In the nervous system, synapses are special and pervasive structures between axonal and dendritic terminals, which facilitate electrical and chemical communications among neurons. Extensive studies have been conducted in mice and rats to explore the RNA pool at synapses and investigate RNA transport, local protein synthesis, and synaptic plasticity. However, owing to the experimental difficulties of studying human synaptic transcriptomes, the full pool of human synaptic RNAs remains largely unclear. We developed a new machine learning method, called PredSynRNA, to predict the synaptic localization of human RNAs. Training instances of dendritically localized RNAs were compiled from previous rodent studies, overcoming the shortage of empirical instances of human synaptic RNAs. Using RNA sequence and gene expression data as features, various models with different learning algorithms were constructed and evaluated. Strikingly, the models using the developmental brain gene expression features achieved superior performance for predicting synaptically localized RNAs. We examined the relevant expression features learned by PredSynRNA and used an independent test dataset to further validate the model performance. PredSynRNA models were then applied to the prediction and prioritization of candidate RNAs localized to human synapses, providing valuable targets for experimental investigations into neuronal mechanisms and brain disorders. How people memorize, learn, and process external information largely depends on the sophisticated connections between neurons . Unlike With highly polarized morphology, neurons offer a great model for studying RNA localization . Subcell2+/calmodulin-dependent protein kinase II (CaMKIIa) targets the mRNA to dendrites for local translation [CaMKIIa mRNA altered its distribution in dendrites, resulting in reduced accumulation of CaMKIIa in postsynaptic densities (PSD) and impairments of synaptic plasticity and spatial memory [Aplysia [Previous studies suggest that neuronal mRNAs may carry regulatory elements that affect mRNA localization, stability, and translation ,20, whernslation . The losl memory . The 5′ [Aplysia . These fSince many neuronal proteins are involved in synaptic plasticity and higher-order brain functions such as learning and memory , synaptiWith the growing size and complexity of genomic data, machine learning techniques have been increasingly used to extract hidden knowledge regarding a specific biological problem. One intriguing problem is RNA subcellular localization, which plays an important role in modulating protein distributions and cellular functions of various classes of RNAs transcribed from the genome . To dateIn this study, we developed a new machine learning method named PredSynRNA to predict human synaptically localized RNAs. We compiled a training dataset from previous studies and used RNA sequence and developmental brain gene expression data as features to construct various models with different learning algorithms. Interestingly, the Support Vector Machine (SVM) model using the expression features achieved the best performance. PredSynRNA was then employed to predict and prioritize candidate RNAs, including 1070 mRNAs and 330 lncRNAs, which might be localized to human synapses.Considering the lack of training instances of human dendritic and somatic RNAs, we first collected those from respective lists published in five rodent studies that utilized RNA-sequencing techniques ,18,19,20k-mer frequencies of concatenated 5′ and 3′ UTR of an mRNA transcript . Protein-coding transcript sequences were downloaded from the GENCODE GRCh38 release 33 [k-mer combinations  were examined for model construction  transformation. The expression and sequence features were also normalized using the min–max method.The gene expression features for each RNA instance were extracted from the BrainSpan Atlas of the Developing Human Brain . The BraThe high dimensionality of sequence and expression features might lead to model overfitting. Feature selection could be utilized to remove redundant and irrelevant features . It was Various machine learning algorithms, including logistic regression (LR), support vector machine (SVM), random forest (RF), XGBoost (XGB), and artificial neural network (ANN), were tested for model construction. LR is a statistical method that finds the best fitting model to describe the relationship between the logit of outcome and a set of independent variables. SVM is a learning algorithm that aims to distinguish two classes by a hyperplane with the maximal margin . RF is aDuring model construction, PredSynRNA performance was evaluated by five repetitions of 10-fold cross-validations, in which the training dataset was randomly divided into 10 equal-sized subsets: one holdout subset for testing and the remaining nine subsets for training . For theFurthermore, an independent test dataset was collected from a previous study on the somato-dendritic localization of mRNAs in mouse hippocampus  and usedThe performance metrics used in this study are as follows:True positives (TP), true negatives (TN), false positives (FP), and false negatives (FN) are tabulated and used to calculate the performance metrics shown above. The Matthews correlation coefficient (MCC) measures the correlation between the predicted and actual classifications on a scale of 0 ≤|MCC| ≤ 1 . The recAfter model validation, PredSynRNA was applied to the prediction of synaptically localized candidate RNAs from a list of brain-expressed RNAs, including 7046 mRNAs and 3331 lncRNAs. The top three PredSynRNA models with the best performance in cross-validations and on the independent test dataset were used to predict the probability of a given RNA transcript being synaptically localized, with the default probability threshold of 0.5. The positive predictions shared by all the three models were referred to as the high-confidence list of candidate RNAs.To understand the biological processes or cellular functions in which the high-confidence candidates might be involved, we performed functional annotation clustering analysis using DAVID Bioinformatics Resources 6.8 with the list of brain-expressed genes as the background . High clThe high-confidence list of candidate RNAs was also compared with the SynGO gene list, which included 1112 synaptic genes based on gene ontology (GO) annotations and published, expert-curated evidence . GSEAPrek-mer frequencies of RNA transcript sequences and the developmental brain gene expression profiles from the BrainSpan Atlas of the Developing Human Brain [The machine learning task in this study can be defined as a binary classification problem, and our method, PredSynRNA, is illustrated in an Brain  were usek-mer frequencies. We first constructed and evaluated various machine learning models using sequence features in terms of Next, we built different machine learning models using developmental brain gene expression features. Based on the ROC and PR curves from 10-fold cross-validations, the expression-based models clearly outperformed the sequence-based models . ParticuFeature selection was performed in this study to potentially improve model performance and to identify the most relevant features for predicting synaptically localized RNAs. We first computed the importance score of each feature using the RF-based method and then utilized the top-ranked expression or sequence features to build various machine learning models. However, when compared with using the full feature sets, feature selection did not significantly improve the performance of the expression or sequence-based models . For theWe examined the expression features, which included a series of developmental time points and brain tissue types of the samples in the BrainSpan dataset. As shown in To further evaluate the predictive performance of the models, we compiled an independent test dataset with 613 positive instances and 925 negative instances, which were not included in the training dataset. Notably, almost all the tested models achieved comparable performance on the independent test dataset as in cross-validations . The perTo identify synaptically localized candidate RNAs, we applied the SVM, ANN, and RF models trained with the full expression features to classify a list of 10,377 brain-expressed RNAs, including 7046 mRNAs and 3331 lncRNAs. Overall, 2747, 1348, and 2777 mRNAs were predicted to be synaptically localized mRNAs by the SVM, ANN, and RF models, respectively . ParticuTo characterize the high-confidence candidates, we performed DAVID functional annotation clustering analysis . As showp-value is estimated to be zero . A list of 82 SynGO synaptic genes showing core enrichment is provided in To further examine the functional association with synapses, we compared the candidate RNAs with a set of 1112 human synaptic genes curated by the SynGO database . The lisRNA localization to synapses is not only regarded as one of the driving forces for developmental changes in the brain but is also implicated in neurological diseases. While machine learning methods have been developed for predicting RNA localization to multiple cellular compartments ,33,34,35k-mer frequencies did not further improve model performance. This is rather surprising as many previous studies attempted to identify potential localization elements present in the untranslated regions, mostly the 3′ UTRs of mRNA transcripts in neurites. Since these elements can be heterogeneous to a great extent in size and structure, it may be hard to predict and deduce the consensus sequence or structural motifs [k-mer frequencies may not be able to delineate the complex patterns of RNA localization to synapses.Despite the limited and noisy training data, PredSynRNA models using the developmental brain gene expression features achieved relatively high performance for predicting synaptically localized RNAs. However, the addition of RNA sequence features in terms of l motifs . Moreovel motifs ,20,66,67Nevertheless, the results do not necessarily mean that RNA transcript sequences do not contain relevant information for predicting synaptically localized RNAs. In future studies, state-of-the-art deep learning techniques may be utilized to uncover the sequence patterns that determine RNA localization to synapses. It is noteworthy that deep learning techniques have been used to identify sequence motifs for mRNA subcellular localization to the nucleus, cytosol, endoplasmic reticulum, and exosome ,32. RNATRPL8, MZT2B, RPS20, TMEM219, and HBB  associated with several brain disorders, including schizophrenia, seizure, and Alzheimer’s disease [SNHG8 and MALAT1 (Since the robust performance of PredSynRNA was demonstrated by cross-validations and using an independent test dataset, the models were then utilized to predict and prioritize candidate RNAs, mostly mRNAs, which might be localized to human synapses. Interestingly, the top five candidate mRNAs include  and HBB . RPL8 ha disease . RPS20 hchildren . The TME disease ,72,73. H disease ,75,76,77d MALAT1 . As the In this study, we developed a new machine learning method, PredSynRNA, to predict the synaptic localization of human RNAs. The PredSynRNA model utilized developmental brain gene expression data as features to achieve relatively high performance during cross-validations and on an independent test dataset. Our results also suggest that the model is capable of capturing relevant expression features and can be used to predict and prioritize candidate RNAs localized to human synapses. In the future, PredSynRNA model performance may be further improved by compiling and curating a more comprehensive and high-quality training dataset for this difficult machine learning task.


