate could therefore be independently or simultaneously oxidized. The discovery of this versatile bacterium demonstrates that methanotrophy and thiotrophy are compatible in a single microorganism and underpins the intimate interactions of methane and sulfur cycles in oxic-anoxic interface environments.

Shifts in benthic bacterial communities associated with farming stages and a microbiological proxy for assessing sulfidic sediment conditions at fish farms.

To assess the aquaculture-induced sediment conditions associated with sulfur cycles, shifts in bacterial communities across farming stages were investigated. The sulfate reduction rate (SRR), and concentrations of acid volatile sulfide (AVS) and H2S were significantly higher at the mid- and post-farming stages than at the early stage, indicating that the aquaculture effects persist even after harvest. Incomplete organic carbon-oxidizing sulfate-reducing bacteria (IO-SRB) affiliated with Desulfobulbaceae, and gammaproteobacterial sulfur oxidizing bacteria (SOB) (Thiohalobacter, Thioprofundum, and Thiohalomonas) were dominant during the early stage, whereas fermenting bacteria (Bacteroidetes and Firmicutes) and complete oxidizing SRB (CO-SRB) belonging to Desulfobacteraceae, and epsilonproteobacterial SOB (Sulfurovum) dominated during the mid- and post-stages. The shift in SRB and SOB communities well reflected the anoxic and sulfidic conditions of farm sediment. Especially, the Sulfurovum-like SOB correlated highly and positively with H2S, AVS, and SRR, suggesting that they could be relevant microbiological proxies to assess sulfidic conditions in farm sediment.

Gradual decorrelation of CA3 ensembles associated with contextual discrimination learning is impaired by Kv1.2 insufficiency.

The associative network of hippocampal CA3 is thought to contribute to rapid formation of contextual memory from one-trial learning, but the network mechanisms underlying decorrelation of neuronal ensembles in CA3 is largely unknown. Kv1.2 expressions in rodent CA3 pyramidal cells (CA3-PCs) are polarized to distal apical dendrites, and its downregulation specifically enhances dendritic responses to perforant pathway (PP) synaptic inputs. We found that haploinsufficiency of Kv1.2 (Kcna2+/-) in CA3-PCs, but not Kv1.1 (Kcna1+/-), lowers the threshold for long-term potentiation (LTP) at PP-CA3 synapses, and that the Kcna2+/- mice are normal in discrimination of distinct contexts but impaired in discrimination of similar but slightly distinct contexts. We further examined the neuronal ensembles in CA3 and dentate gyrus (DG), which represent the two similar contexts using in situ hybridization of immediate early genes, Homer1a and Arc. The size and overlap of CA3 ensembles activated by the first visit to the similar contexts were not different between wild type and Kcna2+/- mice, but these ensemble parameters diverged over training days between genotypes, suggesting that abnormal plastic changes at PP-CA3 synapses of Kcna2+/- mice is responsible for the impaired pattern separation. Unlike CA3, DG ensembles were not different between two genotype mice. The DG ensembles were already separated on the first day, and their overlap did not further evolve. Eventually, the Kcna2+/- mice exhibited larger CA3 ensemble size and overlap upon retrieval of two contexts, compared to wild type or Kcna1+/- mice. These results suggest that sparse LTP at PP-CA3 synapse probably supervised by mossy fiber inputs is essential for gradual decorrelation of CA3 ensembles.

Eco-engineering approaches for ocean negative carbon emission.

The goal of achieving carbon neutrality in the next 30-40 years is approaching worldwide consensus and requires coordinated efforts to combat the increasing threat of climate change. Two main sets of actions have been proposed to address this grand goal. One is to reduce anthropogenic CO2 emissions to the atmosphere, and the other is to increase carbon sinks or negative emissions, i.e., removing CO2 from the atmosphere. Here we advocate eco-engineering approaches for ocean negative carbon emission (ONCE), aiming to enhance carbon sinks in the marine environment. An international program is being established to promote coordinated efforts in developing ONCE-relevant strategies and methodologies, taking into consideration ecological/biogeochemical processes and mechanisms related to different forms of carbon (inorganic/organic, biotic/abiotic, particulate/dissolved) for sequestration. We focus on marine ecosystem-based approaches and pay special attention to mechanisms that require transformative research, including those elucidating interactions between the biological pump (BP), the microbial carbon pump (MCP), and microbially induced carbonate precipitation (MICP). Eutrophic estuaries, hypoxic and anoxic waters, coral reef ecosystems, as well as aquaculture areas are particularly considered in the context of efforts to increase their capacity as carbon sinks. ONCE approaches are thus expected to be beneficial for both carbon sequestration and alleviation of environmental stresses.

