Neural models of approach-avoidance (AA) conflict behavior and its dysfunction have focused traditionally on the hippocampus, with the assumption that this medial temporal lobe (MTL) structure plays a ubiquitous role in arbitrating AA conflict. We challenge this perspective by using three different AA behavioral tasks in conjunction with optogenetics, to demonstrate that a neighboring region in male rats, perirhinal cortex, is also critically involved but only when conflicting motivational values are associated with objects and not contextual information. The ventral hippocampus, in contrast, was found not to be essential for object-associated AA conflict, suggesting its preferential involvement in context-associated conflict. We propose that stimulus type can impact MTL involvement during AA conflict and that a more nuanced understanding of MTL contributions to impaired AA behavior  is required. These findings serve to expand upon the established functions of the perirhinal cortex while concurrently presenting innovative behavioral paradigms that permit the assessment of different facets of AA conflict behavior. Approach-avoidance (AA) conflict is elicited when an organism experiences competition between incompatible motivations of being attracted to and repelled by the same goal stimulus. Its effective resolution is vital for survival and everyday decision-making, while its dysfunction manifests across a spectrum of psychiatric disorders including anxiety and addiction . Since tGiven the focus on the role of the HPC in AA conflict processing, very limited empirical work has explored the potential contributions of the surrounding medial temporal lobe (MTL) cortices. This is somewhat surprising given the intricate anatomical and functional relationships between MTL structures. Moreover, a revised formulation of the BIS postulated the involvement of the entorhinal cortex and perirhinal cortex (PRC) in the detection and resolution of AA conflict , a suggeHere, we designed two rodent AA tasks using objects as target stimuli to test the hypothesis that differential MTL recruitment occurs during AA conflict processing in a stimulus-type specific manner in rodents. In both tasks, rats were trained to associate object pairs with either appetitive or aversive outcomes and were subsequently presented with these objects in a conflict arrangement (simultaneous presentation of appetitive and aversive objects) while the PRC or vHPC (CA3 subfield) was optogenetically inhibited. However, each task was conducted in a different apparatus, specifically a runway or shuttle box, to elicit a different type of avoidance behavior (passive vs. active). The PRC was also inhibited while animals completed an established ‘contextual’ AA task, known to be dependent on the vHPC . Across Rats were first surgically infused with inhibitory archaerhodopsin T (ArchT) or green fluorescent protein only (GFP) and implanted bilaterally with optic fibers into the PRC . Rats wep2 = 0.95). Rats also exhibited a significant reduction in goal box time during aversive test session between the two acquisition tests =16.86, p<0.001, ηp2 = 0.46) =1.1, p=0.34, ηp2 = 0.05; test × construct: F=0.2, p=0.66, ηp2 = 0.01).Acquisition of object valences was assessed after four (test 1) and eight (test 2) conditioning sessions, without laser treatment. PRC groups acquired the object-outcome associations successfully by test 2, with rats spending the most time in the goal box after exposure to the appetitive object pair (p<0.001), and the least time after aversive object pair exposure (p<0.001), compared to goal box time in neutral trials =396.99, p<0.001, η = 0.46) . The patp2 = 0.67; all post hoc: p<0.001) (p2 = 0.55), although there was no effect on the number of goal box entries  = 3.12, p=0.09, ηp2 = 0.14) =0.47, p=0.63, ηp2 = 0.02), nor did it have an effect on the exploration of appetitive and aversive objects  = 3.10, p=0.09, ηp2 = 0.13; laser × construct × object: F=0.15, p=0.70, ηp2 = 0.007) (p2 = 0.05)  or low  level of motivational conflict, or no conflict  . When anp<0.001) , indicat = 0.14)  during t= 0.007) . Collect = 0.05) .p2 = 0.37) (p2 = 0.02) or retreats from =0.54, p=0.47, ηp2 = 0.04) the goal box during the choice period =0.21, p=0.81, ηp2 = 0.01), or valence of the object pairing  = 2.38, p=0.15, ηp2 = 0.17) =0.02, p=0.88, ηp2 = 0.001), with comparable object exploration between valence pairings  = 1.64, p=0.22, ηp2 = 0.09; valence × construct: F=3.73, p=0.07, ηp2 = 0.19) , their time in the goal box increased =9.37, p=0.007, η = 0.37) , but thee period . An anal = 0.17) . There w = 0.19) .p2 = 0.94; laser × valence × construct: F=1.14, p=0.3, ηp2 = 0.07), with all animals spending significantly more time in the goal box for appetitive-neutral pairings than aversive-neutral =229.64, p<0.001, ηp<0.001, . Furtherp<0.001, , indicatp2 = 0.95; pairing: F=1.67, p=0.22, ηp2 = 0.11; valence × pairing: F=0.27, p=0.61, ηp2 = 0.02; p2 = 0.01; pairing × construct: F=0.04, p=0.86, ηp2 = 0.003).To rule out the possibility that the observed effects of PRC inhibition were driven by a failure to respond to novel recombinations of object pairings rather than conflict processing, we repeated the runway task in which animals were trained to associate a new set of four object pairs with either appetitive or aversive outcomes (two pairs each) . Animalsp2 = 0.007; laser × construct: F=0.01, p=0.92, ηp2 = 0.001) =0.05, p=0.82, ηp2 = 0.004). PRC inhibition also had no effect on the number of entries into =0.06, p=0.82, ηp2 = 0.004) nor retreats from the goal box =0.15, p=0.71, ηp2 = 0.01), and furthermore, all PRC-inhibited rats made fewer entries (p<0.001) and exhibited more retreat behavior (p<0.0001) when presented with an aversive pairing compared with an appetitive pairing =0.30, p=0.74, ηp2 = 0.02) regardless of the valence of the object pairing =2.99, p=0.11, ηp2 = 0.18); nor did it have an effect on the exploration of appetitive and aversive object pairs =1.07, p=0.32, ηp2 = 0.07; laser × construct × object: F=0.07, p=0.80, ηp2 = 0.005), with comparable object exploration between valence pairings =3.67, p=0.08, ηp2 = 0.21; valence × construct: F=0.39, p=0.55, ηp2 = 0.03; Rats were then administered a within-valence ‘no conflict’ recombination test . PRC inhibition did not lead to significant changes in the time spent in the goal box after exposure to novel appetitive or aversive object pairs =0.10, p=0.76, η= 0.001) . An anal pairing . PRC inhp2 = 0.57; laser × conflict: F=5.43, p=0.0093, ηp2 = 0.25) , we analyzed the amount of time and proportion of total goal box time that ArchT animals spent in a demarcated ‘dispenser zone’  during the high (App/Av), low (App/Neu), and no conflict (App/App) test sessions. When faced with high conflict, PRC inhibition led to animals spending significantly more time by the sucrose dispenser compared to high conflict test sessions without optogenetic inhibition (p<0.0001), whereas an increase in time spent by the dispenser elicited by low conflict stimuli approached significance =24.34, p<0.0001, η = 0.25) . As expep2 = 0.30, conflict: F=11.19, p=0.00038, ηp2 = 0.41, laser × conflict: F=3.34, p=0.048, ηp2 = 0.17) (p2 = 0.38), which was likely driven by the PRC inhibition group data =3.92, p=0.065, ηp2 = 0.20; laser × construct × box × conflict: F=0.2, p=0.82; p2 = 0.40). Object exploration was also comparable between high vs. low conflict conditions, with PRC inhibition having no impact on the durations of object exploration =0.003, p=0.96, ηp2 = 0.0001; laser: F=0.034, p=0.86, ηp2 = 0.002; laser × construct: F=0.056, p=0.82, ηp2 = 0.003).When considering the time spent by the dispenser as a proportion of the total time in the goal box, PRC-inhibited rats spent a higher proportion of time at the dispenser during high conflict trials compared with low conflict (p=0.0008) and no conflict (p=0.0012) trials, as well as high conflict trials completed with the laser off =7.80, p=0.012, η = 0.17) . Laser oCollectively, these findings indicate that the PRC inhibition-induced increase in approach behavior during the high and low conflict tests arises from alterations in conflict processing, rather than impairments in valence retrieval or novel stimulus recombination processing. Furthermore, in the absence of the normal PRC functioning, animals spent more time by the sucrose dispenser and traversed through the runway more quickly, reflecting a greater anticipation for a reward outcome.after object exploration) and prior to the choice period  was trained on the runway paradigm, wherein the optogenetic inhibition was applied when the animal entered the neutral box . The pattern of valence acquisition was comparable between ArchT and GFP animals =1.09, p=0.3, ηp2 = 0.07; valence × construct: F=1.53, p=0.23, ηp2 = 0.10; test × construct: F=1.64, p=0.22, ηp2 = 0.10).All rats acquired the object-outcome associations successfully by test 2, with rats spending the most time in the goal box after exposure to the appetitive object pair (p<0.001), and the least time after aversive object pair exposure (p<0.001), compared to goal box time in neutral trials =231.3, p<0.001, ηp2=0.85; all post hoc: p<0.001) =0.06, p=0.81, ηp2=0.002; laser timing × construct: F=0.12, p=0.7, ηp2=0.005), suggesting that PRC mediates object-based motivational conflict rather than encoding or retrieving representations of highly conflicting objects. The difference score of the two conflict test sessions (laser on–laser off) revealed that laser treatment increased goal box time for ArchT animals only  = 9.02, p<0.001).When animals were exposed to a high conflict object pair, optogenetic inhibition of PRC prior to goal box access significantly increased time spent in the goal box compared to trials without inhibition, and compared to GFP control animals =81.3, p<0.001, ηp<0.001) . Turningp2 = 0.73), while having no effect on the number of goal box entries =0.88, p=0.37, ηp2 = 0.06) =0.26, p=0.77, ηp2 = 0.02), or exploration of appetitive and aversive objects  = 0.007, p=0.93, ηp2 = 0.001; laser × construct × object: F=0.05, p=82, ηp2 = 0.004)  = 38.11, p<0.001, η = 0.06) , the LTE= 0.004) . PRC inhp2=0.53) =0.24, p=0.64, ηp2=0.05) =0.11, p=0.11, ηp2=0.07), or the exploration of the App-Neu and Av-Neu object pairings =1.42, p=0.25, ηp2=0.09; laser × construct × object: F=0.1, p=0.92, ηp2=0.001), with comparable object exploration between the valanced pairs =2.36, p=0.15, ηp2=0.14; valence × construct: F = 0.001, p=0.97, ηp2<0.0001) =15.77, p<0.005, ηp2=0.53) . The lasp2=0.05) . PRC inh<0.0001) .p2 = 0.95; laser × valence × construct: F=1.0, p=0.34, ηp2 = 0.07), with all animals spending significantly more time in the goal box for appetitive-neutral pairings than aversive-neutral =253.86, p<0.001, ηp<0.001, . Furtherp<0.001, . Thus, vp2 = 0.95; laser × conflict: F=10.70, p=0.014, ηp2 = 0.61) =24.41, p=0.002, ηp2 = 0.78; laser × conflict: F=5.95, p=0.045, ηp2 = 0.46) =1.48, p=0.24, ηp2 = 0.1), with all rats exhibiting overall longer latencies to enter all boxes during the high conflict test compared with the low conflict test =9.99, p=0.007, ηp2 = 0.42; construct: F=0.17, p=0.69, ηp2 = 0.01; conflict × construct: F=0.28, p=0.60, ηp2 = 0.02; laser × construct × box × conflict: F=0.16, p=0.85).Finally, PRC-inhibited ArchT animals spent significantly more time by the sucrose dispenser after encountering both high conflict (p<0.001) and low conflict (p<0.01) stimuli, compared to test sessions without optogenetic inhibition =130.11, p<0.0001, η = 0.61) . PRC-inh = 0.46) . An analp2 = 0.78; valence × test: F=16.17, p=0.001, ηp2 = 0.55) =4.22, p=0.06, ηp2 = 0.25; test × construct: F=2.69, p=0.13, ηp2 = 0.17). PRC rats also spent more time in the outcome-associated area (stay behavior) when exposed to the appetitive object pair compared with the aversive object pair =456.04, p<0.0001, ηp2 = 0.97; valence × test: F=21.18, p<0.001, ηp2 = 0.62) =8.17, p=0.013, ηp2 = 0.39; test × construct: F=1.65, p=0.22, ηp2 = 0.11).To investigate whether the observed increase in goal box preference time exhibited by PRC-inhibited rats was specific to the conditions elicited by