Adolescence is an important developmental period associated with increased risk for excessive alcohol use, but also high rates of recovery from alcohol use-related problems, suggesting potential resilience to long-term effects compared to adults. The aim of this systematic review is to evaluate the current evidence for a moderating role of age on the impact of chronic alcohol exposure on the brain and cognition. We searched Medline, PsycInfo, and Cochrane Library databases up to February 3, 2021. All human and animal studies that directly tested whether the relationship between chronic alcohol exposure and neurocognitive outcomes differs between adolescents and adults were included. Study characteristics and results of age-related analyses were extracted into reference tables and results were separately narratively synthesized for each cognitive and brain-related outcome. The evidence strength for age-related differences varies across outcomes. Human evidence is largely missing, but animal research provides limited but consistent evidence of heightened adolescent sensitivity to chronic alcohol’s effects on several outcomes, including conditioned aversion, dopaminergic transmission in reward-related regions, neurodegeneration, and neurogenesis. At the same time, there is limited evidence for adolescent resilience to chronic alcohol-induced impairments in the domain of cognitive flexibility, warranting future studies investigating the potential mechanisms underlying adolescent risk and resilience to the effects of alcohol. The available evidence from mostly animal studies indicates adolescents are both more vulnerable and potentially more resilient to chronic alcohol effects on specific brain and cognitive outcomes. More human research directly comparing adolescents and adults is needed despite the methodological constraints. Parallel translational animal models can aid in the causal interpretation of observed effects. To improve their translational value, future animal studies should aim to use voluntary self-administration paradigms and incorporate individual differences and environmental context to better model human drinking behavior. Alcohol use disorder (AUD) is the most prevalent substance use disorder worldwide . Most AUIn humans, the salience and reinforcement learning network as well as the central executive network are involved in the development and maintenance of AUD , 14. TheWhile most of our knowledge on the effects of alcohol on the brain and cognitive outcomes is based on research in adults, several recent reviews have examined the effects of alcohol on the brain and cognition in adolescents and young adults specifically –25. Heavbetween adolescents and adults? To address this question, we systematically reviewed human and animal studies that included both age groups and used a factorial design that would allow for the comparison of the effects of chronic alcohol use on cognitive and brain-related outcomes across age groups. We specifically highlight outcomes from voluntary self-administration paradigms when available and discuss the translational quality of the animal evidence base. We conclude with a discussion of prominent knowledge gaps, future research directions, and clinical implications.Although previous reviews underscore the potential negative consequences of heavy alcohol use on the brain and cognition in adolescence, they do not typically address the question of whether adolescents are differentially vulnerable compared to adults to the effects of alcohol on these outcomes. Explicit comparisons between adolescents and adults are crucial to identify potential risk and resilience factors. In the current review, we aimed to extend previous work by systematically examining this critical question: does the relationship between chronic alcohol use and neurocognitive outcomes differ We followed the PRISMA guidelines for the current systematic review . An initial MedLine, Cochrane Library, and PsycInfo search was conducted during September of 2018 with terms related to alcohol, cognition, adolescence/adulthood, and study type (see Appendix for full search strategy and syntax). Two search updates using the same search strategy were conducted on 31 March 2020 and 3 February 2021. For all searches, the identified citations were split into batches and at least two of the following assessors  conducted a blinded review to determine whether articles met the inclusion criteria. In the first phase of screening, only titles and abstracts were screened and articles that clearly did not meet the inclusion criteria were excluded. In the second phase, the remaining articles received a full-text review and those that did not meet all inclusion criteria were excluded. The first inclusion criterion that was not adhered to was recorded as the reason for excluding. If there was a discrepancy between authors after initial and full-text screening process, the reviewing authors discussed the article and a consensus was reached.during adolescence or adulthood, not retrospectively ; (6) Primary quantitative data collection ; (7) Solely looking at alcohol-related factors as the independent variables ; (8) Written in English; (9) Published in a peer-reviewed journal before February 3, 2021  Human samples including both adolescents younger than 18 and adults older than 18 and animal samples including adolescent  25–42 for rodents) and adult [The definitions for adolescence are variable, hampering the direct comparison of human and rodent research. In rodents, the end of early-mid adolescence is considered to be approximately PND 42 when rats reach sexual puberty. By contrast, the boundaries for the onset of early adolescence are less clear. Based on the notion that most age-typical physiological changes that are characteristic of adolescence emerge from PND 28 , the conThe exact boundaries of human adolescence are similarly nebulous. From a neurodevelopmental perspective, adolescence is now often thought of as continuing until approximately age 25 because of the continuing maturation of the brain . HoweverNotably, we excluded studies that assessed alcohol exposure retrospectively  because age of onset variables are often inaccurate, with reported age of alcohol onset increasing with both historical age  and currIn humans, we defined cognition as any construct that typically falls within the umbrella of neuropsychological testing, as well as brain-based studies. We also included more distal constructs of cognition, like craving and impulsivity, because they play a prominent role in addictive behaviors , 36. In Our searches identified 7229 studies once duplicates were removed. A total of 6791 studies were excluded after initial review of abstracts. Then, 434 studies received a full-text review and 371 were excluded for failing to meet all inclusion criteria. See Fig. Conditioned alcohol aversion or preference: Lower sensitivity to alcohol’s aversive effects  but higher sensitivity to alcohol’s rewarding effects has been hypothesized to underlie the higher levels of alcohol use, especially binge-like behavior, in adolescents compared to adults [o adults . SeveralThe conditioned taste aversion (CTA) paradigm is widely used to measure perceived aversiveness of alcohol in animals. Repeated high-dose ethanol injections are paired with a conditioned stimulus . The reduction in CS consumption after conditioning is used as an index of alcohol aversion. Two studies examined CTA in mice , 49 and Overall, three studies found support for lower sensitivity to alcohol’s aversive effects in adolescents, whereas one observed no differences. Future research should employ intragastric as opposed intraperitoneal exposure to better mimic human binge-like drinking in order to increase the translational value of the findings.To measure differences in alcohol’s motivational value, conditioned place preference (CPP) paradigms have been used. This involves repeated pairings of ethanol injections with one compartment and saline injections with another compartment of the testing apparatus. On test days, CPP is assessed by measuring how long the animal stays in the compartment paired with ethanol relative to saline injections. Four studies examined CPP, with two studies observing age-related differences –55. In tOverall, there is inconsistent evidence for age-related differences in the motivational value of ethanol. One study found support for increased sensitivity to the rewarding effects of ethanol in adolescents, whereas one found support for adults being more sensitive and two observed no differences.Fear conditioning and retention: Pavlovian fear conditioning paradigms are used to investigate associative learning and memory in animals. These paradigms are relevant for addiction because fear and drug-seeking behavior are considered conditioned responses with overlapping neural mechanisms [chanisms . Rodentschanisms –59, and chanisms . Five stchanisms –64 and ochanisms .Only one of the four studies observed age-related differences in tone fear conditioning. Bergstrom et al.  found evTwo of the three studies observed age-related differences in contextual fear conditioning –64. In tOnly one study examined fear extinction, and found no effect of ethanol exposure  on extinction after tone conditioning. However, adults had higher levels of contextual fear extinction compared to mid-adolescents while late adolescents performed similar to adults . MoreoveIn sum, there is limited but fairly consistent evidence for adolescent-specific impairments in hippocampal-linked contextual fear conditioning across two rat studies, while no age differences emerged in context-based fear retention in one study of mice. In contrast, only one of the four studies found evidence of impaired tone fear conditioning in adolescents , with most finding no effect of alcohol on tone fear conditioning regardless of age. With only one study examining medial PFC-linked fear extinction, no strong conclusions can be drawn, but initial evidence suggests context-based fear extinction may be diminished in mid-adolescents compared to adults and late adolescents. Research on age-related differences on the effect of alcohol on longer-term fear memory is largely missing.Spatial learning and memory: The Morris Water Maze (MWM) is commonly used to test spatial learning and memory in rodents. Across trials, time to find the hidden platform in a round swimming pool is used as a measure of spatial learning. Spatial memory can be tested by removing the platform and measuring the time the animal spends in the quadrant where the escape used to be. The sand box maze (SBM) is a similar paradigm in which animals need to locate a buried appetitive reinforcer.Six rat studies examined spatial learning and memory using these paradigms. Three of the six studies observed age-related differences. Four examined the effects of repeated ethanol challenges 30 minutes prior to MWM training, showing mixed results –68. WhilTwo studies examined the effects of chronic ethanol exposure prior to training with or without acute challenges , 70. MatIn sum, evidence for impaired spatial learning and memory after acute challenges is mixed across six studies. Two studies found support for ethanol having a larger impact in adolescents compared to adults, whereas