IgSF11 homophilic adhesion proteins promote layer-specific synaptic assembly of the cortical interneuron subtype.

The most prominent structural hallmark of the mammalian neocortical circuitry is the layer-based organization of specific cell types and synaptic inputs. Accordingly, cortical inhibitory interneurons (INs), which shape local network activity, exhibit subtype-specific laminar specificity of synaptic outputs. However, the underlying molecular mechanisms remain unknown. Here, we demonstrate that Immunoglobulin Superfamily member 11 (IgSF11) homophilic adhesion proteins are preferentially expressed in one of the most distinctive IN subtypes, namely, chandelier cells (ChCs) that specifically innervate axon initial segments of pyramidal neurons (PNs), and their synaptic laminar target. Loss-of-function experiments in either ChCs or postsynaptic cells revealed that IgSF11 is required for ChC synaptic development in the target layer. While overexpression of IgSF11 in ChCs enlarges ChC presynaptic boutons, expressing IgSF11 in nontarget layers induces ectopic ChC synapses. These findings provide evidence that synapse-promoting adhesion proteins, highly localized to synaptic partners, determine the layer-specific synaptic connectivity of the cortical IN subtype.

Environmental Distribution of Styrene Oligomers (SOs) Coupled with Their Source Characteristics: Tracing the Origin of SOs in the Environment.

Despite growing concerns regarding plastic additives, their environmental fate coupled with leaching from source materials are not well known. Styrene oligomers (SOs), which are unintended additives in expanded polystyrene (EPS), are estrogenic micropollutants. Here, we identified the effects of their potential sources (i.e., EPS buoy and its leachate) and environmental dilution on SO distribution within coastal sediments. SO content in fresh EPS particles was 0.1% (w/w), dominated by 2,4,6-triphenyl-hexene (ST-1), while 2,4-diphenyl-1-butene (SD-2) accounted for most of the SOs in EPS leachate, indicating its faster leachability. In lake and offshore environments, the SO composition profiles from their terrestrial inputs and inner sites were similar to those of EPS leachate; meanwhile, the exponentially decreasing SO concentration and increasing styrene trimers (STs) fraction with distance from the inner to outer sites were evident. These profiles indicated continuous SO leaching from their potential sources in the inland, followed by a change in SOs due to environmental dilution. SOs in beach sediment implied the presence of micro-sized EPS particles. We suggest the ST-1 to SD-2 ratio as an index to differentiate among freshly leached SOs (∼0.02), environmentally diluted SOs after leaching (∼0.1), SOs in fresh EPS (∼1.2), and SOs in aged EPS (> 2).

Impact of finfish aquaculture on biogeochemical processes in coastal ecosystems and elemental sulfur as a relevant proxy for assessing farming condition.

We conducted experiments to investigate the effects of finfish aquaculture and to propose appropriate proxies for assessing their environmental impact. Due to enhanced fish feed input, sulfate reduction (SR) and the resulting metabolic products (H2S, NH4+, PO43-) were significantly greater at the farm than at the control site. Benthic release of dissolved inorganic nitrogen (DIN) and phosphorus (DIP) from farm sediment accounted for 52-837% and 926-1048%, respectively, of the potential DIN and DIP demand for phytoplankton production. The results suggest that excess organic loading in fish farms induces deleterious eutrophication and algal blooms in coastal ecosystems via benthic-pelagic coupling. Direct SR measurement provided the most useful information of all the parameters on organic contamination in fish farms. However, given its abundance, relatively lower chemical reactivity and relative ease of analysis, elemental sulfur was regarded as the most appropriate proxy for assessing the environmental impacts of finfish aquaculture.

Distinct temporal dynamics of planktonic archaeal and bacterial assemblages in the bays of the Yellow Sea.