the runway task, the same PRC rats completed another novel behavioral paradigm conducted in a modified two-way active avoidance ‘shuttle box’ apparatus . In the  = 0.55) . The pat = 0.62) . This pap2 = 0.59) (p2 = 0.07), PRC-inhibited animals spent significantly longer in the outcome-associated area containing the conflict object pair compared with trials completed without inhibition , and compared to GFP control animals (p<0.005) with laser on and off =12.32, p=0.004, ηp2 = 0.49) , and increased cumulative duration in the outcome-associated area  for ArchT animals compared with GFP.When animals were next exposed to a high conflict pairing, optogenetic inhibition of PRC (laser on) significantly prolonged escape latencies compared with trials completed without inhibition , and compared to GFP control animals (p<0.005) with laser on and off =18.97, p=0.001, η = 0.59) , indicat = 0.49) . An analp2 = 0.06; laser × construct: F=0.15, p=0.71, ηp2 = 0.01; valence × construct: F=0.04, p=0.84, ηp2 = 0.003), with all rats exhibiting significantly shorter escape latencies for aversive than appetitive trials =84.84, p<0.001, ηp2 = 0.87; construct: F=0.06, p=0.82, ηp2 = 0.004) =0.003, p=0.96, ηp2 < 0.001; valence × construct: F=0.22, p=0.65, ηp2 = 0.02), with all rats spending significantly less time in the cued side during aversive than appetitive trials =470.47, p<0.0001, ηp2 = 0.97; construct: F=0.55, p=0.47, ηp2 = 0.04) =0.88, p=0.37, η= 0.004) . Similar = 0.04) . Collectp2 = 0.9; valence × test: F=116.05, p<0.001, ηp2 = 0.89) =0.04, p=0.85, ηp2 = 0.003; test × construct: F=1.06, p=0.32, ηp2 = 0.07). Rats also spent more time in the outcome-associated area (stay behavior) when exposed to the appetitive object pair compared with the aversive object pair =419.32, p<0.0001, ηp2 = 0.97; valence × test: F=140.61, p<0.0001, ηp2 = 0.91) =0.35, p=0.56, ηp2 = 0.02; test × construct: F=0.95, p=0.35, ηp2 = 0.06).As with the runway task, a separate cohort of animals received optogenetic inhibition immediately after object exploration  . The pat = 0.91) . This pap2 = 0.77) (p2 = 0.13), PRC-inhibited animals spent significantly longer in the outcome-associated area containing the conflict object pair compared with trials completed without inhibition , and compared to GFP control animals (p<0.005) with laser on and off =14.98, p=0.002, ηp2 = 0.52)  during the choice period significantly prolonged escape latencies compared with trials completed without inhibition , and compared to GFP control animals (p<0.001) with laser on and off =47.74, p<0.001, η = 0.77) , indicat = 0.52) . An analTo investigate whether the observed alteration in object-based AA conflict processing extended to conflict represented by ‘context-like’ cues, we administered an established Y-maze AA task that is vHPC-dependent, with large vHPC lesions and ventral CA3 (vCA3) or dentate gyrus inactivation increasing approach behavior in the face of high AA conflict =24.26, p<0.0001, ηp2 = 0.6), regardless of construct =0.17, p=0.69, ηp2 = 0.01; test × construct: F = 0.01, p=0.92, ηp2 = 0.001; valence × construct: F=1.51, p=0.24, ηp2 = 0.09) by spending significantly more time in the appetitive arm (both tests p<0.001) and less time in the aversive arm (both p≤0.003) compared with the neutral arm =1.06, p=0.32, ηp2 = 0.06) =0.31, p=0.58, ηp2 = 0.02; arm × construct: F=2.47, p=0.14, ηp2 = 0.13). Moreover, PRC inhibition did not significantly alter the number of entries made into or retreats from either the conflict or neutral arms =0.001, p=0.98, ηp2 <0.001; construct: F=3.36, p=0.09, ηp2 = 0.17; laser × construct: F=0.07, p=0.8, ηp2 = 0.004; retreats: laser: F=0.03, p=0.86, ηp2 = 0.002; construct: F=0.5, p=0.49, ηp2 = 0.3; laser × construct: F=0.2, p=0.66, ηp2 = 0.12) =0.001, p=0.98, ηp2 <0.001; retreats: arm: F=1.19, p=0.29, ηp2 = 0.07) for both ArchT and GFP animals =0.07, p=0.8, ηp2 = 0.004; retreats: arm × construct: F=0.43, p=0.52, ηp2 = 0.03). Thus, while PRC is critical for AA conflict processing associated with discrete objects, it may play a minimal role in contextual AA conflict.Rats first learned the valences of three visuotactile cues  that spanned the length of three different maze arms . Analysitral arm . Rats th = 0.06) , and bot = 0.12) . The nump2 = 0.93; valence × test: F=23.73, p<0.0001, ηp2 = 0.65) =0.20, p=0.82, ηp2 = 0.02) with the exception that ArchT rats spent less time in the goal box compared to GFP rats during acquisition test 1 =7.13, p=0.019, ηp2 = 0.35; test 1 construct: F=10.94, p=0.006, ηp2 = 0.46). ArchT and GFP animals did not, however, differ at the end of training =2.21, p=0.16, ηp2 = 0.15).To investigate whether the vHPC plays a role in object-associated AA conflict processing, rats with either ArchT or GFP and optical fiber implants in the vCA3  were adm = 0.65) , and at p2 = 0.18; construct: F=0.001, p=0.99, ηp2 <0.001; laser × construct: F=0.93, p=0.60, ηp2 = 0.02) (p2 = 0.44). vCA3 inhibition also did not impact other behavioral measures (p2 = 0.003), entries made into the goal box =0.58, p=0.46, ηp2 = 0.04), and LTE =0.37, p=0.55, ηp2 = 0.03). vCA3 inhibition also did not affect time spent in the goal box when rodents were presented with the neutral object pair =0.03, p=0.90, ηp2 = 0.03; laser × construct: F=2.36, p=0.15, ηp2 = 0.18) =2.92, p=0.11, η = 0.02) . Indeed,measures  includin = 0.18) .p2 = 0.008; laser × construct: F=0.29, p=0.60, ηp2 = 0.02) (p2 = 0.17). All vCA3 animals could readily discriminate between valences as reflected by time spent in the goal box =550.18, p<0.0001, ηp2 = 0.98; valence × construct: F=0.25, p=0.63, ηp2 = 0.02) and retreats =13.80, p=0.003, ηp2 = 0.52). There was, however, no effect of vCA3 inhibition on entries into and retreats from the goal box ≤0.55, p≥0.47, ηp2≤ 0.09) =0.11, p=0.75, η = 0.02)  and cruc2≤ 0.09) .p2 = 0.97; pairing: F=0.006, p=0.94, ηp2 <0.001; valence × pairing: F=0.19, p=0.67, ηp2 = 0.02; pairing × construct: F=0.14, p=0.72, ηp2 = 0.01) =0.040, p=0.85, ηp2 = 0.003) (p2≤ 0.2) =0.03, p=0.88, ηp2 = 0.002). In sum, vCA3 inhibition had no impact on any aspect of performance on the object AA runway task.Following successful acquisition of a new set of appetitive and aversive object pairs =361.37, p<0.0001, η = 0.01) , vCA3 in= 0.003) , with enp2≤ 0.2) . Both Arp2 = 0.90; valence × test: F=92.12, p<0.0001, ηp2 = 0.88; construct: F=1.43, p=0.25, ηp2 = 0.10; valence × construct: F=1.18, p=0.30, ηp2 = 0.08; test × construct: F=0.03, p=0.87, ηp2 = 0.002) =9.41, p=0.0090, ηp2 = 0.42; construct: F=0.01, p=0.98, ηp2 < 0.001; valence × construct: F=0.68, p=0.42, ηp2 = 0.05; test × construct: F=1.28, p=0.28, ηp2 = 0.09) =119.71, p<0.0001, η= 0.002) . All vCA = 0.09) .p2 = 0.04; construct: F=0.03, p=0.87, ηp2 = 0.002; laser × construct: F=0.29, p=0.87, ηp2 = 0.02) (p2 = 0.39). vCA3 inhibition also had no effect on the time spent in the outcome-associated area containing the conflict object pair =0.26, p=0.62, ηp2 = 0.02; construct: F=0.12, p=0.74, ηp2 = 0.009; laser × construct: F=0.020, p=0.89, ηp2 = 0.002) (p2 = 0.27).In contrast to PRC rats, there was no effect of vCA3 inhibition on escape latency behavior when exposed to the high conflict object pairing =0.48, p=0.5, η = 0.02) . An over= 0.002) . Similarp2 = 0.04; laser × construct: F=3.13, p=0.1, ηp2 = 0.19; valence × construct: F=0.25, p=0.63, ηp2 = 0.02), with all rats exhibiting significantly shorter escape latencies for aversive than appetitive trials =826.68, p<0.0001, ηp2 = 0.99; construct: F=0.03, p=0.88, ηp2 = 0.002) =0.04, p=0.84, ηp2 = 0.003; valence × construct: F=0.69, p=0.42, ηp2 = 0.05), with all rats spending significantly less time in the cued side during aversive than appetitive trials =565.77, p<0.0001, ηp2 = 0.98; construct: F=0.30, p=0.59, ηp2 = 0.02) =0.51, p=0.49, η= 0.002) . Similar = 0.02) .In sum, these results indicate that vCA3 inhibition had no impact on object-associated motivational behavior whether under AA conflict or the presence of appetitive or aversive cues alone.To confirm optogenetic inhibition of cellular activity in the PRC and vCA3, cFos immunohistochemistry was performed following a PRC-dependent task: novel object recognition (NOR), in which rats explored an open maze containing a novel and a familiar object; or a vHPC-dependent elevated plus maze (EPM), in which rats explored two anxiogenic open arms and two ‘safe’ closed arms.p2 = 0.57, construct: F=28.46, p<0.0001, ηp2 = 0.46; laser × construct: F=39.63, p<0.0001, ηp2 = 0.54) =14.42, p=0.003, ηp2 = 0.57; laser × construct: F = 7.1, p=0.022, ηp2 = 0.39) compared with laser-off Arch T and vCA3-GFP control animals   revealed that laser-treated PRC ArchT animals exhibited a significant NOR impairment =45.6, p<0.0001, η = 0.54)  comparedp≤0.001) .p2 = 0.74; construct: F=56.49, p<0.0001, ηp2 = 0.62; laser × construct: F=64.82, p<0.0001, ηp2 = 0.66) or vCA3 =5.81, p=0.035, ηp2 = 0.35; construct: F=3.77, p=0.07, ηp2 = 0.26; laser × construct: F=12.78, p=0.004; ηp2 = 0.54), compared with ArchT animals that completed the tasks with laser off and GFP control animals   or EPM (vCA3) tasks with the laser on prior to sacrifice demonstrated a significant reduction in cFos labeling in the PRC =98.11, p<0.0001, ηp≤0.001) . Thus, lFinally, histological analysis confirmed robust bilateral expression of GFP/ArchT and optic fiber tip placement immediately dorsal to the viral injection site in all PRC animals . Thus, nUsing a set of original object-based AA paradigms, we found that optogenetic inhibition of the rodent PRC, but not the vCA3, resulted in a robust increase in approach bias during motivational conflict elicited by the presentation of discrete object pairs associated with non-matching affective values. In contrast, PRC inhibition did not disrupt behavior during a contextual vHPC-dependent AA task. Critically, PRC inhibition did not disrupt AA behavior in response to neutral stimuli or novel re-configurations of objects with the same valence. Furthermore, selective optogenetic inhibition of the PRC continuously throughout the entire test session or prior to the first goal box choice (and therefore post-object exploration) induced the same approach bias in choice behavior, suggesting that the observed impact of PRC inhibition on object-associated AA conflict processing was unlikely to be driven by impairments in mnemonic functioning.Our finding that optogenetic inhibition of PRC, but not vCA3, disrupted object-associated motivational conflict behavior contrasts with a plethora of studies detailing a role for the rodent vHPC in the resolution of AA conflict . HoweverWe suggest that the observed approach bias in the face of object-associated motivational conflict during PRC inhibition reflects both a robust increase in approach behaviour and decrease in avoidance behaviour. The latter is reflected in the fact that PRC inhibition induced an increase in the time spent in the ‘Av-Neu low conflict’ condition of the runway task and led to a prolonged ‘escape latency’ when rodents were first exposed to conflicting object pairs. PRC-inhibited rats also spent a greater amount of time in the conflict cued side in the shuttle box task, further lending support to the idea that there was a decrease in avoidance. On the other hand, we also observed that PRC-inhibited animals spent a disproportionate amount of time by the sucrose dispenser in the runway task, seeking out reward under extinction conditions in the face of motivational conflict, which we interpret as an increase in approach behaviour.Although the observed involvement of the PRC in the arbitration of AA conflict may, at first sight, be unexpected given that this area is traditionally associated with mnemonic function, its involvement in motivational processes is reasonable considering its reciprocal connectivity with limbic centers of the brain, including the amygdala and orbitofrontal, prelimbic and infralimbic cortices . The nonAn intriguing question is why PRC inhibition potentiated approach, as opposed to avoidance, of an uncertain outcome. In the present study, the excitatory/principal neuronal population  in PRC was targeted and silenced. These neurons receive powerful