one study found the opposite and three studies did not observe any differences. Differences in ethanol doses stress responses may partially explain the discrepancies across studies. Importantly, given the sparsity of studies addressing the effects of long-term and voluntary ethanol exposure, no conclusion can be drawn about the impact of age on the relation between chronic alcohol exposure and spatial learning and memory.Non-spatial learning and memory: Non-spatial learning can also be assessed in the MWM and SBM by marking the target location with a pole and moving it across trials, measuring time and distances traveled to locate the target. By assessing non-spatial learning as well, studies can determine whether learning is more generally impaired by ethanol or whether it is specific to hippocampal-dependent spatial learning processes. A total of six studies assessed facets of non-spatial learning and memory. Two of the six studies observed age-related differences.In the four studies that examined non-spatial memory using the MWM or SBM in rats, none found an effect of alcohol regardless of dose, duration, or abstinence period in either age group , 67, 70.Across facets of non-spatial memory, there is little evidence for age-related differences in the effect of chronic alcohol, with four of the six studies finding no age differences. For memory of visually cued target locations in the MWM and SBM paradigms, alcohol does not alter performance in either age. Also, both adolescents and adults appear similarly vulnerable to alcohol-induced impairments in reward-related learning based on the one study. Only in the domain of object memory did any age-related differences emerge, with adolescents and not adults showing reduced novel object recognition after binge-like alcohol exposure in one study. However, more research into object recognition memory and reward-related learning and memory is needed to draw strong conclusions in these domains.Human evidence clearly suggests that alcohol is related to learning and memory impairments, both during intoxication  and afteWorking memory: Working memory refers to the limited capacity system for temporarily storing and manipulating information, which is necessary for reasoning and decision-making [n-making . In the n-making , some ofn-making  spontanen-making , 80, 83.Chronic binge-like alcohol exposure had no effects on spontaneous alterations after prolonged abstinence  , 80 in rIn sum, there is little evidence for impairments in working memory function in rats after chronic ethanol exposure, with four of the five studies observing no difference between age groups. While acute intoxication impairs working memory function in both ages, there is evidence from only one study that adolescents may make more working memory errors.Attentional processes: Attentional processing refers to the selection of information that gains access to working memory [g memory . PPI is g memory . For exag memory , 89. OneSlawecki and Ehlers  observedIn sum, there is limited and mixed evidence from two studies of age-related differences in the pre-attentional process of sensorimotor gating. Only one study found support for adolescent sensitivity to ethanol effects.Cognitive flexibility: Cognitive flexibility refers to the ability to update information based on environmental factors r changing goals in order to adaptively guide decision-making and is linked to the inability to reduce or abstain from drinking [drinking . Three sdrinking –81. Two In two rat studies, cognitive flexibility was assessed using reversal learning paradigms , 80. In In another study, Labots et al.  used a cOverall, there is limited evidence for adolescent resilience to the effects of chronic alcohol on cognitive flexibility. Two studies found support for adolescent resilience to ethanol’s effect on behavioral flexibility, whereas another study found no differences between adolescents and adults.Impulsivity: Impulsivity is a multi-faceted behavioral trait that encompasses impaired response inhibition, preference for an immediate reward over a larger but delayed reward, and premature expression of behaviors which may be maladaptive or in conflict with conscious goals. Impulsivity is a risk-factor for the development of addiction and may also be a consequence of sustained substance use [ance use . Pharmacance use . Two stuance use , 78. BotOne study examined impulsive behavior using a delay-discounting task in which choices are made between immediate small rewards and larger delayed rewards . RegardlIn sum, both studies found support for ethanol having a larger impact on adolescent compared to adults on impulsive behavior.Goal-directed behavior: Goal-directed behavior refers to when actions are sensitive to both the outcome value  and contingency between the behavior and the outcome [reflexively having a drink when seeing an item that is often associated with the rewarding effects of alcohol  despite not consciously desiring the alcohol ‘reward’ is an example of how habitual behavior (possibly driven by sign-tracking) can initiate the behavior as opposed to an intentional goal [ outcome . Two stu outcome , 77. One outcome . In huma outcome . In the nal goal . Omissional goal , 77. OneIn summary, one study found support for adolescents being less sensitive to ethanol effects on goal-directed behavior compared to adults, whereas one study found no effect of ethanol in either age group.Across the domains of executive function, there is some evidence that adolescents may be more vulnerable to impairments in certain executive and higher-order cognitive functions following chronic alcohol exposure, with increased risky decision-making after prolonged abstinence , impulsiExecutive functions are a domain of cognitive processes underlying higher-order cognitive functions such as goal-directed behavior. Executive functions can include but are not limited to working memory, attentional processes, cognitive flexibility, and impulse control or inhibition . A core Anxiety: AUD is highly comorbid with anxiety disorders [isorders , especiaisorders . While aisorders , 98. Manisorders . In the isorders . The opeisorders . In lighisorders . Anxietyisorders . In the isorders , anxietyisorders .Eleven studies examined anxiety-like behavior in rodents with mixed results across paradigms , 106–111Two studies used the OFT, finding no effects of voluntary  or forced (12/day vapor) ethanol exposure on anxiety-like behavior in adolescents or adult rats during withdrawal (7–9 h)  or afterFour studies used the elevated maze paradigm with mixed results. Only one study observed age-related differences in mice after chronic exposure (8–10w vapor) . AdolescThree rat studies used a startle paradigm to assess anxiety. Two observed reduced acoustic startle responses after ethanol exposure (12 h/d vapor) in both age groups during acute withdrawal periods (7–10 h) and following more sustained abstinence (6d) , 89. In Two studies used the light-dark box paradigm with mixed results , 111. OnIn summary, there is inconsistent evidence for age-related differences in the effect of chronic ethanol exposure on anxiety outcomes in rodents. The substantial differences across studies in how anxiety was elicited and measured make it challenging to draw strong conclusions. In the five studies that found age-related differences, adults tend to show higher levels of anxiety, particularly during early withdrawal; however, the opposite was found in the one study examining anxiety in social interactions. Six studies did not observe any age-related differences. Overall, adolescents may be less sensitive to the anxiety-inducing effects of chronic alcohol exposure.Social behavior: Two studies were identified that examined the effects of chronic ethanol exposure on social behavior in rats [ in rats , 113, wi in rats , while t in rats . In contThe findings from these two studies paint a complicated and inconsistent picture of the effects of ethanol on social behavior in adults and adolescents warranting further research. One study found support for a larger effect of chronic ethanol on adolescent social behavior compared to adults, while the other did not observe effects of ethanol in either group. One study found support for a larger effect of chronic plus acute ethanol intoxication on social behavior, with the opposite observed in the other.Glutamate is the brain’s main excitatory neurotransmitter and plays a crucial role in synaptic plasticity . Glutamatergic transmission plays an important role in the formation and maintenance of addictive behaviors and the nucleus accumbens (NAc) is considered an important hub in this, receiving glutamatergic input from cortical-limbic areas and dopaminergic input from the midbrain . Seven sThree studies investigated glutamate-related processes in the NAc , 118. TwTwo studies investigated glutamate-related processes in the  amygdala , 116. InAlcohol’s effects on frontal cortex functioning is thought to be mediated by alterations in NMDA receptor subunit expression , 120. TwConrad and Winder  assessedTwo studies looked at glutamate receptor subunit expression in the hippocampus , 115. PiIn sum, there is limited and inconsistent evidence for age-related differences in glutamate function across seven studies. The direction of the observed age-related differences varies across regions, with evidence of both increased and decreased sensitivity to ethanol effects in adolescents compared to adults in the four studies that observed age-related differences.A receptors are a primary mediator of alcohol’s pharmacological effects [GABA is the brain’s main inhibitory neurotransmitter. GABA effects . A total effects , 123. ThA α1 and GAD67 (enzyme that converts Glutamate to GABA) mRNA expression in adult rats only, 60 days after 18-days ethanol exposure [One study investigated GABA-related processes in the  amygdala, showing reduced GABAexposure .A receptor expression [A receptor functioning was not affected by 1 month of chronic intermittent ethanol exposure, GABAA receptors were less sensitive to the neurosteroid THDOC in adolescents. This neuromodulatory effect was not found in adults and did not persist after 33 days of abstinence. However, these results indicate that neurosteroids may play an indirect role in age differences in the GABAA receptor’s response to alcohol.Two studies looked at the rat cortex as a whole , 122. Vapression . Grobin A receptor functioning. Adolescent rats showed decreased tonic inhibitory current amplitudes after ethanol exposure, which was not the case for young adult and adult rats. Also, only the adolescents showed greater sensitivity to (ex vivo) acute ethanol exposure induced enhanced GABAergic tonic currents. The specificity of these effects to adolescent exposure might indicate adolescent vulnerability to ethanol-induced effects on the hippocampus; however, Van Skike et al. [A receptor expression in the hippocampus.Two studies focused on the rat hippocampus , 124. Fle et al.  did not In sum, given the limited number of studies and lack of replicated effects, no clear conclusions can be drawn about the role of age on the effects of alcohol on GABAergic neurotransmission. Age-specific effects appear to be regionally distinct. The only available study found support for