The Yellow Sea features unique characteristics due to strong tides and nutrient-enriched freshwater outflows from China and Korea. The coupling of archaeal and bacterial assemblages associated with environmental factors at two bay areas in the Yellow Sea was investigated. Temporal variations of the archaeal and bacterial assemblages were shown to be greater than the spatial variations based on an analysis of the 16S rRNA gene sequences. Distinct temporal dynamics of both planktonic archaeal and bacterial assemblages was associated with temperature, NO2-, and chlorophyll a ([chl-a]) concentrations in the bays of the Yellow Sea. The [chl-a] was the prime predictor of bacterial abundance, and some taxa were clearly correlated with [chl-a]. Bacteroidetes and Alpha-proteobacteria dominated at high [chl-a] stations while Gamma-proteobacteria (esp. SAR86 clade) and Actinobacteria (Candidatus Actinomarina clade) were abundant at low [chl-a] stations. The archaeal abundance was comparable with the bacterial abundance in most of the October samples. Co-dominance of Marine Group II (MGII) and Candidatus Nitrosopumilus suggests that the assimilation of organic nitrogen by MGII could be coupled with nitrification by ammonia-oxidizing archaea. The distinct temporal dynamics of the archaeal and bacterial assemblages might be attributable to the strong tides and the inflow of nutrient-rich freshwater.

Impacts of typhoon-induced heavy rainfalls and resultant freshwater runoff on the partitioning of organic carbon oxidation and nutrient dynamics in the intertidal sediments of the Han River estuary, Yellow Sea.

Occurrence of typhoons accompanied by heavy precipitation has increased for the past 40 years in northeast Asia. To elucidate the impact of three consecutive typhoon-induced heavy rainfall events and resultant freshwater runoff on the partitioning of organic carbon (Corg) oxidation and nutrient dynamics, we investigated the geochemical constituents, the rate of anaerobic Corg oxidation, sulfate reduction (SR), iron reduction (FeR) and P speciation in the intertidal mud flat of the Han River estuary, Yellow Sea. Corg oxidation by SR and FeR and their metabolic products (∑CO2, NH4+, H2S, Fe2+) decreased significantly (P < 0.05) during and immediately after the heavy rainfall. Additional mesocosm experiments demonstrated that potential N2 production rates increased up to 2.4 times with increased nitrate concentrations during freshwater runoff. The results suggest that denitrification becomes a significant Corg oxidation pathway substituting for SR during high-nitrate freshwater runoff, which may remove substantial portion of the N introduced into the estuary. P speciation analysis further revealed that the concentrations of iron bound P decreased by 2.2 fold during the heavy rainfall compared to that measured before the rainfall. The results suggest that an excess supply of riverine Si keeps P from binding to Fe, thereby stimulating P release. Taking projections of enhanced rainfall events in the future into account, our results suggest that the intensified storm events and resultant riverine runoff induces a shift of Corg oxidation pathways in the sediments, which ultimately alters C-N-P-S-Fe dynamics and may deepen N-limiting conditions in coastal ecosystems of the Yellow Sea.

Correction to: Genomic and metatranscriptomic analyses of carbon remineralization in an Antarctic polynya.

Following publication of the original article [1], the authors identified wrong citations in the maintext.

Intracellular Zn2+ Signaling Facilitates Mossy Fiber Input-Induced Heterosynaptic Potentiation of Direct Cortical Inputs in Hippocampal CA3 Pyramidal Cells.