excitatory inputs from the basolateral amygdala, lateral amygdala, and medial prefrontal cortex , which cFinally, given the well-established role of the PRC in recognition memory and the processing of novelty signals , it is iTo conclude, we demonstrate that rodent PRC is involved in the resolution of AA conflict, particularly when the goal stimulus is object-based. Thus, stimulus type may fundamentally alter how the brain represents motivational conflict, which may in turn recruit differential MTL structures for the purpose of AA conflict resolution. Our findings also have implications for our understanding of mental disorders in which AA behavior is awry and suggest that a singular focus within the MTL on HPC dysfunction may be inadequate. Further work is needed on delineating the network of structures involved in various aspects of the conflict process, along with examining how a changing stimulus gradient  may recruit different brain structures to resolve conflict.Subjects were 53 male Long Evans rats  weighing between 320 and 380 g at the start of the experiment. Following surgery, rats were individually housed to prevent damage to the implanted optic fiber, and maintained at a constant room temperature of 22°C under a 12 hr light/dark cycle (lights on at 7:00am). Water was available ad libitum, and animals were fed ad libitum up until food-motivated experimental training commenced. Food-restricted animals were maintained at 85–90% of their baseline free-feeding weight. All experiments were conducted during the light cycle and were done in accordance with the regulations of the Canadian Council of Animal Care and approved by the University and Local Animal Care Committee of the University of Toronto (Protocol no. 20012479). We anticipated a moderate to large effect size (0.6–1.1) based on unpublished behavioral data from our laboratory obtained from three previous cohorts of animals. These values were used for an a priori power analysis to compute the required sample size for the current experiment, in which a within-subjects design for optogenetic inhibition was used .unpublished observations) that demonstrated refined viral expression patterns and optimal fiber implantation localized to the PRC and vCA3 regions.Rats were randomly assigned to receive viral infusions to either the PRC  or the vCA3 region of the HPC . Rats were anesthetized with isoflurane  and secured in a stereotaxic frame  with the incisor bar set at –3.3 mm below the interaural line. An incision along the midline of the skull was made and the fascia retracted by small skin clips to reveal bregma. For PRC surgeries, the temporalis muscle was carefully peeled back from the temporal ridge to access lateral injection sites. Small burr holes were created directly over the injection sites using a dental drill, and 0.5 µl of virus  was infused bilaterally with a 1 µl Hamilton syringe into either the PRC  or the vCA3  over 5 min, with the needle left in situ for a further 5 min. Optic fibers were then implanted 0.5 mm dorsal to the infusion site, and were secured in place with dental cement anchored to jeweler screws implanted in the skull. Rats were given at least 7 days to recover in their home-cages before beginning behavioral experiments. The viral infusion and optic fiber implantation coordinates were chosen based on two previous cohorts of animals (Testing was conducted in three Y-maze arms (each 77 cm [L] × 11.5 cm [W] × 35 cm [H]) arranged to form a continuous ‘runway’ (231 cm [L]), with the sides of the apparatus were wrapped in red cellophane to minimize reliance on extra-maze cues, with the top remaining exposed to permit optogenetic tethering and free mobility of the compartment door. The runway was segmented into four compartments  which were separable by stainless-steel guillotine door inserts. The entire stainless-steel grid floor was covered in opaque black Plexiglas, except for the goal box, which contained exposed grid flooring connected to a shock generator . A stainless-steel dish connected to a sucrose dispenser was presented at the end of the grid flooring , which the animals were required to traverse in order to obtain reward.Behavioral testing was conducted in a modified active avoidance ‘shuttle box’ apparatus (54 cm [L] × 26 cm [W] × 32.5 cm [H]) , which was divided into two equally halved compartments (27 cm [L] × 26 cm [W] × 32.5 cm [H]) separated by a central guillotine door, with the entire apparatus wrapped in red cellophane to minimize reliance on extra-maze cues. Two removable panels (40 cm [L] × 25 cm [W]), one opaque and one transparent, were inserted in each side of the apparatus, which served to prevent recognition of and access to the object pairs, respectively. The transparent panel contained two rows of holes (each 4 cm apart), positioned at the rat’s eye level, to permit olfactory sampling. Two stainless-steel dishes connected to sucrose dispensers were presented at opposing ends of the apparatus, located behind both panels.Behavioral testing was conducted in a three-arm Y-maze apparatus, as previously described . BrieflyBehavioral testing was conducted in a transparent open-field apparatus (50 cm [L] × 50 cm [W] × 50 cm [H]), wrapped with black plastic to reduce anxiety. The apparatus was lined with home-cage bedding, which was replaced on days of testing. The bedding was agitated after each trial to eliminate potential odor traces.Given the established role for the PRC in the resolution of feature ambiguity between highly similar objects , we atteA set of visuotactile cues were used in the Y-maze task, which consisted of wood panel inserts (46.5 cm [L] × 9.6 cm [H]) affixed to the length of each arm with hook-and-loop fasteners. Three sets of bar cues were used, with two sets wrapped in either duct tape or a denim cloth material, and the last set being an unwrapped, varnished wooden material. Bar cues were wiped down with a 70% ethanol solution between trials.PRC rats and VCA3 rats first completed testing in the object-based AA runway task . They thAll rats were given 4 days of habituation to permit exposure to the runway apparatus and the object cues. On day 1, rats were confined to the start box for 30 s, and then permitted to explore the entire apparatus for 3.5 min. On day 2, following a 30 s confinement to the start box, rats could proceed into and were confined within the object box (without any objects) for 1 min, with re-entry to the start box prohibited by a stainless-steel guillotine door. Rats could then proceed to the neutral box, in which they were confined for 1 min, with re-entry to the object boxes prohibited. Following this, they were confined to the goal box for 30 s before being removed from the apparatus. Days 3 and 4 introduced the rats to the six object cues used for the object-based AA task; each rat performed three daily trials, with each trial presenting two object cues, and thus rats were exposed to all six object cues daily. The order that the objects appeared for each rat was counterbalanced between animals and habituation sessions, and the time spent exploring the objects was recorded. Object habituation began with 30 s confinement to the start box, and rats were then permitted 1 min to explore the two object cues during a given session. Rats then entered the neutral box, in which they were confined for 1 min, followed by the goal box, in which they were confined for 30 s. The affective valence of the object cues for subsequent conditioning sessions was determined based on exploration latencies during habituation 3 and 4. The two most-explored object cues were assigned as the aversive cue pair, the two least-explored as the appetitive cue pair, and the remaining two as the neutral cue pair.The animals were trained to associate the three object pairs with either appetitive (sucrose), aversive (mild foot shock), or neutral (no event) outcomes over nine daily conditioning sessions. Each rat completed three trials per day, one for each affective valence. During a trial, the time spent exploring each object cue was recorded, and the rat could enter the neutral box after 1 min elapsed. After 10 s, rats could enter the goal box where they were confined to for 30 s during which the outcome assigned to the explored object pair was administered . The magnitude of shock was deliberately kept at a level to elicit avoidance behaviour but preserve the animals’ exploratory drive . After 30 s elapsed, rats were removed from the apparatus and returned to their home-cage in preparation for the next conditioning trial. If rats did not readily enter the next box within 30 s, they were gently ushered in by the experimenter, and object, neutral, and goal boxes LTE data was also recorded as a potential measure of preference/aversion of expected outcome. The shock level was calibrated for each rat during the first aversive conditioning session and fixed at a level which elicited a mild startle response and defensive treading behavior, but not freezing. The order of presentation of the object cues and the order in which rats completed each trial were changed daily.Acquisition tests performed under extinction conditions were conducted after conditioning days 4 and 8 in order to assess learning of the object cue-outcome associations. The experimental procedure was identical to that of cue conditioning training, except that rats were not confined to the goal box upon entry and were permitted 2 min to freely move between the neutral box and goal box during this time. Successful acquisition was indicated by the rats spending more time in the goal box for appetitive trials than aversive and neutral trials, and rats spending more time in the neutral box for aversive trials than appetitive and neutral trials. Following the second acquisition test, rats were given a ‘refresher’ conditioning day prior to proceeding to the AA conflict test.On the day of AA conflict testing, rats were bilaterally tethered to the laser and placed into the start box. The laser was then turned on for animals completing the entire task under PRC inhibition, before the start box guillotine door was removed exposing the animals to the object box, to which they were confined upon entry. One appetitive object cue and one aversive object cue were presented in recombination as the ‘high conflict object pair’, which animals could freely explore for 1 min. Identical to conditioning sessions, rats were then given 30 s to freely enter the neutral box, after which they were gently ushered in by the experimenter, and confined therein for 10 s. Rats were then permitted to enter the goal box. At this point, rats were given 3 min to freely move between the neutral box and goal box, after which the laser was turned off and the animal was removed from the apparatus and returned to its home-cage in preparation for the next trial. All rats completed two conflict test sessions, one with the laser on and one with the laser off, each with a different set of conflict object cues. The order of the laser treatment, the assigned combination of the conflict object cues, and whether the aversive or appetitive object was presented first in the runway were all counterbalanced.The time spent in the neutral and goal boxes, the number of entries into the goal box, and LTE each of the boxes  in the runway were recorded during the tests. As an additional measure of AA behavior, the number of full-body entries into and retreats from the goal box were recorded. A full-body entry occurred when the animal’s hind limbs stepped on to the grid floor of the goal box. To be consistent with our previous work using the same Y-maze task , a retreTo ensure that PRC-inhibited could still discriminate stimuli that they were trained on during the conditioning session, a test with the neutral object pair was conducted on the same day as the two AA conflict tests, during which half of the rats  completed the test with the laser on, and half with the laser off. . Unlike the high approach-avoidance conflict test, the neutral object test was conducted in a between-subjects design since we deemed that a fully counterbalanced experimental design with 2 conflict