heightened adult sensitivity to ethanol in the amygdala. In contrast, one study found support for greater adolescent sensitivity in the hippocampus and whole cortex, whereas the other found no age-related differences.The mesocorticolimbic dopamine system, with dopaminergic neurons in the ventral tegmental area (VTA) projecting to the NAc and prefrontal cortex, plays a key role in AUD, particularly through reward and motivational processes . Only twCarrara-Nascimento et al.  investigThese results suggest reduced alcohol-induced dopamine reactivity in adolescents in the PFC and NAc based on the two available studies, but more studies are warranted for a more detailed understanding of the relationship between age and dopamine receptor expression following chronic ethanol exposure.Acetylcholine is a known neuromodulator of reward and cognition-related processes . The comChronic alcohol consumption is thought to lead to brain damage by influencing processes involved in neurodegeneration and neurogenesis. The formation of addictive behaviors is paralleled by the formation of new axons and dendrites, strengthening specific neuronal pathways . While bHuang et al.  showed rLooking at hippocampal neurogenesis, ethanol exposure has been shown to initially reduce hippocampal neurogenesis in adult rodents, recovering after 1-month abstinence . CompareCyclin-dependent kinase 5 (CDK5) is involved in axon, dendrite, and synapse formation and regulation. CDK5 is overexpressed in the prefrontal cortex and the NAc following exposure to substances of abuse including alcohol . MoreoveIn sum, strong conclusions cannot be drawn due to the limited number of studies and lack of replicated effects. However, preliminary evidence points to adolescent vulnerability to damage in the cortex, reduced neurogenesis, and increased neurodegeneration in the hippocampus and the cortex as a whole based on four of the five studies. In contrast, one study found support for adult vulnerability to ethanol’s effects axon, dendrite, and synapse formation and regulation.Brain-derived neurotrophic factor (BNDF) and nerve growth factor (NGF) are involved in brain homeostasis and neural recovery , 138. WhIn rats, 30 weeks of chronic ethanol exposure reduced prefrontal mBDNF and β-NGF regardless of age, despite adolescents consuming more ethanol . MoreoveIn sum, the results of the four available studies suggest lower prefrontal BDNF during chronic alcohol use that recovers after abstinence regardless of age. However, the rate of recovery may be influenced by age with slower recovery in adolescents. In the two available studies, no age-related differences were observed in BDNF expression in the hippocampus.The transcription factors cFos and FosB are transiently upregulated in response to substance use, and ΔFosB accumulates after chronic exposure, particularly in striatal and other reward-related areas . Two stuAfter chronic ethanol exposure , adolescent compared to adult rats showed increased prefrontal H3 and H4 acetylation of the cFos promotor region and increased H4 acetylation and H3 dimethylation of FosB promotor regions after acute abstinence . MoreoveEthanol is known to trigger immune responses in the brain , causing inflammation and oxidative stress –145. ThrMicroglia remove damaged brain tissue and infectious agents and are key to the brain’s immune defense. Only one study investigated microglia levels . AlthougIn sum, two of the studies found support for increased immune responses after ethanol exposure in adults compared to adolescents, whereas the one other study found no difference between the age groups.Chronic stress and HPA-axis functionality have been associated with the maintenance of AUD  . Two stuFalco et al.  found deNo conclusions can be drawn. One study observed found support for reduced effects of ethanol on HPA-axis functionality compared to adults, whereas the other observed no difference between the age groups. Future studies using different (voluntary) exposure paradigms are needed to further investigate the effects of alcohol on HPA activity in relation to age of alcohol exposure.Neuropeptides are a diverse class of proteins that have a modulatory function in many different processes, including but not limited to neurotransmission, stress, immune responses, homeostasis, and pain –153. OnlSlawecki et al.  specificThe first metabolite of ethanol is acetaldehyde, which has been theorized to mediate the effects of ethanol on both brain and behavior . Only onRhoads et al. showed that despite the fact that adolescent rats consumed more alcohol brain catalase levels after 3-weeks of ethanol exposure (no abstinence) did not differ between adolescents and adults . AlthougWhile the previously described studies focused on specific factors involved in neurotransmission, brain health, and plasticity, proteomics allows for the study of the full proteome in a specific region or tissue type. One study investigated the impact of age on ethanol-induced changes in the hippocampal proteome, observing age-related differences . In thisEthanol-induced molecular changes may eventually change neuronal activity. Three studies investigated neuronal activity and functioning , 160 usiGalaj et al.  assessedIA) conductance was measured. Differences emerged between age groups : EtOH-exposed adolescents and adults both showed lower IA mean peak amplitude compared to the respective control groups. However, adolescents also showed reduced IA density and increased mean decay time, which decreased in adults. Furthermore, only adolescents showed increased depolarization required for activation compared to controls, which can result in higher interneuron firing rates in the CA1 region that could affect learning processes. Additional research is needed to connect these findings to behavioral measures of learning and memory.Li et al.  examinedSlawecki et al.  was the Overall, strong conclusions cannot be drawn given the disparate paradigms and outcomes utilized. While adolescents and adults appear to differ in the effect of ethanol on neuronal firing, the meaning of these differences is not clear given the lack of connection between these findings and behavioral outcomes.Four studies examined age-related differences of the effect of alcohol on brain or cognition in humans –165.Müller-Oehring et al.  examinedThree studies examined age effects in alcohol-related implicit cognitions, specifically attentional bias , 165, alRooke and Hine  found evCousijn et al.  also invIn summary, human evidence is largely missing, with no studies comparing more severe and dependent levels of alcohol use between adolescents and adults. The preliminary evidence is too weak and heterogeneous to draw conclusions, warranting future studies investigating the impact of age.The current systematic review assessed the evidence for the moderating role of age in the effects of chronic alcohol use on the brain and cognition. The identified 59 rodent studies Table  and 4 huAge-related differences in learning and memory-related processes appear to be highly domain specific. There is limited but fairly consistent evidence for adolescent-specific impairments in contextual fear conditioning, which could be related to hippocampal dysfunction. Results for other hippocampus-related memory processes such as spatial memory are mixed and largely based on forced exposure with acute challenge studies rather than voluntary long-term exposure to alcohol. The evidence base is currently insufficient to draw conclusions about the role of age in alcohol’s effects on non-spatial types of learning and memory. Alcohol generally did not impact performance in the non-spatial variants of the MWM and SBM paradigms or in reward-learning, but the results of the limited studies in the object-learning domain highlight potential impairments and the importance of age therein. For example, adolescents but not adults demonstrated impaired object memory in the only study using the novel object recognition task . Acute cHigher rates of adolescent alcohol use, especially binge drinking, may be facilitated by a heightened sensitivity to the rewarding properties of alcohol in combination with a reduced sensitivity to the negative effects of high doses . In lineRegarding executive functioning, there is limited but fairly consistent evidence from animal studies that adolescents are more vulnerable to long-term effects of chronic exposure on decision-making and are more impulsive than adults during acute intoxication and after prolonged abstinence following chronic exposure. Impulsivity is associated with functional alterations of the limbic cortico-striatal systems , with inOnly a few studies have investigated age-related differences in cognitive functioning in humans. These studies focused on mostly non-dependent users and studied different outcomes, including cognitive biases and implicit and explicit alcohol-related cognitions. Overall, there was limited but consistent evidence that age does not affect alcohol attentional or approach biases, with heavy drinkers in both age groups allocating more attention to alcohol cues compared to controls , 165. InRegarding anxiety-related outcomes, results are inconsistent across studies and paradigms. When age-differences are observed, adolescents often show reduced anxiety compared to adults during both acute withdrawal and sustained abstinence following chronic ethanol exposure. However, the direction of age-related effects of alcohol may also be anxiety-domain specific. In social settings, adults show reduced anxiety compared to adolescents. Research on the neurocircuitry of anxiety processes implicates the extended amygdala, especially the BNST, in anxiety behaviors with an emphasis on the role of GABAergic projections to the limbic, hindbrain, and cortical structures in rodents . DespiteThe reviewed studies revealed a high degree of variability in study designs and outcomes, hindering integration and evaluation of research findings. We were unable to differentiate our conclusions based on drinking patterns . The prevalence of binge-drinking in adolescence is very high and is associated with neurocognitive alterations . StudiesIt is also important to acknowledge the limitations of the choice of adolescent and adult age ranges in our inclusion criteria. Rodent studies had to include an adolescent group exposed to alcohol between the ages of PND 25–42 and an adult group exposed after age PND 65. Ontogenetic changes may still be occurring between PND 42–55, and this period may more closely correspond to late adolescence and emerging adulthood in humans . Studies that compared animals in this post-pubertal but pre-adulthood age range were not reviewed. Studies investigating age-related differences in the effects of ethanol on brain and cognitive outcomes in emerging adulthood are also translationally valuable given the high rates and risky patterns of drinking observed during this developmental period . Indeed,Furthermore, we did not conduct a risk of bias assessment to examine the methodological quality of the animal studies. The applicability and validity of the risk of bias tools for general animal intervention studies, such as the SYRCLE risk of bias tool , remain Moreover, most work is done in male rodents and is based on forced ethanol