Repetitive action potentials (APs) in hippocampal CA3 pyramidal cells (CA3-PCs) backpropagate to distal apical dendrites, and induce calcium and protein tyrosine kinase (PTK)-dependent downregulation of Kv1.2, resulting in long-term potentiation of direct cortical inputs and intrinsic excitability (LTP-IE). When APs were elicited by direct somatic stimulation of CA3-PCs from rodents of either sex, only a narrow window of distal dendritic [Ca2+] allowed LTP-IE because of Ca2+-dependent coactivation of PTK and protein tyrosine phosphatase (PTP), which renders non-mossy fiber (MF) inputs incompetent in LTP-IE induction. High-frequency MF inputs, however, could induce LTP-IE at high dendritic [Ca2+] of the window. We show that MF input-induced Zn2+ signaling inhibits postsynaptic PTP, and thus enables MF inputs to induce LTP-IE at a wide range of [Ca2+]i values. Extracellular chelation of Zn2+ or genetic deletion of vesicular zinc transporter abrogated the privilege of MF inputs for LTP-IE induction. Moreover, the incompetence of somatic stimulation was rescued by the inhibition of PTP or a supplement of extracellular zinc, indicating that MF input-induced increase in dendritic [Zn2+] facilitates the induction of LTP-IE by inhibiting PTP. Consistently, high-frequency MF stimulation induced immediate and delayed elevations of [Zn2+] at proximal and distal dendrites, respectively. These results indicate that MF inputs are uniquely linked to the regulation of direct cortical inputs owing to synaptic Zn2+ signaling.SIGNIFICANCE STATEMENT Zn2+ has been mostly implicated in pathological processes, and the physiological roles of synaptically released Zn2+ in intracellular signaling are little known. We show here that Zn2+ released from hippocampal mossy fiber (MF) terminals enters postsynaptic CA3 pyramidal cells, and plays a facilitating role in MF input-induced heterosynaptic potentiation of perforant path (PP) synaptic inputs through long-term potentiation of intrinsic excitability (LTP-IE). We show that the window of cytosolic [Ca2+] that induces LTP-IE is normally very narrow because of the Ca2+-dependent coactivation of antagonistic signaling pairs, whereby non-MF inputs become ineffective in inducing excitability change. The MF-induced Zn2+ signaling, however, biases toward facilitating the induction of LTP-IE. The present study elucidates why MF inputs are more privileged for the regulation of PP synapses.

A large artificial dyke greatly alters partitioning of sulfate and iron reduction and resultant phosphorus dynamics in sediments of the Yeongsan River estuary, Yellow Sea.

We investigated sediment geochemistry, partitioning of organic carbon (Corg) oxidation by iron reduction (FeR) and sulfate reduction (SR), and benthic phosphorus (P) release, together with the P speciation in the sediments to elucidate the P dynamics in two contrasting sediments (i.e., estuarine vs. limnetic) separated by a large dyke in the Yeongsan River estuary of the Yellow Sea. In the sediments of the Yeongsan River estuary (St. YE), SR dominated the Corg oxidation pathway, accounting for 81.7% of total anaerobic Corg oxidation. Under the SR-dominated condition, H2S derived from SR reacts quickly with iron oxides to form iron sulfides, which ultimately release the P bound to Fe(III) into the pore water. The enhanced benthic P flux (0.24 mmol m-2 d-1) at the YE site accounted for 80% of the P required for primary production in the water column. In contrast, in the limnetic sediments of the Yeongsan Lake (St. YL), where high levels of CH4 accumulated, most P was bound to Fe and Al, which resulted in a low benthic P flux (0.03 mmol m-2 d-1). The results suggest that the frequent discharge of relatively P-depleted freshwater into the estuary via the artificial dyke may result in relatively P-limiting conditions in estuarine ecosystems. As a result, benthic P release from the SR-dominated estuarine sediment is a significant internal source of P in the coastal ecosystem. Our results indicate that the construction of a large dyke at a river mouth greatly alters Corg oxidation pathways and P dynamics in coastal ecosystems.

Genomic and metatranscriptomic analyses of carbon remineralization in an Antarctic polynya.