trials and 2 neutral trials might lead to unreliable data during the last trial owing to extinction-related confounds. We therefore prioritized maximizing the reliability of the data collected from the 2 conflict trials. The animals were allocated into the Laser on/off groups randomly, after it was confirmed that baseline performance during the conflict trials was comparable across animals.To investigate whether reducing the level of object-associated motivational conflict might change PRC-mediated behavior, both PRC and vCA3 rats were given a ‘refresher’ conditioning session prior to administration of a ‘low conflict’ recombination test, during which either an appetitive or aversive object cue was paired with a neutral object cue. PRC and vCA3 rats were each given two trials per test, an appetitive-neutral object pairing, and an aversive-neutral pairing. Each rat was tested twice, once with the laser on throughout the entire test session, and once with the laser off. The order of laser treatment, the assigned combination of the object cues, and whether the appetitive/aversive or neutral object was presented first were counterbalanced within and between each test session.In a separate cohort of animals , all AA conflict tests (high and low) and neutral object test were administered exactly as described above, with the exception that the laser was turned on after the animals entered the neutral box, and prior to the door to the goal box being raised . The laser then remained on for the duration of the goal box exploration (3min).Finally, to control for the novelty of the recombined object pair presented during both the ‘high’ and ‘low’ conflict tests, along with an additional measure to confirm that PRC rats could still discriminate stimuli under optogenetic inhibition, all rats (except for PRC ArchT-expressing rats undergoing pre-choice inhibition) completed a ‘no conflict’ test, during which a novel recombination of objects of the same valence were presented. Rats were first trained to associate a novel set of eight objects with either appetitive  or aversive outcome across four daily conditioning sessions, followed by an acquisition test and a ‘refresher’ conditioning session prior to testing. On the day of testing, rats  were given two trials per test, an appetitive-appetitive object pairing, and an aversive-aversive pairing. Each rat was tested twice, once with the laser on throughout the entire test session, and once with the laser off.All rats were given 3 days of habituation the runway apparatus. On day 1, rats could freely explore both sides of the apparatus for 5 min, with the central guillotine door raised. On day 2, the rats were confined to one side of the apparatus with the guillotine door and following 2.5 min of exploration, the door was raised and animals were permitted to shuttle to the opposite side of the apparatus. Upon entry, the door was lowered and the animals were confined for a further 2.5 min. On day 3, the rats were confined to one side of the apparatus which now contained two sets of removable panels – one opaque and one transparent, preventing access to the sucrose dishes located at the end of the two compartments. After 10 s the opaque panel was removed, providing visual but not tactile access to the sucrose dispenser. After a further 30 s, the transparent panel was removed and the central guillotine door was simultaneously raised. The animals could then freely explore the empty sucrose dish, and were permitted to shuttle to the opposite side, which contained another set of opaque and transparent panels. Once animals shuttled to the opposite side, the door was lowered, and the habituation procedure was repeated in the other compartment.The animals were trained to associate the two object pairs with either appetitive (sucrose) or aversive (mild foot shock) outcomes over nine daily conditioning sessions. Each rat completed four trials per day, two for each valence. During a trial, rats were placed between the central guillotine door and the opaque panel, confining them to one side of the apparatus. Following 10 s the opaque panel was removed and the animals were permitted to visually sample the object pair for 1 min, which remained behind the transparent panel. After 1 min elapsed, the transparent barrier was removed, the central door was simultaneously raised, and the outcome delivered . Animals were given 1 min to shuttle to the opposite side of the apparatus before they were gently ushered in by the experimenter. Upon shuttling, the central door was lowered and the next trial began. The time spent visually sampling the objects, defined as the amount of time the rats directed their nose toward the object immediately behind the transparent panel, and escape latency data were collected. The order of presentation of the object cues and the order in which rats completed each trial were changed daily.Acquisition tests performed under extinction conditions were administered after conditioning days 4 and 8 to assess learning of the object cue-outcome associations. The experimental procedure was identical to that of cue conditioning training, except that the central guillotine door was not lowered upon the first shuttle response, and rats were permitted to re-enter the cued side of the apparatus and given 3 min to freely move between the cued and uncued sides. Similar to conditioning sessions, the uncued side contained an opaque panel. Successful acquisition was indicated by the rats exhibiting significantly shorter escape latencies for aversive trials than appetitive trials, and spending less time in the cued side during aversive trials than appetitive trials. Following the second acquisition test, rats were given a ‘refresher’ conditioning day prior to proceeding to the conflict test.On the day of AA conflict testing, rats were bilaterally tethered to the laser and rats were placed between the central guillotine door and the opaque panel, confining them to one side of the apparatus. The laser was then turned on, and remained illuminated for the entire duration of the test. The opaque panel was then removed after 10 s, exposing the animals to the object pairs behind the transparent panel. One appetitive object cue and one aversive object cue were presented in recombination as the ‘conflict object pair’, which animals could visually sample for 1 min. Once 1 min had elapsed, the transparent panel was removed and the central door was simultaneously raised. At this point, rats were given 3 min to freely move between the ‘conflict-cued’ side of the apparatus and the uncued side, after which the animal was gently ushered into and confined to the uncued side by the experimenter. The laser was then turned off. All rats completed two conflict test sessions under extinction conditions, one with the laser on and one with the laser off, each with a different set of conflict object cues. The escape latency and time spent in the ‘conflict-cued’ side of the apparatus were recorded, along with the number of re-entries into the cued side; a re-entry was defined when the animal’s hind limbs crossed the threshold of the central door of the cued side. The order of the laser treatment and the assigned combination of the conflict object cues were all counterbalanced.In a separate cohort of animals , the conflict test was administered exactly as described above, with the exception that the laser was turned on as the transparent door was removed after visual inspection of the object pair. The laser then remained on for the duration of the object exploration (3 min).Following the conflict test, animals were given a ‘refresher conditioning’ session, prior to completing two sets of conditioned cue acquisition tests, one with the laser on and one with the laser off. The procedure of the tests was identical to the conditioned cue acquisition test described above, which were conducted under extinction conditions. On the day of testing, the laser was turned on for animals completing the task under inactivation conditions before completing one appetitive and one aversive acquisition test. On the next day all rats were given another ‘refresher conditioning’ session before completing another set of acquisition tests the following day, with the laser turned off for the animals completing the task without inactivation conditions.PRC rats underwent four, 5 min habituation sessions as previously described . BrieflyPRC rats were trained to associate three sets of visuotactile cues with appetitive (sucrose), aversive (shock), or neutral (no event) outcomes over nine daily conditioning sessions. The rat was placed in the central hub for 30 s after which one guillotine door was raised permitting entry to an arm, followed by confinement in the arm for 2 min. During this time the outcome assigned to the cue was administered . After 2 min elapsed, the door was raised and rats were permitted to return to the central hub, and this process was repeated for the remaining two arms. The assignment of each cue to a given arm and the order in which each cue and arm were presented was changed daily. While the relative shape of the maze was held constant between sessions (Y-maze configuration), the maze was rotated either clockwise or counter-clockwise by 60° for each training session to prevent the use of spatial cues.Acquisition tests performed under extinction conditions were conducted after the fourth and eighth training sessions. The test procedure was identical to day 2 of habituation in that rats were given 5 min to explore all three arms simultaneously, but with cue inserts under extinction conditions. Successful acquisition of conditioned behavior was indicated by the rats spending more time in the appetitive arm than aversive and neutral arms, and rats spending less time in the aversive arm than the appetitive and neutral arms. Following the second acquisition test, rats were given a ‘refresher’ conditioning session prior to proceeding to the AA conflict test.The procedure for this test was identical to day 3 of habituation, with rats being permitted 3 min under extinction conditions to explore two arms presented simultaneously, one containing the neutral cue and one containing a superimposition of the appetitive and aversive cues. In addition to the time spent in each arm, the number of entries and retreats into both arms was also recorded. All rats completed two test sessions on the same day, one with the laser on for the entire 3 min test period, and one with the laser off. The order of laser treatment and the assignment of the ‘conflict’ and neutral cues to a given arm were counterbalanced within and between test sessions.2 , which exceeds the minimum amount needed to produce opsin activation (Five hundred and thirty-two nm (green) laser light was continuously applied for the length of time specified in each experiment. Laser illumination was delivered to an implanted optic fiber attached with plastic sleeves  via two steel-braided fiber-optic cables , which received light through a bifurcating rotary joint (Doric Lenses) secured with an FC connector. The rotary joint was connected to a class 3-B diode-pumped solid-state laser  via an FC connector. The light output of the optic fiber was adjusted to approximately 15 mW, and based on previous measurements  incorportivation .Prior to sacrifice, rats either completed a NOR task (PRC n=38) or an EPM test (vCA3 n=15) to endogenously increase c-Fos labeling in the respective brain areas. Previous work has demonstrated that c-Fos is consistently increased in the rat PRC following exposure to novel visual stimuli . SimilarThe NOR task was conducted in the open-field apparatus (50 cm [L] × 50 cm [W] × 50 cm [H]). Rats were first given two daily 5 min habituation sessions of the apparatus, without any object stimuli. On the day of testing, the bedding in the apparatus was changed, and rats were placed in the open-field facing away from two identical objects (A1 and A2), which they were permitted to ‘sample’ for 5 min. Rats were subsequently returned to their home-cage for a 15 min delay interval. During the test phase, rats were returned to the open field, which