exposure regimes. In a recent opinion article, Field and Kersbergen  questionThe intermittent voluntary access protocol resembles the patterns of alcohol use observed in humans, and also result in physiologically relevant levels of alcohol intake –184. OnlOn a practical level, rodents mature quickly and choice-based exposure paradigms are more complex and time-consuming than most forced exposure paradigms. Consequently, by the time final behavioral measurements are recorded, both the adolescent and adult exposure groups have reached adulthood. To combat this, many of the included studies use forced ethanol exposure, such as ethanol vapor, to quickly expose rodents to very high doses of ethanol. Although the means and degrees of alcohol exposure may not directly translate to human patterns of alcohol use, such studies do allow for the assessment of the impact of high cumulative doses of ethanol within a relatively short period of time which allows for more time in the developmental window to test age-related differences in the outcomes. When considering the translational value of a study, it is therefore important to evaluate studies based on the goal, while not ignoring the practical constraints.While human research is challenging due to the lack of experimental control and the inherent confounds in observational studies between age and alcohol exposure history, large-scale prospective longitudinal studies offer a gateway towards a better understanding. Comparisons of different trajectories of drinking from adolescence to adulthood  could offer insight into the associated effects on cognitive and brain-related outcomes. Of course, different drinking trajectories are likely confounded with potentially relevant covariates which limits causal inference. Direct comparisons of low and heavy adolescent and adult drinkers, supported by a parallel animal model can help to bolster the causality of observed age-related differences in human studies. In addition, changes in legislation around the minimum age for alcohol consumption in some countries provide a unique opportunity to investigate how delaying alcohol use to later in adolescence or even young adulthood impacts cognitive functioning over time. Importantly, future studies investigating the moderating role of age in humans should carefully consider the impact of psychiatric comorbidities. While adolescence into young adulthood is the period in which mental health issues often emerge , 190, thboth similarities and differences in the processes underlying alcohol use and dependence between adolescents and adults. However, due to the large degree of heterogeneity in the studies included in sample, designs, and outcomes, we were unable to perform meta-analytic synthesis techniques.The aim of this systematic review was to extend our understanding of adolescent risk and resilience to the effects of alcohol on brain and cognitive outcomes compared to adults. In comparison to recent existing reviews on the impact of alcohol on the adolescent brain and cognition , 22, 23,In conclusion, while the identified studies used varying paradigms and outcomes, key patterns of results emerged indicating a complex role of age, with evidence pointing towards both adolescent vulnerability and resilience. The evidence suggests adolescents may be more vulnerable than adults in domains that may promote heavy and binge drinking, including reduced sensitivity to aversive effects of high alcohol dosages, reduced dopaminergic neurotransmission in the NAc and PFC, greater neurodegeneration and impaired neurogenesis, and other neuromodulatory processes. At the same time, adolescents may be more resilient than adults to alcohol-induced impairments in domains which may promote recovery from heavy drinking, such as cognitive flexibility. However, in most domains, the evidence was too limited or inconsistent to draw clear conclusions. Importantly, human studies directly comparing adolescents and adults are largely missing. Recent reviews of longitudinal human research in adolescents, however, revealed consistent evidence of alterations to gray matter, and to a lesser extent white matter, structure in drinkers , 18, butAppendix


Temporomandibular disorders (TMD) represent a group of musculoskeletal conditions involving the temporomandibular joints (TMJ), the masticatory muscles and associated structures. Painful TMD are highly prevalent and conditions afflict 4% of US adults annually. TMD include heterogenous musculoskeletal pain conditions, such as myalgia, arthralgia, and myofascial pain. A subpopulations of TMD patients show structural changes in TMJ, including disc displacement or degenerative joint diseases (DJD). DJD is a slowly progressing, degenerative disease of the TMJ characterized by cartilage degradation and subchondral bone remodeling. Patients with DJD often develop pain , but do not always have pain (TMJ osteoarthrosis). Therefore, pain symptoms are not always associated with altered TMJ structures, which suggests that a causal relationship between TMJ degeneration and pain is unclear. Multiple animal models have been developed for determining altered joint structure and pain phenotypes in response to various TMJ injuries. Rodent models of TMJOA and pain include injections to induce inflammation or cartilage destruction, sustained opening of the oral cavity, surgical resection of the articular disc, transgenic approaches to knockout or overexpress key genes, and an integrative approach with superimposed emotional stress or comorbidities. In rodents, TMJ pain and degeneration occur during partially overlapping time periods in these models, which suggests that common biological factors may mediate TMJ pain and degeneration over different time courses. While substances such as intra-articular pro-inflammatory cytokines commonly cause pain and joint degeneration, it remains unclear whether pain or nociceptive activities are causally associated with structural degeneration of TMJ and whether structural degeneration of TMJ is necessary for producing persistent pain. A thorough understanding of the determining factors of pain-structure relationships of TMJ during the onset, progression, and chronification by adopting novel approaches and models should improve the ability to simultaneously treat TMJ pain and TMJ degeneration. Orofacial pain is a highly prevalent condition. Approximately 15% of the general population experiences various orofacial pain symptoms, such as dento-alveolar pain, tenderness of the temporomandibular joint (TMJ) or masticatory muscles, pain with TMJ biomechanics or pre-auricular pain . TemporoTMD also include joint disorders, such as disc displacement and degenerative joint disease (DJD) . DJD is Degenerative changes in the TMJ are highly prevalent in TMD patients –10. RadiPatients with TMJOA show poorer oral health-related quality of life than healthy individuals . Pain frRadiographic studies have supported the association of condyle morphology and destructive condylar changes with pain symptomatology, with condylar flattening being most highly correlated with general pain complaints . CondylaHowever, there are studies suggesting that the extent of degenerative changes in the TMJ is not associated with pain-related variables , 30, 31.vice versa. However, studying the relationship between pain and degenerative changes remains important. Identifying definitive causal factors for joint degeneration and pain can serve as a biomarker of disease onset and progression, which will enable detection and treatment during the early phase of the disease. Persistent pain can adversely impact the prognosis of the treatment of degenerative conditions  , rodent TMD patients with pain are heterogeneous in terms of psychosocial and sensory phenotypes and systemic comorbid conditions . In addi2+ channels reduces knee arthritis-induced pain, but increases joint destruction  decreases both mechanical allodynia and structural changes in a mouse model of knee OA . Transfotruction . Neuropetruction . Identiftruction , 52, 53,The etiology of TMJOA is complex and multifactorial, and may be secondary to disc displacement, trauma, parafunctioning, unstable occlusion, or functional overloading . These fMethods of measuring pain-related outcomes from TMJ have been developed in rodents to mimic various pain conditions frequently reported by TMD patients . These aIntra-TMJ injections of CFA induces mechanical hyperalgesia on the TMJ, which peaks after 1–4 days and lasts approximately 10–14 days in mice and rats , 57. Intvia the spreading of nociceptive signals within the trigeminal ganglia (TG) through soluble mediators and gap junction channels involving satellite glia  produced mechanical hyperalgesia of the masseter muscle, which began as early as 1 day after the 1st opening event and lasted for 32 days afterwards . The amoMechanisms of TMJ structural degeneration after forced mouth opening in mice were studied in a sustained mouth-opening model that used a spring to maintain an open mouth by 14 mm and delivered 2 N force for 3 h per day for 5 days . Four weRodent forced mouth opening models can be useful for determining structural changes in the TMJ that mimic loading and trauma pathogenesis in humans. The model has translational potential since the associations of TMJ trauma with the development of painful TMD or TMJ degeneration is known , 118. DeOf note, the non-physiological range of excessive occlusal interferences can lead to pain and TMJ degeneration in animals as reviewed previously , 130. HoA surgical approach to destabilize the knee joint by transection of the medial meniscotibial ligament has been widely used in rodents to produce slow progression of structural changes and pain-like behaviors from 4 weeks following surgery , 134. DePartial or total removal of the disc in the TMJ of rats and mice results in early-onset OA-like changes , 144. PaSurgical models of TMJOA are primarily used for studying TMJ degeneration rather than pain phenotypes, and information regarding the correlation or association of pain and TMJ structure is unavailable. Since surgical manipulation itself can cause inflammation inside the affected joint, segregating the effects of acute, post-operational changes on TMJ structure from the effects of surgical manipulation of the disc is difficult. Interestingly, unilateral, partial discectomy not only leads to degeneration in the surgical-side of the TMJ, but also initiates early-onset articular cartilage degeneration in the contralateral, non-surgical side of the TMJ in mice . TherefoMice carrying spontaneous or targeted mutations of multiple genes naturally develop TMJ degeneration in a non-invasive manner and without the need for injecting agents or inducing trauma . These mvia overexpression of IL1β is prevented by the overexpression of the IL1 receptor antagonist in the brainstem. Consistently, conditional overexpression of IL1β in the medullary dorsal horn driven GFAP promoter is sufficient to induce TMJ degeneration, albeit to a lesser extent than the overexpression of IL1β in the TMJ. Furthermore, facial grooming behaviors induced by the overexpression of IL1β in the TMJ is attenuated by the overexpression of the IL1 receptor antagonist in the brainstem . However, most studies are only focused on