Anxiolytic-like effects of milk proteins.

Milk varieties and specific proteins exhibit anxiolytic-like actions in mice and rats exposed to several tests, the most prominent being the elevated plus-maze. Administrations of αs1-casein, its 91-100 (α-casozepine), 91-97, 91-93, and 91-92 fragments, the 60-69 fragment of ß-casein, lactoferrin, ß-lactotensin, wheylin-1, wheylin-2, and α-lactalbumin have been reported to increase open arm exploration relative to enclosed arm exploration. Anxiolytic-like actions have also been described for 91-93 and 91-92 fragments of αs1-casein, wheylin-1, α-lactalbumin, and lactoferrin in the open-field. Some effects appear to be mediated by the GABAA receptor complex, since antagonists mitigated the anxiolytic-like actions of αs1-casein, the 91-92 fragment of αs1-casein, and wheylin-1. Other neurotransmitters purported to affect such behaviors include 5HT, dopamine, and neurotensin. Further research is needed to identify their neuropharmacological actions.

Tree adaptive growth (TAG) model: a life-history theory-based analytical model for post-thinning forest stand dynamics.

Background: Understanding stand dynamics is essential for predicting future wood supply and associated ecosystem services for sustainable forest management. The dynamics of natural stands can be characterized by age-dependent growth and yield models. However, dynamics in managed stands appear somewhat different from that of natural stands, especially with difficulties in explaining the phenomenon of post-thinning overcompensation, based upon some long-term observations. Though overcompensation is an ideal outcome for the forest sector, it had been largely treated as an outlier and thus ignored or dismissed as "out-of-the-ordinary". Methodology: We developed a life history theory-based, state-dependent model of Tree Adaptive Growth (TAG) to investigate this phenomenon and verified that overcompensation should be a common outcome in post-thinning forest stands when the stand growth over time is sigmoid shaped. TAG posits that individual trees will invest proportionately more into growth following thinning because it is evolutionarily adaptive to do so. Results: Our investigation of the model's behavior unearthed diverse stand growth patterns similar to that which is observed in the empirical datasets and predicted by a statistics-based Tree's Compensatory Growth (TreeCG) model. Conclusion: A simple, theory-driven, analytical model, TAG, can reproduce the diverse growth patterns in post-thinning stands and thus assist addressing silviculture-related issues. The model can be applied to various jurisdictions even without detailed regional growth and yield relationships and is capable of incorporating the effects of other time sensitive factors like fertilization, pruning, and climate change.

The Neuroanatomical Basis of the 5-HT Syndrome and Harmalineinduced Tremor.

The 5-HT syndrome in rats is composed of head weaving, body shaking, forepaw treading, flat body posture, hindlimb abduction, and Straub tail. The importance of the brainstem and spinal cord for the syndrome is underlined by findings of 5,7-dihydroxytryptamine (5,7-DHT)-induced denervation supersensitivity in response to 5-HT-stimulant drugs. For head weaving and Straub tail, supersensitivity occurred when the neurotoxin was injected into the cisterna magna or spinal cord, for forepaw treading in cisterna magna, and for hindlimb abduction in the spinal cord. Although 5,7- DHT-related body shaking increased in the spinal cord, the sign decreased when injected into the striatum, indicating the modulatory influence of the basal ganglia. Further details on body shaking are provided by its reduced response to harmaline after 5-HT depletion caused by intraventricular 5,7-DHT, electrolytic lesions of the medial or dorsal raphe, and lesions of the inferior olive caused by systemic injection of 3-acetylpyridine along with those found in Agtpbp1pcd or nr cerebellar mouse mutants. Yet the influence of the climbing fiber pathway on other signs of the 5-HT syndrome remains to be determined.

pIGLET: Safe harbor landing sites for reproducible and efficient transgenesis in zebrafish.

Standard methods for transgenesis in zebrafish depend on random transgene integration into the genome followed by resource-intensive screening and validation. Targeted vector integration into validated genomic loci using phiC31 integrase-based attP/attB recombination has transformed mouse and Drosophila transgenesis. However, while the phiC31 system functions in zebrafish, validated loci carrying attP-based landing or safe harbor sites suitable for universal transgenesis applications in zebrafish have not been established. Here, using CRISPR-Cas9, we converted two well-validated single insertion Tol2-based zebrafish transgenes with long-standing genetic stability into two attP landing sites, called phiC31 Integrase Genomic Loci Engineered for Transgenesis (pIGLET). Generating fluorescent reporters, loxP-based Switch lines, CreERT2 drivers, and gene-regulatory variant reporters in the pIGLET14a and pIGLET24b landing site alleles, we document their suitability for transgenesis applications across cell types and developmental stages. For both landing sites, we routinely achieve 25-50% germline transmission of targeted transgene integrations, drastically reducing the number of required animals and necessary resources to generate individual transgenic lines. We document that phiC31 integrase-based transgenesis into pIGLET14a and pIGLET24b reproducibly results in representative reporter expression patterns in injected F0 zebrafish embryos suitable for enhancer discovery and qualitative and quantitative comparison of gene-regulatory element variants. Taken together, our new phiC31 integrase-based transgene landing sites establish reproducible, targeted zebrafish transgenesis for numerous applications while greatly reducing the workload of generating new transgenic zebrafish lines.