now contained a third identical copy of the sample object (A3) and a novel object (B1), which they were permitted to explore for 3 min. Between the sample and test phases, the apparatus was not cleaned, nor the bedding agitated, and no other rats were placed in the apparatus during this time. Half of the rats received laser treatment (n=19 [ArchT = 9]) during the sample and test phases, and half completed the task with the laser off.The EPM apparatus consisted of a central area (10 cm [L] × 10 cm [W]) with four maze arms (43.2 [L] × 10 [W] × 43.2 [H]) forming a plus shape. Two maze arms, directly across from one another, were enclosed by high walls (24.8 cm [H]), while the other two arms were unenclosed or ‘open’. Rats were placed into the central area facing the ‘open’ arm, and were given 10 min to freely explore the apparatus. Half of the rats (n=8 [ArchT = 5]) completed the task with the laser on, while the other half with the laser off.Ninety minutes after completing the test phase in the NOR task or the EPM test, rats were administered a terminal dose of sodium pentobarbital  and were intracardially perfused with phosphate-buffered saline (PBS), followed by 4% paraformaldehyde (PFA). Brains were removed and stored in 4% PFA for 24 hr before sectioning. Fifty µm coronal sections were obtained with a vibratome , mounted onto glass slides with a Flouroshield mounting medium containing DAPI . Optic fiber placement and viral injection site targeting were confirmed at ×10 and ×20 magnification under an Eclipse Ni-U epifluorescence microscope  using a FITC filter.2O2 in PBS for 30 min. Sections were then incubated in 0.5% TNB blocking buffer for 1 hr, followed by rabbit anti-cFos , and left overnight at 4°C on a shaker. The next day, sections were first incubated for 1 hr in donkey anti-rabbit , and then in diluted Rhodamine tyramide signal amplification (TSA) solution  and wrapped in foil to prevent photobleaching for 30 min. Sections were mounted onto glass slides and treated with a mounting medium . Labeling of c-Fos proteins in the PRC and vCA3 were confirmed at ×10 magnification under the Eclipse Ni-U epifluorescence microscope  using FITC and TexasRed filters, with cell counting performed by an automated counting software  and incubated in 1% Hre Fiji; . cFos quAll behavioral testing was recorded and tracked using Noldus EthoVision XT . Data were analyzed using SPSS statistical package version 26.0 . For the object-based AA task, conditioned cue acquisition test data for the time spent in the goal box, the LTE, number of entries and retreats, and object exploration latencies were analyzed by separate 3×2 repeated-measures ANOVA with a within-subjects factor of valence , and a between-subjects factor of group. Analyzed data for the high conflict test were identical to the acquisition test (including object exploration data) and were subject to a 2×2 repeated-measures ANOVA with within-subjects factors of laser treatment, and a between-subjects factor of group. Each rat only completed one session of the neutral test, with between-subjects factors of laser treatment and group, and data were analyzed by univariate ANOVA. Data for the low conflict and no conflict control tests were each first analyzed by separate 2×2×2 repeated-measures ANOVA with within-subjects factors of valence and laser treatment, and a between-subjects factor of group. Activity analysis by the sucrose dispenser and generation of heatmap figures were completed using Noldus EthoVision XT, in which an 18 cm (L) × 11.5 cm (W) region of interest was demarcated within the goal box , and could reliably contain an entire rat.For the Y-maze AA task, conditioned cue acquisition data for the time spent in each of the cued arms, as well as the number of entries and retreats were analyzed by a 3×2 repeated-measures ANOVA with a within-subjects factor of valence  and a between-subjects factor of group. Analyzed data for the conflict test were identical to the acquisition test and were subject to a 2×2×2 repeated-measures ANOVA with within-subjects factors of laser treatment and trial type , and a between-subjects factor of group. For the EPM test data, 2 x 2×2 repeated-measure ANOVAs were conducted to compare the time spent in the open and closed arms, as well as the number of entries made into both, with a within-subjects factor of arm type (open vs closed) and between-subjects factors of laser treatment and group.2 = For the NOR task, a 2×2 repeated-measures ANOVA was conducted on the ‘discrimination ratio’  were blindly scored by a second experimenter. None of the data collected were excluded in the final analysis. In this important study the authors combined innovative object-based conflict assays with optogenetic silencing to probe the role of the perirhinal cortex in motivational conflict. The evidence provided by the authors is convincing and provides new insight into how conflicting motivation is processed. This paper will interest neuroscientists studying the neuronal mechanisms underlying approach-avoidance decisions. public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.Our editorial process produces two outputs: (i) Decision letter after peer review:eLife. Your article has been reviewed by 3 peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Kate Wassum as the Senior Editor. The following individuals involved in the review of your submission have agreed to reveal their identity: Christian Bravo-Rivera (Reviewer #2); Lukas I Schmitt (Reviewer #3).Thank you for submitting your article "Rodent perirhinal cortex, but not ventral hippocampus, inhibition induces approach bias under object-based approach-avoidance conflict" for consideration by The reviewers have discussed their reviews with one another, and the Reviewing Editor has drafted this to help you prepare a revised submission.Essential revisions:Reviewer #1 (Recommendations for the authors):General1. Inhibition of the PRC altered conflict processing in the first and second tasks  but not in the third (Y-maze). How do we know that the failure to detect an effect of PRC inhibition in the third conflict task is due to the fact that it requires context/spatial processing as opposed to reduced efficacy of the repeated PRC inhibition?2. How did animals interact with the object pairs in conflict trials? That is, did they preferentially explore one or the other object, and did PRC inhibition alter this exploration in a manner that correlates with its impact on test performance? I.e., did it create a bias towards the exploration of the object that signaled the appetitive outcome? The authors indicate that the exploration data were recorded; their inclusion in the manuscript could help to clarify how inhibition of the PRC has the effects that it does in the conflict test.3a. L151. "When animals were exposed to a high conflict pairing, optogenetic inhibition of PRC (laser on) significantly increased time spent in the goal box compared with trials completed without inhibition (laser off) and compared to GFP control animals with laser on and off  = 29.03, p < 0.001, ηp2 = 0.65; all post hoc: p < 0.001) , indicative of an increase in approach behaviour under motivational conflict. Furthermore, PRC inhibition led to a significant decrease in the number of retreats from the goal box  = 16.80, p < 0.001, ηp2 = 0.51), although there was no effect on the number of goal box entries  = 1.92, p = 0.19, ηp2 = 0.11)  during the choice period." Does the fact that PRC inhibited rats stay longer in the goal box indicate an increase in approach behaviour? These rats enter the goal box just as readily as the controls (entry and latency data) but stay longer when neither outcome occurs; so perhaps they are less sensitive to the absence of sucrose than the controls? The fact that PRC inhibition also increased time in the goal box on trials that included aversive-neutral object pairs (~L169) suggests that it may generally reduce avoidance of an aversive outcome. Is this a fair description of the conflict results? That is, PRC inhibition may have resulted in an appetitive response bias by reducing sensitivity to (or decreasing avoidance of) an aversive outcome…3b. L490. "Using a set of original object-based AA paradigms, we found that optogenetic inhibition of the rodent PRC, but not the vCA3, resulted in both a robust increase in approach behaviour and decrease in avoidance behaviour during motivational conflict elicited by the representation of discrete object pairs associated with unmatching affective values." What is the evidence of an increase in appetitive behaviour and decrease in avoidance behaviour?Design1. How were the rats allocated to groups for the Neu-Neu test shown in Figure 2c? Why was this testing conducted between-subjects (resulting in ns of just 5 per group) rather than within-subjects, as in the previous high conflict test? Did the 5 rats in each ArchT group (laser on vs laser off) and each GFP group (laser on vs laser off) exhibit the same profile of responding in the previous high conflict test? An obvious concern is that the allocation of rats to groups biased against detection of differences among the ArchT groups.2a. Why are there so few rats in the "Low Conflict-GFP" conditions? That is, why weren't rats in the "High Conflict-GFP" condition carried forward to these additional tests? It renders analyses of the Low and NO conflict conditions problematic: as there are 8 rats in the ArchT group and 4 rats in the GFP group, how can these be submitted to any sort of common analysis via ANOVA?2b. How many rats were in the ArchT and GFP groups for the novel recombination tests in the runway experiment? The figure again suggests 8 and 4, which again renders the ANOVA problematic.2c. The query in relation to unequal ns and application of ANOVA also applies to the results of the shuttle box experiment shown in Figure 3f-i ; and to the combination of PRC and vHPC data that is reported to have been analysed together near line 379. Can this be legitimized in some way?3. L640, 652, 664. How were rats tethered to the lasering in the AA conflict test as "the entire apparatus [was] wrapped in red cellophane to minimize reliance on extra-maze cues"?Data/stats1. L551: "An intriguing question is why PRC inhibition potentiated approach, as opposed to avoidance, of an uncertain outcome." I understand that there is no effect of the laser on latency to enter any of the runway sections in the high and low conflict tasks. I assume that this is due to the fact that latency data is notoriously variable and the ns are relatively low. As such, I would be interested in an assessment of whether there is an effect of the laser on latency to enter any of the runway sections in an analysis that combines high and low conflict tasks that include an appetitive component…2. In task 2, are the escape latency and "time in cued side" data correlated? If so, they are potentially two measures of the same thing, which would warrant some correction in the α rate to control the chance of a Type 1 error. This may have consequences for claims based on these data .3. L765. "The number of entries into and 'retreats' from the outcome box were recorded; an entry was defined when the animal's hind limbs stepped onto the grid floor of the outcome box whereas 'retreats' were defined as partial entry into the outcome box followed by an immediate exit or backward treading into the neutral box." Why is there an asymmetry in the definition of entries and retreats? That is, the definition of entry is very clearly defined in terms of hind limbs making contact with the grid floor whereas the definition of retreat is more subjectively defined as "partial entry to the outcome box followed by an immediate exit or backward treading into the neutral box." What is a partial entry: i.e., what criteria were used to define a partial entry? Are 'retreats' capturing something that could be referred to as 'hesitancy'?Reviewer #2 (Recommendations for the authors):1) The authors should include histological reports of their implants for all optogenetic experiments.2) There is no stated justification for the exclusion of female subjects. The