the time course of the disease and biological factors involved in degenerative changes within the TMJ and, therefore, do not pay much attention to pain-related phenotypes. Hence, determining pain phenotypes using the various assays discussed above during the time course of pathogenesis of TMJOA in these genetic models is warranted.International Association for the Study of Pain defines pain as “an unpleasant sensory and emotional experience associated with, or resembling that associated with, actual or potential tissue damage, or described in terms of such damage”, meaning pain is not a simple sensation but involves emotional aspects. One prospective study showed that multiple psychosocial variables including somatic symptoms, general psychological symptoms, a negative mood, and multiple measures of stress are risk factors for the onset of painful TMD . Furthervia transplantation of fecal microbiota from resveratrol-treated mice into mice with intra-TMJ CFA (Streptococcus is associated with pain and inflammation in knee OA (The mechanisms of TMD and comorbid pain conditions, such as irritable bowel syndrome, have also been studied using rodent models. Masseter inflammation followed by swim stress induces months-long visceral hypersensitivity and concomitant cutaneous hyperalgesia in rats, which is mediated by ERK1/2 in the spinal cord , 173. Su-TMJ CFA , which s knee OA . While avice versa. Considering the broad impact of the brain on skeletal homeostasis (Studies that have focused on the mechanisms of TMJ pain and structural degeneration using different models indicate that TMJ pain and degeneration occur over a partially overlapping time course. There are molecules that commonly cause pain and joint degeneration, e.g., intra-articular pro-inflammatory cytokines. However, it is unclear whether pain is causally associated with structural degeneration and whether structural degeneration is necessary for producing persistent pain. To experimentally address the pain-structure relationship, it will be important to determine whether the activity of peripheral and central pathways involved in TMJ pain contributes to TMJ degeneration or eostasis , it is l


Forest restoration has been proposed as a scalable nature-based solution to achieve global environmental and socio-economic outcomes and is central to many policy initiatives, such as the Bonn Challenge. Restored forests contain appreciable biodiversity, improve habitat connectivity and sequester carbon. Incentive mechanisms  have been a focus of forest restoration efforts for decades. Yet, there is still little understanding of their role in promoting restoration success. We conducted a systematic literature review to investigate how incentive mechanisms are used to promote forest restoration, outcomes, and the biophysical and socio-economic factors that influence implementation and program success. We found that socio-economic factors, such as governance, monitoring systems and the experience and beliefs of participants, dominate whether or not an incentive mechanism is successful. We found that approximately half of the studies report both positive ecological and socio-economic outcomes. However, reported adverse outcomes were more commonly socio-economic than ecological. Our results reveal that achieving forest restoration at a sufficient scale to meet international commitments will require stronger assessment and management of socio-economic factors that enable or constrain the success of incentive mechanisms.This article is part of the theme issue ‘Understanding forest landscape restoration: reinforcing scientific foundations for the UN Decade on Ecosystem Restoration’. To helpTo drive restoration at scale, a plethora of multilateral environment agreements have spawned over the past decade. These started with the Aichi target 15 of the Convention on Biological Diversity (CBD), setting, in 2010, a goal to restore 15% of degraded ecosystems , followeDespite the globally recognized benefits of achieving restoration at scale, doing so cost-effectively remains a challenge . GovernmIdeally, incentive mechanisms should compensate landholders and land users for the foregone opportunities associated with active restoration and natural regrowth in agricultural landscapes. Incentives, such as PES and technical assistance, are also widely used to promote the adoption of sustainable agricultural practices  and can Here, we conduct a systematic literature review to address three main research gaps: (i) What types of incentive mechanisms are used for forest restoration and how are these financed? (ii) What biophysical and socio-economic factors affect the success of incentive mechanisms to promote forest restoration? (iii) What have been the ecological and social outcomes of incentive mechanisms for promoting forest restoration? On the basis of our findings, we discuss the challenges and potential of using incentive mechanisms as pathways to promote large-scale, long-term restoration, while maximizing ecological and social benefits.. 2 (a)We conducted a literature review of peer-reviewed articles published between January 2010 and October 2020. This time frame was selected to understand the current trends in incentive mechanisms for forest restoration, as preliminary literature searches showed that few papers on incentives for restoration were published before 2010, compared to more recent years, with a strong focus on the Reducing Emissions from Deforestation and Forest Degradation (REDD+) incentives. We also identified that few studies have examined the role of disincentive mechanisms, such as penalties, fines and quotas. For this reason, we decided to focus on incentives for this literature review. The literature search was conducted via: (i) database search using key terms; (ii) screening of studies resulting from the initial search; and (iii) selection of papers for final analysis based on eligibility criteria. Key words searched include incentive mechanism terms (first filter) and forest restoration and regrowth terms (second filter), such as ‘payment* for ecosystem services’ and ‘ecological restoration’, respectively. Electronic supplementary material, figure S1 details the selection of studies for the analysis in a PRISMA diagram. A detailed description of the literature search, search terms and paper selection is also provided in the electronic supplementary material . Searches returned 1421 papers that were imported to the software Covidence . After t (b)To answer the first research question, through full-text reading of each paper and using 15 structured questions , we gathered descriptive data on three main aspects of the evaluated incentive mechanism: (i) general characteristics: country, time frame of intervention at the time of evaluation, spatial scale of implementation, land-tenure regimes in the area of implementation and primary land use; (ii) governance characteristics: type of proponent organizations, type of incentive, type of financing mechanism, source of funding, type of governance and targeted recipients; and (iii) the restoration approaches incentivized (e.g. active forest restoration and agroforest plantation).To answer the second and third research questions, we conducted a thematic analysis using NVivo software . We used. 3 (a)c). This increasing publication rate reflects the growing interest in incentive mechanisms and forest restoration. The publications covered 10 different types of incentives, offered alone or in combination (a), across 24 countries (e and S3), followed by forest conservation or sustainable forest management  and adoption of sustainable land use practices . Natural forest regrowth and afforestation  and were implemented at a spatial scale of 1000 km2 or greater .In total, 73 papers met the eligibility criteria , with 25 (34%) of these published since 2019  were tha). By contrast, for other types of incentive mechanism, only 29% of the studies involved agricultural production as the primary use. At the landscape scale, overall, public-led governance was the most common mechanism among the reviewed studies (65%). The public sector was the proponent (the organization responsible for the implementation) for 64% of initiatives offering direct payments . Similarly, there was a dominance of public-led initiatives among the other incentive types (71%). For non-public-led initiatives, such as third-sector  and private-led initiatives, the incentives were almost exclusively direct payments, except for one third-sector initiative using access to trading markets as an incentive for forest restoration. On the other hand, land-tenure arrangements did not influence the type of incentive mechanisms applied. Private land-tenure regimes were the most frequent among the studies, for both direct payments (38% of studies) and all other incentives considered together (36%), while other tenure arrangements were broadly distributed among all incentive types .We found that the types of incentive mechanisms implemented varied according to some governance characteristics at the plot and landscape scale, such as the target area's primary land use and the incentive's institutional governance type . At the plot scale, more than 65% of the direct payments incentivized restoration where agricultural production was the primary land use Our review revealed that social factors—including incentive governance and target beneficiary characteristics—were the most commonly reported influences on program success. A broad range of socio-economic factors were associated with the success or failure of incentive mechanisms for forest restoration , table 1Constraints at the institutional scale included communication issues , lack of beneficiary engagement , legal uncertainty , weak participatory component in the decision-making  and lack of transparency . Late payments and short duration of incentives were also mentioned in some cases . At the individual scale, constraints consisted of, among other aspects, lack of access to land or to markets , socio-cultural values or norms related to the traditional use of land  and scepticism or lack of trust in institutions involved in the project governance . The most frequent constraints related to the economic context and incentive characteristics were competing economic activities  and small payments offered , respectively. Biophysical constraints were less frequently identified and included property characteristics such as small area size available and susceptibility to climate hazards .We found that enablers at the institutional scale included partnerships among stakeholders , appropriate legal frameworks  and non-bureaucratic agreements . Enabling factors at the individual scale included motivational aspects such as the perceived benefit of planting trees , social norms and values influencing decisions  and access to information and level of education . Commonly, the same factors were mentioned as enablers and constraints in different studies, depending on the implementation context . (c)a). A similar proportion of unmet objectives were identified for ecological (in 26% of studies) and socio-economic aspects (27%). Finally, perverse outcomes were more commonly reported for socio-economic aspects (38% of studies) than ecological (15%). These ecological and socio-economic outcomes were summarized within five and six main classes, respectively (b).Out of the three analysed outcomes resulting from incentive mechanisms , beneficial outcomes dominated reporting for both ecological (reported in 49% of studies) and socio-economic aspects (50%) a. A simiectively b.FigureThe most mentioned beneficial ecological outcomes were increased ecosystem services and natural capital, cited in 41% of studies. Within those, increased forest cover (29%), carbon sequestration (7%) and improved water quality and availability (7%) are among the most frequent outcomes reported. Studies reporting low or no additionality of incentives  to ecological aspects (26%) include low additionality to the provision of ecosystem services or natural capital (11%), and lack of evidence of effectiveness of restoration incentives to ecological impacts (11%). Perverse ecological outcomes were linked to spillover  and reduced ecosystem services (11%).c. Among the socio-economic factors, governance beneficial outcomes were reported in 18% of the studies, with increased stakeholder engagement (8%) being the most mentioned outcome. Insufficient benefits for income generation (12% of studies) and unfulfilled participants’ expectations (15%) were the primary unmet socio-economic objectives. Perverse governance outcomes and inequity outcomes included reduction of engagement (one study) and unequal distribution of benefits , respectively. The beneficial and perverse outcomes of incentives for livelihoods assets  are detailed in figure 4. 