The claustrum is required for reward acquisition under high cognitive demand

The claustrum is proposed to mediate a variety of functions ranging from sensory binding to top-down cognitive control of action, but direct functional assessments of this telencephalic nucleus are lacking. Here we employ the guanine nucleotide-binding subunit beta-4 cre driver line in mice to selectively monitor and manipulate claustrum projection neurons. Using fiber photometry, we find elevated claustrum activity prior to an expected cue during correct performance on a cognitively demanding five-choice response assay relative to a less-demanding one-choice version of the task. Claustrum activity during reward acquisition is also enhanced when cognitive demand is higher. Furthermore, we use optogenetic inhibition of claustrum prior to the expected cue to demonstrate that claustrum is critical for accurate performance on the five-choice, but not the one-choice, task. These results suggest the claustrum supports a cognitive control function necessary for reward acquisition under cognitively demanding conditions.


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Across species, the claustrum is widely connected with the neocortex including 49 sensory, motor, association and executive cortices (Crick and Koch 2005;Mathur 50 2014). This connectivity motivates a number of functional hypotheses (Remedios et al. percepts (Crick and Koch 2005). Direct analysis of claustrum function is historically 54 intractable, which necessitated indirect functional assessments of this structure. For 55 instance, a recent study leveraged the dense anterior cingulate cortex input to 56 claustrum (Smith and Alloway 2010; Wang et al. 2017;White et al. 2017) to show that 57 activity of this circuit rises with, and is required for, optimal performance on a five-choice 58 response task (White et al. 2018). 59 Novel transgenic tools, such as cre recombinase driver lines, now provide 60 genetic access to the claustrum (Wang et al. 2017). In this study, we use the guanine 61 nucleotide binding protein beta 4 cre driver line (GNB4-cre) for monitoring and 62 manipulation of claustrum projection neurons in awake, freely moving mice. Given the 63 role of the anterior cingulate cortex input to the claustrum on five-choice response task 64 performance, we herein examine the role of the claustrum itself on this task. GNB4-positive (+) neurons by performing whole-cell recordings from labeled neurons 79 using recording pipettes filled with AlexaFluor®-594. We found that GNB4+ neurons 80 were spiny ( Figure 1D), consistent with a projection neuron identity (Braak and Braak 81 1982;Hur and Zaborszky 2005;Watakabe et al. 2014). Basic membrane properties of 82 these neurons are shown in Figure 1E and representative responses to current injection 83 steps are shown in Figure 1F. Because capacitance delineates two claustrum projection 84 neurons (White and Mathur 2018b), this membrane property can further identify GNB4+ 85 neurons. In particular, we find a wide range of GNB4+ capacitance values (89 to 176 86 pF) consistent with sampling from both subtypes (type I = 118 ± 16 pF; type II = 158 ± 9 87 pF [mean ± SD]; White and Mathur 2018b). 88 To determine if claustrum is critical for the five-choice serial reaction time task 89 (5CSRTT) performance, we injected cre-dependent halorhodopsin (AAV-DIO- Experimental and control mice were trained to perform the 5CSRTT and subsequently a 95 one-choice stimulus-response task (1CSRTT; Figure 2C). The 1CSRTT was used to 96 control for the basic sensory and motor components of the 5CSRTT. The claustrum was 97 exposed to 470 nm light during the inter-trial interval (ITI) pseudo-randomly on 33% of 98 5CSRTT and 1CSRTT trials ( Figure 2D). This protocol was derived from our previous 99 work showing ACC input to claustrum is critical for optimal task performance within 1 s AAV-DIO-eNPhR3.0 mice, we found that accuracy deficits on inactivation trials during 106 the 1CSRTT were less than those on the 5CSRTT ( Figure 2H). For AAV-DIO-eYFP 107 mice, no differences in accuracy deficits were observed between the two tasks ( Figure   108 2H).

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To further exclude possible motor or reward-related effects elicited by claustrum 110 inactivation, we performed a real-time place preference (RTPP) assay. We found that Claustrum activity is sensitive to task load 119 We used a custom-made in vivo fiber photometry system to monitor calcium-120 dependent activity of claustrum in GBN4-cre mice injected with a virus expressing 121 GCaMP6f in a cre-dependent manner (AAV-FLEX-GCaMP6f; Figure 3A). The system 122 and data processing were designed to minimize sources of noise, such as fluctuations 123 in excitation laser intensity, motion-related artifacts, and bleaching artifacts. Fluctuations 124 in excitation laser intensity were controlled for by exciting a stable fluorophore, bleaching during 30 min 5CSRTT sessions, signals were examined in 10 trial bins.

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During 5CSRTT performance, claustrum activity was elevated on correct and 132 omission trials in the early phase of the ITI relative to incorrect trials ( Figure 3B). In the 133 late phase of the ITI, there were no differences in claustrum activity among the different 134 trial types ( Figure 3B). We next aligned claustrum activity to correct and incorrect nose 135 pokes during 5CSRTT performance. Average claustrum activity around the time of nose 136 poke was significantly greater for correct nose pokes compared to incorrect nose pokes 137 ( Figure 3C). To assess if claustrum activity bears any relationship with movement, we 138 7 monitored activity during free movement in an open field ( Figure 3D). Claustrum activity 139 was weakly and negatively correlated with movement velocity (Average r = -0.0062 140 Figure 3E). We next examined if claustrum activity during correct 5CSRTT performance 141 reflects task load. To this end, we first compared claustrum activity during the ITI 142 between correctly performed 5CSRTT and 1CSRTT trials ( Figure 4A) and found 143 relatively enhanced claustrum activity during the early ITI on the 5CSRTT but no 144 differences during the late ITI ( Figure 4B). We next compared the two tasks by aligning 145 activity to the correct nose pokes ( Figure 4C). We did not observe any activity 146 differences between the two tasks immediately prior to or subsequent to nose pokes 147 ( Figure 4D). Lastly, we aligned claustrum activity to the acquisition of sucrose pellets 148 ( Figure 4E). Activity was greater for the 5CSRTT relative to the 1CSRTT immediately 149 before and after acquisition of the sucrose pellet ( Figure 4F).

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Our results suggest that claustrum is required for optimal performance on the 153 cognitively-demanding 5CSRTT but not the 1CSRTT or open field movement.

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Claustrum activity on the 5CSRTT is higher on accurate compared to inaccurate task 155 performance. In addition, relative to the 1CSRTT, claustrum activity on the 5CSRTT is  It is important to note that our photometry analysis detects population level 161 claustrum projection neuron activity. Therefore, it is difficult to determine, for example, 162 whether increased activity-dependent calcium signals reflect recruitment of more 163 claustrum neurons, an increase in firing of a subset of claustrum neurons, synchrony of 164 claustrum neurons, or some combination of these possibilities. As such, future studies 165 will need to assess if selective activation of functionally distinct subpopulations may 166 explain our finding that claustrum activity on omission trials, as with correct trials, is 167 elevated relative to incorrect trials during the 5CSRTT ITI, for example.

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Claustrum activity may also be driven by retrosplenial cortex, which is heavily