authors should qualify their results indicating that they characterized the male rodent brain; this should be reflected in the title and abstract.Reviewer #3 (Recommendations for the authors):1. A major concern is the possibility that the perirhinal cortex is important for the identification of the conflicting stimuli, rather than in processing the conflicting motivation per se. This confound arises because optogenetic suppression occurs during the exposure to the objects. To address this concern, then, the authors should include an additional control for their first experimental paradigm in which they start suppression only after the entrance to the Neutral box or Goal box. If the perirhinal cortex is responsible for mediating motivational conflict, this should be sufficient to produce the originally observed effect. Although some of the experiments partially address this issue, as the authors argue in the discussion, the controls included don't address the possibility that encoding and retrieval specifically for the more complicated conflict stimuli. Given the importance of this concept to the overall message of the paper, this possibility requires additional direct interrogation.2. Some of the results are confusing as presented and would benefit from additional analysis. In particular, the lack of difference in the number of entries between laser and non-laser for the first task along with a substantial difference in the number of retreats in Figure 2b is at least superficially confusing. I assume that this is across multiple trials  but this should be clarified in the text and the number of trials should be included in the figure legend. A latency to retreat metric similar to the latency to enter would also be useful to include.3. While optogenetic effects are observed for comparisons of the laser and non-laser trials across mice in the ArchT groups for the PRC suppression, it would be useful to assess this difference within mice, for instance by subtracting time-spent in goal box in laser and non-laser conditions. This would allow easier direct comparison between ArchT and GFP groups. Other metrics, such as the escape latency included in Figure 3f, could also be compared in this way. On a related note, the number of animals used appears to vary across experiments  but I do not see this noted in the figure legend or text. This should be clarified.4. The difference in the Laser ON and Laser OFF for the example of c-fos staining with ArchT suppression of the entorhinal cortex shown in Figure 7d is not very convincing, which is surprising given the quantification in the previous panel. This should be addressed. Essential revisions:Reviewer #1 (Recommendations for the authors):General1. Inhibition of the PRC altered conflict processing in the first and second tasks  but not in the third (Y-maze). How do we know that the failure to detect an effect of PRC inhibition in the third conflict task is due to the fact that it requires context/spatial processing as opposed to reduced efficacy of the repeated PRC inhibition?after the Y-maze task, and observed that the laser application led to behavioural effects consistent with PRC inhibition, namely reduced novel object preference. In addition, subsequent immunohistochemical analysis revealed a significant reduction in cFos labeling in ArchT animals only following laser stimulation during this test.The reviewer has a valid concern regarding the use of repeated laser application. However, we are confident that the observed failure to detect an effect of PRC inhibition in our Y maze task is a meaningful outcome, based on the fact that we conducted another behavioural test (Novel Object Recognition task) 2. How did animals interact with the object pairs in conflict trials? That is, did they preferentially explore one or the other object, and did PRC inhibition alter this exploration in a manner that correlates with its impact on test performance? I.e., did it create a bias towards the exploration of the object that signaled the appetitive outcome? The authors indicate that the exploration data were recorded; their inclusion in the manuscript could help to clarify how inhibition of the PRC has the effects that it does in the conflict test.We did indeed record object exploration data and found that there were no differences between App and Av object exploration during any of the conflict tests. The object exploration data have now been included in the revised version of the manuscript .3a. L151. "When animals were exposed to a high conflict pairing, optogenetic inhibition of PRC (laser on) significantly increased time spent in the goal box compared with trials completed without inhibition (laser off) and compared to GFP control animals with laser on and off  = 29.03, p < 0.001, ηp2 = 0.65; all post hoc: p < 0.001) , indicative of an increase in approach behaviour under motivational conflict. Furthermore, PRC inhibition led to a significant decrease in the number of retreats from the goal box  = 16.80, p < 0.001, ηp2 = 0.51), although there was no effect on the number of goal box entries  = 1.92, p = 0.19, ηp2 = 0.11)  during the choice period." Does the fact that PRC inhibited rats stay longer in the goal box indicate an increase in approach behaviour? These rats enter the goal box just as readily as the controls (entry and latency data) but stay longer when neither outcome occurs; so perhaps they are less sensitive to the absence of sucrose than the controls? The fact that PRC inhibition also increased time in the goal box on trials that included aversive-neutral object pairs (~L169) suggests that it may generally reduce avoidance of an aversive outcome. Is this a fair description of the conflict results? That is, PRC inhibition may have resulted in an appetitive response bias by reducing sensitivity to (or decreasing avoidance of) an aversive outcome…3b. L490. "Using a set of original object-based AA paradigms, we found that optogenetic inhibition of the rodent PRC, but not the vCA3, resulted in both a robust increase in approach behaviour and decrease in avoidance behaviour during motivational conflict elicited by the representation of discrete object pairs associated with unmatching affective values." What is the evidence of an increase in appetitive behaviour and decrease in avoidance behaviour?The reviewer’s interpretation of our runway PRC inhibition data – that the increased approach bias is a result of decreased avoidance of the goal box is indeed a fair assessment, and one that cannot easily be disambiguated from the alternate account that PRC inhibition induces increased approach behaviour on the basis of our runway data. We believe we have evidence for both: the fact that we observed PRC inhibition-induced increase in the time spent in our ‘Av-Neu low conflict’ condition, in addition to our PRC inhibited rats exhibiting a prolonged ‘escape latency’ when first exposed to conflict object pairs and greater time spent in the conflict cued side in the shuttle box task appear to suggest that the decreased avoidance account may hold true. On the other hand, we also have strong evidence that PRC inhibited animals spend a disproportionate amount of time by the sucrose dispenser, seeking out reward under extinction conditions, which speaks to the presence of a robust increase in approach behaviour. We have inserted additional text in the Discussion to touch on this issue .Design1. How were the rats allocated to groups for the Neu-Neu test shown in Figure 2c? Why was this testing conducted between-subjects (resulting in ns of just 5 per group) rather than within-subjects, as in the previous high conflict test? Did the 5 rats in each ArchT group (laser on vs laser off) and each GFP group (laser on vs laser off) exhibit the same profile of responding in the previous high conflict test? An obvious concern is that the allocation of rats to groups biased against detection of differences among the ArchT groups.The reviewer’s point is well taken. Laser application during the Neu-Neu test, although not ideal, was conducted in a between-subjects design since we deemed that a fully counterbalanced experimental design with 2 conflict trials and 2 neutral trials might lead to unreliable data during the last trial owing to extinction-related confounds. We therefore prioritized maximizing the reliability of the data collected from the 2 conflict trials. The animals were allocated into the Laser on/off groups randomly, after it was confirmed that baseline performance during the conflict trials was comparable across animals .2a. Why are there so few rats in the "Low Conflict-GFP" conditions? That is, why weren't rats in the "High Conflict-GFP" condition carried forward to these additional tests? It renders analyses of the Low and NO conflict conditions problematic: as there are 8 rats in the ArchT group and 4 rats in the GFP group, how can these be submitted to any sort of common analysis via ANOVA?2b. How many rats were in the ArchT and GFP groups for the novel recombination tests in the runway experiment? The figure again suggests 8 and 4, which again renders the ANOVA problematic.2c. The query in relation to unequal ns and application of ANOVA also applies to the results of the shuttle box experiment shown in Figure 3f-i ; and to the combination of PRC and vHPC data that is reported to have been analysed together near line 379. Can this be legitimized in some way?We agree that there were issues with unequal group sizes here and in response, we have collected data from an additional 4 animals to add to our GFP group. Importantly, the overall pattern of findings remains unchanged from that reported in our original manuscript.3. L640, 652, 664. How were rats tethered to the lasering in the AA conflict test as "the entire apparatus [was] wrapped in red cellophane to minimize reliance on extra-maze cues"?We apologise that this detail was unclear. The top of the maze was exposed and uncovered to allow for access to the laser machine .Data/stats1. L551: "An intriguing question is why PRC inhibition potentiated approach, as opposed to avoidance, of an uncertain outcome." I understand that there is no effect of the laser on latency to enter any of the runway sections in the high and low conflict tasks. I assume that this is due to the fact that latency data is notoriously variable and the ns are relatively low. As such, I would be interested in an assessment of whether there is an effect of the laser on latency to enter any of the runway sections in an analysis that combines high and low conflict tasks that include an appetitive component…As per the reviewer’s suggestion, we conducted a 4-way ANOVA (Construct (ArchT: GFP) x Laser (on: off) x Conflict (Low: High) x Box ) of the latencies to enter (LTE) all the compartments of the runway across high vs. low conflict trials with an appetitive object. We found that ’laser on’ decreased entry times at both conflict levels, which was driven predominantly by the PRC inhibition group. We also observed that rats exhibited longer latencies to enter all compartments during high conflict trials compared to low conflict trials, which is consistent with the idea that a conflict object pair induced increased ambulatory/hesitant behaviour. These analyses have been included in the revised manuscript , and we believe that they strengthen our account that PRC inhibition potentiates reward approach.2. In task 2, are the escape latency and "time in cued side" data correlated? If so, they are potentially two measures of the same thing, which would warrant some correction in the α rate to control the chance of a Type 1 error. This may have consequences for claims based on these data .Thank you for your comment regarding the correlation between escape latency and "time in cued side" data in task 2 and potential inflation of the α rate. “Escape latency” and “time in cued side” are indeed significantly correlated for both laser on (r=0.8) and laser off trials (r=0.62). However, while they may be measuring aspects of the same underlying construct, the measures are quite different, with the escape latency capturing the first ‘stay’ time in