4Understanding how incentive mechanisms are used for promoting forest restoration and what constrains and enables their success is critical both for upscaling restoration to meet international commitments and achieving multiple targeted benefits. We found that direct payments (e.g. PES), financed mainly through government budgets, carbon offset mechanisms and funds and grants, were the most commonly described incentive mechanisms for forest restoration. In addition, we found that constraints for implementation and long-term success of incentives for restoration are predominantly socio-economic and governance-related, rather than ecological or biophysical. Although ecological factors have been mostly used to assess restoration success ,35, we f (a)We found that ecological aspects were rarely mentioned as constraints or enablers for the implementation of restoration incentives. This is surprising, since ecological constraints of restoration are extensively documented in the literature . They ofWhile most of the studies included in our review evaluated incentive outcomes, only a few used control groups as part of the research design –44. This (b)While the theoretical frameworks for integrating social and ecological systems have been extensively discussed –51, tran (c)Similarly to ecological aspects, the lack of funding is widely recognized as a crucial constraint for restoration  but was  (d)It is crucial to use incentive mechanisms to develop a restoration value chain that translates into long-term financial and livelihood gains, increasing the value of standing forests within agricultural landscapes. As found in our review, this is important because agriculture is often the primary land use in areas targeted by restoration incentives. Many restoration programs offer short-term payment contracts for promoting long-term land use changes. This creates a critical temporal gap between restoration incentives and outcomes, which may undermine landholders’ and users’ confidence in restoration's financial viability . Socio-e (e)No incentive type is universally suitable for all socio-economic contexts; as such, mechanisms to promote restoration should be selected based on local needs and context. Our review revealed the use of diverse incentives, likely to attract different target groups. These included, for example, direct payments (both for ecosystem services and adoption of sustainable practices), funding for community development and allocation of management rights –73. ConsConversely, mechanisms that incentivize restoration by offering land use management rights or alternative livelihoods could appeal more to land users or landholders with informal land-tenure. Despite a dominance of direct payments in our review, it remains unclear to what extent the papers selected simply reflect prevailing research and funding interests or an accurate proportion of types of incentive mechanisms implemented. Additionally, there may be a bias against non-economic incentives, such as institutional support and the offer of extension services. Frequently, these activities are assumed to be ubiquitous and could be underrepresented in the literature on incentives. Nonetheless, the heterogeneity of incentive mechanisms reinforces the need to engage with stakeholders early in the design and implementation process, to ensure that incentive schemes align well with their needs, overcome constraints and safeguard the critical enabling conditions for successful restoration.. 5Although incentive mechanisms, especially when implemented by governments, are essentially seen as policy levers with a strong focus on finance and payment, the original aim of these instruments is to create system-wide change ,76. When


Aromatic amino acids (AAAs) are integral components for protein production and their essentiality goes without saying. Moreover, their derivatives often are used by animal species as neurotransmitters that play some specific functions. The evolvement of specific physiological functions in AAA derivatives is likely due to the relative stability of their bulky planar ring structures of these AAAs. As a result, AAAs are structurally much less flexible than most of other amino acids . AlthougObtaining sufficient amounts of the AAAs from food sources usually is not a major concern in animal species, especially for humans, but regulation of their individual contents and their metabolites is considerably important to human health. Consequently, disorder of their metabolic pathways often leads to medical issues in humans. Among the three commonly considered AAAs , phenylalanine contains a benzene ring, tyrosine has hydroxylated benzene ring, and tryptophan consists of an indole ring. In general, the benzene ring is more stable than indole ring; thus, both phenylalanine and tyrosine are more stable than tryptophan. Because animal species can obtain AAAs from food sources, the ability to breakdown the AAAs or to convert these molecules to other compounds is often important to animal species, particularly humans.m ∼ .5 mM), which prevents phenylalanine from accumulating beyond physiological level, but also leaves adequate phenylalanine content for protein synthesis. It is well known that deficiency of phenylalanine hydroxylase leads to a life-threatening disease, phenylketonuria  functions as a neurotransmitter in the brain and the levels of both dopamine and L-dopa must be tightly controlled to prevent their toxic effects. In fact, the toxic effect of the catechol moiety is visualized by the gray color of the substantial nigra in older animals. It is difficult to completely prevent L-dopa/dopamine oxidation. As time passes, the substantial nigra progressively becomes grayer due to catecholamine oxidation. Parkinson’s disease likely occurs if most of the L-dopa producing cells die due to catecholamine oxidation. When enough attention is paid to maintain adequate reducing capability, balanced diets, etc., some L-dopa producing cells, if not most, should survive and remain functional for the entire lifespan of given species.Tryptophan is the least stable AAA because the indole ring is much less stable than phenyl ring. Tryptophan is relatively rare in proteins and, typically, animals can obtain sufficient quantities from their diet. In humans, majority of tryptophan is degraded through kynurenine pathway. Because it is less abundant, yet necessary for protein production, mammalian species often upregulate tryptophan breakdown during infections as a strategy to starve/eliminate microbial pathogens . Some trAromatic Amino Acid Metabolism, Volume II, there are original research articles discussing some positive relationships between increased AAAs and elevated oxidative stress, which is based on increased levels of products under common oxidative conditions , a review article concerning N-acetylated AAAs in relation to endocannabinoid anandamide production , another review article concerning the role different tryptophan oxidative enzymes play in tryptophan hemostasis , and two other original research articles: one addressing metabolic profile changes in alcohol-dependent inpatients  and another concerning diabetic retinopathy patients . In both disease conditions, noticeable changes in tryptophan metabolites were reported. Results of these studies revealed some intricacy in AAA metabolism on maintaining physiological condition in living species and suggest that we still have much to learn about AAA metabolism. Overall, researchers are continuously making good progress in the area of AAA metabolism and their efforts should eventually lead to a comprehensive understanding AAA metabolism.Although some tyrosine derivatives (such as L-dopa and dopamine) and some tryptophan derivatives (such as 5-hydroxytryptophan and serotonin) are more easily oxidized and may increase oxidative stress, the benefits of their derivatives  to human health and wellbeing far overweigh their potential toxicity under non-health related conditions. In fact, AAAs rarely cause major health issues themselves, but, often, the problems are from disorders in their respective pathways for their biosynthesis, degradation, or modification. In this Research Topic of


