Chemogenetic modulation and single-photon calcium imaging in anterior cingulate cortex reveal a mechanism for effort-based decisions

Hippocampal CA1 place cell spatial maps are known to alter their firing properties in response to contextual fear conditioning-a process called 'remapping'. In the present study, we use chronic calcium imaging to examine remapping during fear retrieval and extinction of an inhibitory avoidance task in mice of both sexes over an extended period of time and with thousands of neurons. We demonstrate that hippocampal ensembles encode space at a finer scale following fear memory acquisition. This effect is strongest near the shock grid. We also characterize the long-term effects of shock on place cell ensemble stability, demonstrating that shock delivery induces a several days of high fear and low between-session place field stability, followed by a new, stable spatial representation that appears after fear extinction. Finally, we identify a novel group of CA1 neurons that robustly encode freeze behavior independently from spatial location. Thus, following fear acquisition, hippocampal CA1 place cells sharpen their spatial tuning and dynamically change spatial encoding stability throughout fear learning and extinction. Significance StatementThe hippocampus contains place cells that encode an animal's location. This spatial code updates, or remaps, in response to environmental change. It is known that contextual fear can induce such remapping; in the present study, we use chronic calcium imaging to examine inhibitory avoidance-induced remapping over an extended period of time and with thousands of neurons and demonstrate that hippocampal ensembles encode space at a finer scale following electric shock, an effect which is 2 enhanced by threat proximity. We also identify a novel group of freeze behavioractivated neurons. These results suggest that, more than merely shuffling their spatial code following threat exposure, place cells enhance their spatial coding with the possible benefit of improved threat localization.

We present a large field of view (FOV) open-source miniature microscope (MiniLFOV) designed to extend the capabilities of the UCLA Miniscope platform to large-scale, single cell resolution neural imaging in freely behaving large rodents and head-fixed mice. This system is capable of multiple imaging configurations, including deep brain imaging using implanted optical probes and cortical imaging through cranial windows. The MiniLFOV interfaces with existing open-source UCLA Miniscope DAQ hardware and software, can achieve single cell resolution imaging across a 3.6 × 2.7 mm field of view at 23 frames per second, has an electrically adjustable working distance of up to 3.5 mm±150 µm using an onboard electrowetting lens, incorporates an absolute head-orientation sensor, and weighs under 14 grams. The MiniLFOV provides a 30-fold larger FOV and yields 20-fold better sensitivity than Miniscope V3, and a 12-fold larger FOV with 2-fold better sensitivity than Miniscope V4. Power and data transmission are handled through a single, flexible coaxial cable down to 0.3 mm in diameter facilitating naturalistic behavior. We validated the MiniLFOV in freely behaving rats by simultaneously imaging >1000 GCaMP7s expressing neurons in the CA1 layer of the hippocampus and in head-fixed mice by simultaneously imaging ∼2000 neurons in the mouse dorsal cortex through a 4 × 4 mm cranial window. For freely behaving experiments, the MiniLFOV supports optional wire-free operation using a 3.5 g wire-free data acquisition expansion board which enables close to 1-hour of wire-free recording with a 400 mAh (7.5 g) on-board single-cell lithium-polymer battery and expands wire-free imaging techniques to larger animal models. We expect this new open-source implementation of the UCLA Miniscope platform will enable researchers to address novel hypotheses concerning brain function in freely behaving animals.

150 words) 37 Prior evidence suggests that the anterior cingulate cortex (ACC) is required for choosing high 38 effort options over less effortful alternatives. Here, rats earned a high-value sucrose reward by 39 lever pressing on a progressive ratio schedule in either the presence (choice) or absence (no-choice) 40 of freely available, low-value lab chow. Disruption of ACC, either via chemogenetic inhibition or 41 excitation, reduced lever pressing for high-value reward in the choice, but not in the no-choice, 42 condition. In vivo calcium imaging revealed cell populations in ACC that selectively responded 43 prior to lever pressing and during sucrose retrieval. These responses were significantly weaker 44 during choice than no-choice sessions, which may have rendered them more susceptible to 45 chemogenetic disruption. Our results suggest that neural responses in ACC encode the relative 46 value of competing outcomes, and that the amount of effort an animal is willing to exert is 47 proportional to the magnitude of this relative value signal. 48 49 photon, calcium imaging 50 51 109 under CaMKIIα in ACC, labeled Gi. (B) Schematic reconstruction of maximum (red) and 110 minimum (pink) viral spread for all rats. Numerals depict +Anterior-Posterior (AP) level relative 111 to Bregma. Scale bars 1mm. (C) Representative photomicrograph showing eGFP (null virus) under 112 CaMKIIα in ACC, labeled GFP. (D) Schematic reconstruction of maximum (dark grey) and 113 minimum (light grey) viral spread for all rats. Numerals depict +Anterior-Posterior (AP) level 114 relative to Bregma. Scale bars 1mm. (E) Representative photomicrograph showing hM3Dq-115 mCherry DREADDs expression in ACC, labeled Gq. (F) Schematic reconstruction of maximum 116 (light green) and minimum (dark green) viral spread for all rats. Numerals depict +Anterior-117 Posterior (AP) level relative to Bregma. Scale bars 1mm. 118 119 Ex vivo electrophysiological validation of DREADDs. We confirmed the efficacy of our 120 DREADDs in slice recordings. A separate group of rats was prepared with Gq DREADDs in ACC 121 (Cg1) using identical surgical procedures to the main experiments (Figure 2A). As described in 122 Methods, a bipolar stimulating electrode was placed near the medial wall in layer I of ACC, and a 123 glass microelectrode filled with ACSF (resistance = 5-10 MΩ) was placed in layer 2/3 of ACC to 124 record field potentials and multiunit responses elicited by layer 1 stimulation. Using similar 125 methods, we have previously reported that application of CNO strongly suppressed evoked field 126 potentials in Gi transfected slices in ACC (Stolyarova et al., 2019). Here, we found that application 127 of CNO induced spontaneous bursting in four out of six Gq transfected slices (spontaneous activity 128rate was increased from 0 to 0.06 Hz ± 0.01 Hz, inter-burst intervals were 18.3 ± 2.6 seconds (mean 129 ± SEM, range: 12.4 -31.6 seconds, n=4 slices from 3 rats) (Figure 2B). In two other Gq transfected 130 slices where no spontaneous bursting was observed...

Imaging large-population, single-cell fluorescent dynamics in freely behaving animals larger than mice remains a key endeavor of neuroscience. We present a large-field-of-view open-source miniature microscope (MiniLFOV) designed for large-scale (3.6 mm × 2.7 mm), cellular resolution neural imaging in freely behaving rats. It has an electrically adjustable working distance of up to 3.5 mm ± 100 μm, incorporates an absolute head orientation sensor, and weighs only 13.9 g. The MiniLFOV is capable of both deep brain and cortical imaging and has been validated in freely behaving rats by simultaneously imaging >1000 GCaMP7s-expressing neurons in the hippocampal CA1 layer and in head-fixed mice by simultaneously imaging ~2000 neurons in the dorsal cortex through a cranial window. The MiniLFOV also supports optional wire-free operation using a novel, wire-free data acquisition expansion board. We expect that this new open-source implementation of the UCLA Miniscope platform will enable researchers to address novel hypotheses concerning brain function in freely behaving animals.