the cued side only, while the ‘time in cued side’ measure captures the time spent across multiple stays in the chamber .Moreover, we would like to clarify that escape latency and "time in cued side" were analysed separately and thus, we do not believe that there is a concern about inflating the α rate or Type 1 errors.3. L765. "The number of entries into and 'retreats' from the outcome box were recorded; an entry was defined when the animal's hind limbs stepped onto the grid floor of the outcome box whereas 'retreats' were defined as partial entry into the outcome box followed by an immediate exit or backward treading into the neutral box." Why is there an asymmetry in the definition of entries and retreats? That is, the definition of entry is very clearly defined in terms of hind limbs making contact with the grid floor whereas the definition of retreat is more subjectively defined as "partial entry to the outcome box followed by an immediate exit or backward treading into the neutral box." What is a partial entry: i.e., what criteria were used to define a partial entry? Are 'retreats' capturing something that could be referred to as 'hesitancy'?Thank you for raising this point. Our usage of the term ‘retreat’ is consistent with how it was originally defined in our previously published approach-avoidance conflict work using a similar entry/retreat paradigm  and can indeed be regarded as an index of hesitancy. The definitions of a ‘partial entry’ and ‘retreat’ have been clarified in the revised manuscript on page 39, para 2.Reviewer #2 (Recommendations for the authors):1) The authors should include histological reports of their implants for all optogenetic experiments.Thank you for this comment. This was amiss on our part, and we have now added a brief report at the end of the Results section  as follows:‘Finally, histological analysis confirmed robust bilateral expression of GFP/ArchT and optic fiber tip placement immediately dorsal to the viral injection site in all PRC animals. Thus, no exclusions were made based on optic fibre placement/viral expression. In the vCA3 group, data from three ArchT-expressing animals were excluded based on the viral expression and optic fibre placement presenting too medially to the CA3 subfield. ‘2) There is no stated justification for the exclusion of female subjects. The authors should qualify their results indicating that they characterized the male rodent brain; this should be reflected in the title and abstract.It is regrettable that we did not include female rats in the present study. The incorporation of females in our experiments has been a more recent commitment in our laboratory, and data collection for this study commenced 5 years ago. The title and abstract have been revised to reflect the use of an exclusively male rat population in the present study.Reviewer #3 (Recommendations for the authors):1. A major concern is the possibility that the perirhinal cortex is important for the identification of the conflicting stimuli, rather than in processing the conflicting motivation per se. This confound arises because optogenetic suppression occurs during the exposure to the objects. To address this concern, then, the authors should include an additional control for their first experimental paradigm in which they start suppression only after the entrance to the Neutral box or Goal box. If the perirhinal cortex is responsible for mediating motivational conflict, this should be sufficient to produce the originally observed effect. Although some of the experiments partially address this issue, as the authors argue in the discussion, the controls included don't address the possibility that encoding and retrieval specifically for the more complicated conflict stimuli. Given the importance of this concept to the overall message of the paper, this possibility requires additional direct interrogation.In order to address the reviewer’s concern, we have conducted a follow-up ‘control’ experiment in an additional cohort of animals , with all rats completing both ‘High’ and ‘Low’ conflict tests in the runway paradigm, along with a ‘High’ conflict test in the shuttle box paradigm. When optogenetic inhibition was applied immediately prior to the decision phase of the task , the behavioural data  did not differ from trials in which optogenetic inhibition was applied throughout the entire trial . Furthermore, PRC-inhibited animals (ArchTLaser ON) in this control experiment also exhibited an increase in ‘reward approach’ behaviour by spending more time by the sucrose dispenser during ‘High’ conflict trials, underlining our suggestion that PRC is responsible for mediating object-based motivation conflict.2. Some of the results are confusing as presented and would benefit from additional analysis. In particular, the lack of difference in the number of entries between laser and non-laser for the first task along with a substantial difference in the number of retreats in Figure 2b is at least superficially confusing. I assume that this is across multiple trials  but this should be clarified in the text and the number of trials should be included in the figure legend. A latency to retreat metric similar to the latency to enter would also be useful to include.We apologize that this was not clear in our original manuscript but given how we define the terms ‘entry’ and ‘retreat’, it is possible that the latter can occur independent of the former, and therefore, it is possible to have more retreats than entries. An entry occurred when the animal’s hind limbs stepped on to the grid floor of the goal box. To be consistent with our previous work using the same Y-maze task , a retreat occurred when the animal exhibited a half-body entry  or head-poke into the goal box followed by an immediate exit or backward treading into the neutral box .3. While optogenetic effects are observed for comparisons of the laser and non-laser trials across mice in the ArchT groups for the PRC suppression, it would be useful to assess this difference within mice, for instance by subtracting time-spent in goal box in laser and non-laser conditions. This would allow easier direct comparison between ArchT and GFP groups. Other metrics, such as the escape latency included in Figure 3f, could also be compared in this way. On a related note, the number of animals used appears to vary across experiments  but I do not see this noted in the figure legend or text. This should be clarified.We thank the reviewer for this suggestion. We have now included a difference score data analysis and presentation (Mean Data + SD from Laser on – Laser off) for a direct comparison of ArchT vs. GFP group data  , alongside our original data. We decided to leave the complete data set in our manuscript for transparency.We have also added more control GFP animals to the experiment and have clarified the Neu-Neu test design and methodology section, explaining the justification for a between-subjects test here  .4. The difference in the Laser ON and Laser OFF for the example of c-fos staining with ArchT suppression of the entorhinal cortex shown in Figure 7d is not very convincing, which is surprising given the quantification in the previous panel. This should be addressed.Higher magnification and resolution photomicrographs have now been included in the manuscript, providing a clearer illustration of the reduction in c-fos labelling following laser application in ArchTPRC tissue.


Doxycycline, an approved aquacultural antibiotic, is extensively used in the treatment of bacterial diseases in fish. Since fish are poikilothermic organisms, their body temperature and metabolic rate are primarily influenced by the temperature of the water. Therefore, temperature may be affected by pharmacokinetic behavior and withdrawal times of drugs. The current study was undertaken to look at the differences in pharmacokinetics, tissue residues, and withdrawal times of doxycycline following oral administration in rainbow trout reared at 10 and 17 °C. The increment of water temperature from 10 to 17 °C decreased the elimination half-life, the body clearance, and the distribution volume of doxycycline and increased plasma concentrations. The withdrawal times for plasma and tissues decreased with the temperature increase. The results contributed to the determination of an optimal dosing regimen and the safe consumption of edible tissues in rainbow trout that were administered doxycycline and reared at different temperatures.The purpose of this study was to compare the pharmacokinetics, tissue residues, and withdrawal times of doxycycline after oral administration in rainbow trout reared at 10 and 17 °C. Fish received a 20 mg/kg oral dose of doxycycline after a single or 5-day administration. Six rainbow trout were used at each sampling time point for plasma and tissue samples, including liver, kidney, and muscle and skin. The doxycycline concentration in the samples was determined using high-performance liquid chromatography with ultraviolet detector. The pharmacokinetic data were evaluated by non-compartmental kinetic analysis. The WT 1.4 software program was used to estimate the withdrawal times. The increase of temperature from 10 to 17 °C shortened the elimination half-life from 41.72 to 28.87 h, increased the area under the concentration–time curve from 173.23 to 240.96 h * μg/mL, and increased the peak plasma concentration from 3.48 to 5.50 μg/mL. At 10 and 17 °C, the doxycycline concentration was obtained in liver > kidney > plasma > muscle and skin. According to the MRL values stated for muscle and skin in Europe and China (100 μg/kg) and in Japan (50 μg/kg), the withdrawal times of doxycycline at 10 and 17 °C were 35 and 31 days, respectively, for Europe and China and 43 and 35 days, respectively, for Japan. Since temperature significantly affected pharmacokinetic behavior and withdrawal times of doxycycline in rainbow trout, temperature-dependent dosing regimens and withdrawal times of doxycycline might be necessary. Fish is important for human health because it contains high levels of protein, unsaturated fatty acids, and essential nutrients and is easier to digest than other meats . Fish coAeromonas hydrophila, Yersinia ruckeri, Edwardsiella tarda, Lactococcus garvieae, Vibrio spp., and Streptococcus spp. cause many diseases that cause economic losses and deaths in rainbow trout such as enteric redmouth disease, hemorrhagic septicemia, meningoencephalitis, and exophthalmos . Both the ≤%(−) 25 and ≥% (+) 25 results were considered to be statistically significant .max and Tmax of doxycycline were 3.48 and 5.50 μg/mL and 4 and 8 h, respectively. The increase in water temperature from 10 °C to 17 °C decreased t1/2ʎz, Tmax, CL/F, and Vdarea/F and increased Cmax and AUC. The percentage AUCextrap for both temperatures was less than 20%.The mean plasma concentrations of doxycycline at different water temperatures are shown in The plasma and tissue concentrations of doxycycline in rainbow trout following daily oral administrations at 20 mg/kg for 5 days at 10 and 17 °C are presented in The WT of doxycycline in plasma, liver, kidney, and muscle and skin was estimated using the WT 1.4 software program. WT values estimated according to MRL values are presented in For the WT calculation, a tolerance limit based on the 95th percentile with a 95% level of confidence was used. The calculated withdrawal time is provided in parenthesis; it was rounded up to the next day.The temperature-dependent plasma pharmacokinetics, tissue residues, and withdrawal times of doxycycline in rainbow trout were examined for the first time in this study. In countries such as China, Japan, India, and the Philippines, it is recommended to use doxycycline at a dose of 20 mg/kg for 3–5 days in fish . No loca1/2ʎz, CL/F, and Vdarea/F were 28.87–41.72 h, 0.08–0.11 L/h/kg, and 3.42–6.82 L/kg, respectively. The t1/2ʎz, CL/F, and Vdarea/F of doxycycline after oral administration at different temperatures (18–28 °C) and different fish species  were reported as 5.81–77.2 h, 0.03–0.72 L/h/kg, and 0.87–5.81 L/kg, respectively [After oral administration of doxycycline to rainbow trout