Bone devitalization is believed to be a critical determinant of complications such as infection or nonunion. However, intraoperative assessment of bone devitalization, particularly in open fractures and infections, remains highly subjective resulting in variation in treatment. Optical imaging tools, particularly dynamic contrast-enhanced fluorescence imaging, can provide real-time, intraoperative assessment of bone and soft tissue perfusion, which informs the tissues' ability to heal and fight infection. We describe a novel technique to apply indocyanine green–based fluorescence imaging, using a device that is frequently used in the operating room to assess skin or flap perfusion in plastic surgery, to assess bone and deep tissue perfusion in three pertinent cases: (1) a chronic infection/nonunion after a Gustilo type 3A tibia fracture (patient 1), (2) an acute Gustilo type 3C tibia open fracture with extensive degloving/soft tissue stripping (patient 2), and (3) an atrophic nonunion of the humerus (patient 3). In all three cases, fluorescence imaging (both time-specific fluorescence and maximum fluorescence) and derived kinetic maps of time-to-peak, ingress slope, and egress slope demonstrated clear spatial variation in perfusion that corresponded to the patient pathogenesis. The impact of this information on patient outcome will need to be evaluated in future clinical trials; however, these cases demonstrate in principle that optical imaging information has the potential to inform surgical practice, reduce the variation in treatment, and improve outcomes observed in these challenging patients. Optical imaging tools are being increasingly applied to a wide array of surgical fields using various targeted and untargeted fluorescent probes to enhance contrast and understand tissue kinetics. In surgical oncology, glioma resections are now routinely guided by 5-aminolevulinic acid visualization, which has improved median survival in high-grade glioma. In bowel anastomoses, indocyanine green fluorescence imaging can provide surgeons with intraoperative data beyond what can be seen and palpated, differentiating between perfused and dysvascular bowel.,9 Recently, it has been observed that certain antibiotics  that are incorporated into bone formation and regeneration processes can be used in humans to identify necrotic bone regions in bisphosphonate-related osteonecrosis of the jaw. In a study of 10 patients, fluorescence imaging was superior to more clinically subjective indicators such as bone bleeding in patients with bisphosphonate-related osteonecrosis of the jaw. There is enormous untapped potential to apply these tools to improve care of musculoskeletal trauma, but unlocking this depends on (1) availability of fluorescence imaging systems, (2) advancements in quantitative analysis, and (3) intraoperative visualization of this information. To improve the objective assessment of bone perfusion, we present our initial, first-in-human experience applying indocyanine green (ICG)–based dynamic contrast-enhanced fluorescence imaging using data processing and analytic strategies customized for bone imaging in three patients to assess bone vascularity in high-energy open fracture, post-traumatic infection, and chronic atrophic nonunion.Dysvascular tissues have limited potential to regenerate, heal, or fight infection due to insufficient delivery of osteoprogenitor cells, growth factors, inflammatory cells, endogenous immune cells, and antibiotics. Because of this, the cornerstone of treatment of contaminated open fracture, established infection, and selected atrophic nonunion is to achieve a thorough debridement of poorly perfused or nonviable bone.–3 To dat,12 (similar to a pulse oximetry probe) was placed on the finger at the start of the procedure to collect a patient-specific arterial input function, which permits measurement of ICG injection parameters. This measurement allows post hoc correction of variation in injection parameters. Video rate recorded images were obtained for 20 seconds before injection of ICG contrast to establish a baseline value. After 20 seconds, baseline measurements were obtained, 0.1 mg/kg of ICG was administered intravenously, and the video rate recording was completed for a total of 4 minutes. White light images of the surgical site were also obtained.In the operating room, the surgical site was exposed and self-retainers were placed into the wound. A sterile drape was then placed over the SPY Elite camera. The SPY Elite system was positioned at a 300-mm working distance from the surgical field. A white light image was obtained, and then, video rate fluorescence images were recorded every 0.3 seconds for 4.5 minutes Fig. . A pulseIn the three patients presented, the surgical debridement proceeded per standard of care without using ICG-based fluorescence imaging. Kinetic parameter maps such as maximum intensity (Imax), time-to-peak (TTP), ingress slope (IS), and egress slope (ES) are shown in a more detailed panel termed “enhanced DCE view” , DICOM images were saved and loaded into in-house developed software. Coregistration between the RGB and the fluorescence stack is performed before the single time-point fluorescence intensity images are displayed in the “SurgeonView” panel. Simple curve analysis is applied to the fluorescence time series, which were first corrected by the arterial input function to account for any variations in how the ICG dye is injected and for allometric variation because the dye is dosed according to weight.w” Figs.  and 5. T in the operating room for 3 patients: patient 1 with a chronic infected nonunion after an open Gustilo type 3A open tibia fracture, patient 2 with an acute Gustilo type 3C open distal tibial fracture, and patient 3 with a chronic atrophic humeral nonunion. The study was approved by the local IRB in accordance with the declarations of the World Medical Association (www.wma.net). All patients provided written informed consent before the procedure.After IRB approval by the Dartmouth-Hitchcock Human Research Protection Program (Study No. 31575 PI: I. L. Gitajn), we report on the results of ICG-based dynamic contrast-enhanced fluorescence imaging (DCE-FI)Patient 1 is a 57-year-old healthy active man involved in a motorcycle collision 2 years prior resulting in an open Gustilo type 3A distal third tibia fracture, which was complicated by acute infection after routine debridement and intramedullary fixation , there were clear trends demonstrating extremely poor vascular perfusion to the infected nonunion site with improving perfusion at each more proximal ROI, suggesting that the patient's failure to heal or clear the infection may be due to an inadequate surgical debridement/bony resection. In the open fracture case (patient 2), there was vascular perfusion at the fracture site, which did improve more proximally/farther away from the fracture. However, the fracture site did ultimately go on to union. In the nonunion case (patient 3) before debridement/acute shortening, perfusion of bone both proximal and distal to the nonunion was clearly poorer than more proximal bone. After bone resection, the poorly perfused bone proximal to the nonunion was resected; however, the bone distal to the nonunion was improved but still seemed deficient compared with surrounding regions, suggesting that the resection may have been inadequate to remove all poorly perfused bone. The differences between injured or clinically suspicious regions and normal regions for each of the kinetic maps and for all three patients are summarized in Table Decision-making about the extent of bone and soft tissue debridement in the setting of complex orthopaedic surgical conditions such as high-energy open traumatic wounds, infections, and select atrophic nonunions remain routine and challenging intraoperative dilemmas. ICG-based dynamic contrast-enhanced fluorescence imaging has been used for more than 50 years in intraoperative settings to assess vascular perfusion of a wide variety of tissues. In this study, we have translated preclinical ICG fluorescence-based perfusion imaging into human patients. Our preclinical data suggest that ICG-based DCE-FI can effectively and quantitatively assess bone viability in real time. patient , there w,16 Changes in bone blood flow is implicated in morbidity associated with tobacco smoke, osteoporosis, osteoarthritis, and diabetes and in bone infection.–26 In the setting of traumatic injury, damaged blood flow to bone and soft tissue is believed to hinder the delivery of antibiotics, endogenous immune cells as well as growth factors, and other building blocks for healing traumatized tissues and preventing infection. In the setting of open fracture, poorly perfused bone then becomes a nidus for biofilm formation from environmental contamination.Bone blood flow is critical to many processes including growth, bone remodeling, maintenance of bony strength, fracture healing, ingrowth after joint replacement, prevention of infection, and treatment of infection. This has profound clinically oriented applications in the context of traumatic injury in which fracture is believed to disrupt endosteal blood flow, and soft tissue stripping is believed to disrupt periosteal blood flow. This has the potential to provide surgeons with a nuanced view of vascular perfusion parameters in surgical tissues in real time in the operating room. However, more work is needed to define which curve-related variables or vascular perfusion parameters are better predictors of outcomes of interest (such as complication or infection) and where thresholds may be for those parameters. Furthermore, histopathology may be used in future research to validate the perfusion-based findings.ICG-based dynamic contrast-enhanced fluorescence imaging is a powerful imaging method which can be used to better understand several components of vascular perfusion reflected in different kinetic curve–related variables such as rate of ingress (wash-in slope), rate of egress (wash-out slope), time needed to reach peak (blood perfusion), and volume of blood getting to tissues (maximum intensity) Fig. . Further Two studies have demonstrated that after nonunion surgery perfusion assessment using dynamic contrast-enhanced magnetic resonance imaging is associated with treatment failure/persistent nonunion,,29 which highlights the importance of blood flow assessment in this patient population. However, there are critical shortcomings associated with both PET/CT and MR techniques for surgical guidance. PET/CT is time intensive and has substantial issues with poor resolution.,31 High spatial resolution is needed to accurately detect regions of poor vascularity surrounded by regions of higher vascularity. DCE-MRI techniques are limited in orthopaedic patients because of metal artifact/signal dropout in association with metal implants. Furthermore, there is substantial cost associated with PET/CT and DCE-MRI, and neither of these imaging modalities are currently feasible for use in the operating room in real time. Owing to analogous goals regarding improving margins in oncologic resections, surgical oncology is increasingly turning to intraoperative real-time fluorescence-guided surgical techniques, and we believe that there is enormous potential in orthopaedic surgical applications as well.More conventional imaging modalities have been evaluated in this context in a relatively limited way. In a small eight-patient case series, positron emission tomography/CT (PET/CT) was applied to patients with chronic osteomyelitis to inform preoperative planning of debridement.In the patients presented here, we demonstrated that ICG-based DCE-FI demonstrates considerable promise in providing surgeons with an objective measure of bone and soft tissue viability, which has the potential to substantially advance management of severe traumatic injury. We believe that providing surgeons with quantitative objective data on vascular perfusion of surgical bone and soft tissues will result in improved ability to assess and perform effective debridement, which may result in decreased variability in treatment and possibly reduced the rate of complication. The use of current commercial fluorescence imaging devices, such as the SPY presented here, requires minimal training to operate, and the per patient cost of contrast agent is less than $100. Indocyanine green has a very strong safety profile and has been used for more than 50 years to determine hepatic function and cardiac output. The use of DCE-FI may be particularly useful for guiding new and inexperienced orthopaedic surgeons or providing care in more remote and austere settings. However, more research is needed to evaluate those questions and to optimize fluorescence imaging methods for these purposes. Based off these results, a larger clinical trial is underway.