BACKGROUND: Polynyas in the Southern Ocean are regions of intense primary production, mainly by Phaeocystis antarctica. Carbon fixed by phytoplankton in the water column is transferred to higher trophic levels, and finally, to the deep ocean. However, in the Amundsen Sea, most of this organic carbon does not reach the sediment but is degraded in the water column due to high bacterial heterotrophic activity. RESULTS: We reconstructed 12 key bacterial genomes from different phases of bloom and analyzed the expression of genes involved in organic carbon remineralization. A high correlation of gene expression between the peak and decline phases was observed in an individual genome bin-based pairwise comparison of gene expression. Polaribacter belonging to Bacteroidetes was found to be dominant in the peak phase, and its transcriptional activity was high (48.9% of the total mRNA reads). Two dominant Polaribacter bins had the potential to utilize major polymers in P. antarctica, chrysolaminarin and xylan, with a distinct set of glycosyl hydrolases. In the decline phase, Gammaproteobacteria (Ant4D3, SUP05, and SAR92), with the potential to utilize low molecular weight-dissolved organic matter (LMW-DOM) including compatible solutes, was increased. The versatility of Gammaproteobacteria may contribute to their abundance in organic carbon-rich polynya waters, while the SAR11 clade was found to be predominant in the sea ice-covered oligotrophic ocean. SAR92 clade showed transcriptional activity for utilization of both polysaccharides and LMW-DOM; this may account for their abundance both in the peak and decline phases. Ant4D3 clade was dominant in all phases of the polynya bloom, implicating the crucial roles of this clade in LMW-DOM remineralization in the Antarctic polynyas. CONCLUSIONS: Genomic reconstruction and in situ gene expression analyses revealed the unique metabolic potential of dominant bacteria of the Antarctic polynya at a finer taxonomic level. The information can be used to predict temporal community succession linked to the availability of substrates derived from the P. antarctica bloom. Global warming has resulted in compositional changes in phytoplankton from P. antarctica to diatoms, and thus, repeated parallel studies in various polynyas are required to predict global warming-related changes in carbon remineralization.

Intersectional monosynaptic tracing for dissecting subtype-specific organization of GABAergic interneuron inputs.

Functionally and anatomically distinct cortical substructures, such as areas or layers, contain different principal neuron (PN) subtypes that generate output signals representing particular information. Various types of cortical inhibitory interneurons (INs) differentially but coordinately regulate PN activity. Despite a potential determinant for functional specialization of PN subtypes, the spatial organization of IN subtypes that innervate defined PN subtypes remains unknown. Here we develop a genetic strategy combining a recombinase-based intersectional labeling method and rabies viral monosynaptic tracing, which enables subtype-specific visualization of cortical IN ensembles sending inputs to defined PN subtypes. Our approach reveals not only cardinal but also underrepresented connections between broad, non-overlapping IN subtypes and PNs. Furthermore, we demonstrate that distinct PN subtypes defined by areal or laminar positions display different organization of input IN subtypes. Our genetic strategy will facilitate understanding of the wiring and developmental principles of cortical inhibitory circuits at unparalleled levels.

The locus coeruleus drives disinhibition in the midline thalamus via a dopaminergic mechanism.

The paraventricular nucleus of the thalamus (PVT) is increasingly being recognized as a critical node linking stress detection to the emergence of adaptive behavioral responses to stress. However, despite growing evidence implicating the PVT in stress processing, the neural mechanisms by which stress impacts PVT neurocircuitry and promotes stressed states remain unknown. Here we show that stress exposure drives a rapid and persistent reduction of inhibitory transmission onto projection neurons of the posterior PVT (pPVT). This stress-induced disinhibition of the pPVT was associated with a locus coeruleus-mediated rise in the extracellular concentration of dopamine in the midline thalamus, required the function of dopamine D2 receptors on PVT neurons, and increased sensitivity to stress. Our findings define the locus coeruleus as an important modulator of PVT function: by controlling the inhibitory tone of the pPVT, it modulates the excitability of pPVT projection neurons and controls stress responsivity.

A calcium- and light-gated switch to induce gene expression in activated neurons.

Despite recent advances in optogenetics, it remains challenging to manipulate gene expression in specific populations of neurons. We present a dual-protein switch system, Cal-Light, that translates neuronal-activity-mediated calcium signaling into gene expression in a light-dependent manner. In cultured neurons and brain slices, we show that Cal-Light drives expression of the reporter EGFP with high spatiotemporal resolution only in the presence of both blue light and calcium. Delivery of the Cal-Light components to the motor cortex of mice by viral vectors labels a subset of excitatory and inhibitory neurons related to learned lever-pressing behavior. By using Cal-Light to drive expression of the inhibitory receptor halorhodopsin (eNpHR), which responds to yellow light, we temporarily inhibit the lever-pressing behavior, confirming that the labeled neurons mediate the behavior. Thus, Cal-Light enables dissection of neural circuits underlying complex mammalian behaviors with high spatiotemporal precision.