at 10 and 17 °C, tectively ,31,34. T1/2ʎz of oral doxycycline to rainbow trout were 41.72 and 28.87 h, respectively. The increase in water temperature in trout significantly decreased the t1/2ʎz of doxycycline. The increase in water temperature did not affect plasma t1/2ʎz of doxycycline in grass carp, but shortened it in tissues such as liver, kidney, and muscle and skin in grass carp [1/2ʎz with temperature may be due to the increase in metabolic rates and cardiac outputs and the acceleration of blood circulation.At 10 and 17 °C, tass carp  and catfass carp . Fish arass carp ,36,37. Iass carp . In thismax was estimated at 3.48 and 5.50 μg/mL at 10 and 17 °C, respectively, with a corresponding Tmax of 8 and 4 h, respectively. The Cmax and AUC values at 17 °C were higher than those at 10 °C. In grass carp, the increase from 18 °C to 24 °C in water temperature caused the high Cmax (from 17.01 to 89.68 μg/mL) and AUC0–last (from 308.66 to 644.78 h * μg/mL) [max, Tmax, and AUC values of marbofloxacin in crucian carp and enrofloxacin in turbot with temperature was similar to our study [max and AUC of doxycycline with an increase in temperature in rainbow trout may be due to increased absorption due to the above-mentioned reasons.After a single oral dose of doxycycline at 20 mg/kg, the C* μg/mL) . In addiur study ,40. Wateur study ,41. The darea/F  values of doxycycline. Due to the increase in metabolic rates, cardiac outputs, and tissue blood supply with the increase in temperature, the values of CL/F and Vdarea/F were expected to increase; however, their decrease was surprising. Similar results were reported in the doxycycline study in grass carp [0–∞ was used for the calculation of CL/F. Although the dose was the same at both temperatures, the AUC0–∞ value was 176.55 and 243.76 h*μg/mL at 10 and 17 °C, respectively. In this study, the increase in AUC0–∞ due to changes in metabolism or absorption with the effect of temperature may be the reason why the CL/F changed. However, to reveal the reason for the decrease in CL with an increase in temperature, it was necessary to determine the CL of doxycycline following IV administration in which the effect of absorption was not observed at temperatures of 10 and 17 °C. The calculation of Vdarea/F was based on the equation Vdarea/F = CL/F/λz. The λz values for 10 and 17 °C were 0.017 and 0.024, respectively. In this study, the decrease in Vdarea/F with temperature may have been due to differences in CL/F and λz.In this study, increasing the water temperature from 10 to 17 °C significantly decreased the CL/F  and Vass carp . Althougass carp , most stass carp ,42. Howemax obtained in plasma and all tissues at 17 °C was higher. The higher Cmax at 17 °C may have been related to increased absorption due to the high temperature. Doxycycline concentrations in the liver and kidney were much greater than in the plasma. Similar findings have been observed in the research on catfish and grass carp [In rainbow trout, after daily oral administration of 20 mg/kg for 5 days at 10 and 17 °C, the highest concentration for plasma, liver, kidney, and muscle and skin was reached at 0.5 days. At 10 °C, the highest concentrations for plasma, liver, kidney, and muscle and skin were 5.93 ± 0.82, 34.98 ± 2.82, 23.21 ± 2.22, and 5.31 ± 0.64 μg/mL, respectively. Doxycycline has a high lipophilicity and permeability that can result in high concentrations in various tissues after oral administration . Compareass carp . Doxycycass carp . In someass carp . The rea1/2ʎz of doxycycline determined at 10 and 17 °C. After oral administration of 20 mg/kg doxycycline for 3 days in grass carp at 24 °C, the WT (100 μg/kg for Europe and China) values for plasma, liver, kidney, and muscle and skin were reported as 25, 56, 42, and 41 days, respectively [In this research, the WT 1.4 software program was used to determine WT values in plasma and tissues. In this study, muscle and skin was preferred because they are the main edible tissues; liver and kidney are the main organs of metabolism and elimination . Many co1/2ʎz, Tmax, CL/F, and Vdarea/F of doxycycline decreased and plasma concentrations increased. In this study, since several important pharmacokinetic parameters changed with temperature, a temperature-dependent dosing regimen of doxycycline might be necessary. To ensure that edible tissues (muscle and skin) are safe for consumption, a WT of doxycycline at 10 and 17 °C after oral administration at 20 mg/kg dose for 5 days in rainbow trout should not be less than 35 and 31 days, respectively, for Europe and China and 41 and 35 days, respectively, for Japan.It can be concluded that temperature has profound effects on the pharmacokinetic behavior of doxycycline. Following the increment of water temperature from 10 to 17 °C, t


To investigate the link between hematopoietic and skeletal tissues in patients with fragility fractures.We retrospectively analyzed the medical records of women older than 40 years who attended the Bone Disease Unit of “Sapienza” University of Rome for their first visit for osteoporosis from January 2020 to June 2022.p = 0.01), and femoral neck BMD (p = 0.007). A red blood cell distribution width (RDW) value higher than 15% was observed four times more in those with fractures compared to non-fractured patients  and was associated with vertebral fracture after adjusting for age, BMI, menopause, nutritional status, smoking, osteoporosis and anemia . Hematocrit was negatively associated with hip fracture also adjusting for age, BMI, menopause, nutritional status, smoking, osteoporosis (p = 0.025).Fragility fractures were found in 61.8% of the sample. In particular, vertebral fractures in 35.5%, femoral fractures in 6.3%, Colles fractures in 16.5% and non-vertebral non-hip in 42.5%. Fractured patients were significantly older compared to non-fractured, had lower mean values of lumbar spine (Our study demonstrates that RDW values were significantly associated with vertebral fracture and hematocrit with hip fracture. Since both parameters are included in the initial evaluation of patients with suspected bone fragility, our results should push doctors to look at these values with no incremental cost for national health services. Complete blood cell count is an integral part of the initial biochemical evaluation of osteoporotic patients. Recently, several investigators examined the possible association of red, white cells and platelets numbers with osteoporosis even though controversial results have been reported –15. In pAs a part of an automated complete blood count, red blood cell distribution width (RDW) represents the variation in size of the red blood cells, which is commonly used in the differential diagnosis of anemia. Recently, RDW is being increasingly recognized as a global marker of chronic inflammation and oxidative stress; elevated values in men have been associated with 2.8 times higher risk of hip fractures than men in the lowest group .C-reactive protein levels  years old, 3.7% of them being premenopausal. Mean age at menopause was 48.6 ± 4.9 years, BMI was in the normal range with a median of 23.2 kg/m2) compared to both osteopenic (24.7 ± 4 kg/m2) and normal subjects (26.3 ± 4 kg/m2), p < 0.001. The osteoporotic patients were more frequently smokers (23.1%), compared to both osteopenic (11.3%) and normal subjects . Mean values of other biochemical parameters were not significantly different in the three groups  of the sample was diagnosed as having osteopenia, 63% (95% CI 58.2–67.6%) osteoporosis and 3.7% (95% CI 2.1–5.8%) had T-score values in the normal range. There were no differences between these three groups concerning anthropometric parameters, except for a mean lower BMI in osteoporotic patients (23 ± 3.7 kg/mFragility fractures were found in 61.8% (95% CI 56.9–66.4%) of the sample and, in particular, vertebral fractures in 35.5% (95% CI 31.0–40.3%), femoral fractures in 6.3% (95% CI 4.3–9.1%), Colles fractures in 16.5% (95% CI 13.2–20.5%) and non-vertebral non-hip fractures  in 42.5% (95% CI 37.8–47.4%).p < 0.001); they had significantly mean lower lumbar spine BMD (p = 0.01), and femoral neck BMD (p = 0.007) compared with those without fractures, as shown in Table Patients with fractures were significantly older 70.35 ± 9.91) compared to those without (65.06 ± 10.76 5 ± 9.91 p = 0.01). Considering the diabetic population only 3 subjects had a RDW value higher than 15%, which is not a statistically significant difference when compared to the number of those with RDW > 15% (n = 22) in the population without diabetes (p > 0.05).Concerning biochemical parameters, the only significant difference was a higher number of patients with RDW > 15% in fractured patients compared to non-fractured ones, as shown in Table p = 0.001). This level of association remained stable when potential confounders were taken into account as shown in Table p = 0.003),  was associated with vertebral fractures , also after adjustment for osteoporosis ; however, it was not significantly associated after adjustment for age, menopause, BMI and nutritional status. A low hematocrit was associated with hip fractures , also after adjustment for age, BMI, menopause, nutritional status, smoking, osteoporosis (Table p = 0.69). Among the diabetic population, a small number of patients were taking SGLTi therapy, without any difference in the number treated in the diabetic fractured group vs the diabetic non-fractured group .Considering other blood parameters, monocyte-to-lymphocyte ratio (MLR) was associated with vertebral fractures (Beta = 2.4, 95% CI 0.24–4.7, We found no association between platelets number and fragility fractures considered as a whole.A number of “in vitro” and “in vivo” studies have consistently shown a two-way influence between skeletal cells and hematopoietic system . RDW, a We showed an association between higher RDW values and vertebral fractures after adjusting for multiple potential confounding variables including smoking which has never been considered in this setting. It is important to consider cigarette smoking, because this life style habit is recognized as an independent risk factor for the development of osteoporosis, and it is linked to an enhanced oxidative stress . Our resOne of the possible hypotheses linking RDW to fractures may be chronic inflammation. In a recent study, RDW independently predicted in-hospital mortality, 90-day mortality, and hospital and ICU length of stay in fractured patients admitted to ICU, highlighting the link between RDW and inflammation leading to a worst outcome . Indeed,We also firstly detected an association between MLR and vertebral fracture. Peripheral blood monocytes can further differentiate into many kinds of cells such as macrophages, dendritic cells and osteoclast cells. MLR could be considered another marker of inflammation linked to vertebral fracture. However, after adjustment for age, BMI and menopause, this association became no longer significant.Our data showed that hematocrit is negatively associated with hip fracture. Although, the exact mechanism by which hematocrit is associated with fracture is still unclear, animal models have shown that osteoblast ablation leads to deficiency in the progenitors of multiple hematopoietic cell lines . InteresOf note, we only included low-energy trauma fractures, while a large population-based cohort in the South Korea, which showed an association of fracture with anemia included all types of fractures, including high energy trauma . We did Our study has some limits. First, this was a single center study, not including men. Second, in our sample, we did not rule out subclinical hypothyroidism, which might influence RDW value. Indeed, RDW has been recently considered a marker of subclinical hypothyroidism . Third, In conclusion, our study demonstrated that RDW values was significantly associated with vertebral fracture and hematocrit with hip fracture. Since both parameters are included in the initial evaluation of patients with suspected bone fragility, our results should push doctors to look at these values with no incremental cost for national health services.