BDNF and Cerebellar Ataxia.

Brain-derived neurotrophic factor (BDNF) has been proposed as a treatment for neurodegeneration, including diseases of the cerebellum, where BDNF levels or those of its main receptor, TrkB, are often diminished relative to controls, thereby serving as replacement therapy. Experimental evidence indicates that BDNF signaling countered cerebellar degeneration, sensorimotor deficits, or both, in transgenic ATXN1 mice mutated for ataxin-1, Cacna1a knock-in mice mutated for ataxin-6, mice injected with lentivectors encoding RNA sequences against human FXN into the cerebellar cortex, Kcnj6Wv (Weaver) mutant mice with granule cell degeneration, and rats with olivocerebellar transaction, similar to a BDNF-overexpressing transgenic line interbred with Cacng2stg mutant mice. In this regard, this study discusses whether BDNF is effective in cerebellar pathologies where BDNF levels are normal and whether it is effective in cases with combined cerebellar and basal ganglia damage.

Probiotic Influences on Motor Skills: A Review.

The effects of probiotics have mostly been shown to be favorable on measures of anxiety and stress. More recent experiments indicate single- and multi-strain probiotics in treating motorrelated diseases. Initial studies in patients with Parkinson's disease and Prader-Willi syndrome are concordant with this hypothesis. In addition, probiotics improved motor coordination in normal animals and models of Parkinson's disease, multiple sclerosis, and spinal cord injury as well as grip strength in hepatic encephalopathy. Further studies should delineate the most optimal bacterial profile under each condition.

One-trial appetitive learning tasks for drug targeting.

One-trial appetitive learning developed from one-trial passive avoidance learning as a standard test of retrograde amnesia. It consists of one learning trial followed by a retention test, in which physiological manipulations are presented. As in passive avoidance learning, food- or water-deprived rats or mice finding food or water inside an enclosure are vulnerable to the retrograde amnesia produced by electroconvulsive shock treatment or the injection of various drugs. In one-trial taste or odor learning conducted in rats, birds, snails, bees, and fruit flies, there is an association between a food item or odorant and contextual stimuli or the unconditioned stimulus of Pavlovian conditioning. The odor-related task in bees was sensitive to protein synthesis inhibition as well as cholinergic receptor blockade, both analogous to results found on the passive avoidance response in rodents, while the task in fruit flies was sensitive to genetic modifications and aging, as seen in the passive avoidance response of genetically modified and aged rodents. These results provide converging evidence of interspecies similarities underlying the neurochemical basis of learning.

Lateral thinking in syndromic congenital cardiovascular disease.

Syndromic birth defects are rare diseases that can present with seemingly pleiotropic comorbidities. Prime examples are rare congenital heart and cardiovascular anomalies that can be accompanied by forelimb defects, kidney disorders and more. Whether such multi-organ defects share a developmental link remains a key question with relevance to the diagnosis, therapeutic intervention and long-term care of affected patients. The heart, endothelial and blood lineages develop together from the lateral plate mesoderm (LPM), which also harbors the progenitor cells for limb connective tissue, kidneys, mesothelia and smooth muscle. This developmental plasticity of the LPM, which founds on multi-lineage progenitor cells and shared transcription factor expression across different descendant lineages, has the potential to explain the seemingly disparate syndromic defects in rare congenital diseases. Combining patient genome-sequencing data with model organism studies has already provided a wealth of insights into complex LPM-associated birth defects, such as heart-hand syndromes. Here, we summarize developmental and known disease-causing mechanisms in early LPM patterning, address how defects in these processes drive multi-organ comorbidities, and outline how several cardiovascular and hematopoietic birth defects with complex comorbidities may be LPM-associated diseases. We also discuss strategies to integrate patient sequencing, data-aggregating resources and model organism studies to mechanistically decode congenital defects, including potentially LPM-associated orphan diseases. Eventually, linking complex congenital phenotypes to a common LPM origin provides a framework to discover developmental mechanisms and to anticipate comorbidities in congenital diseases affecting the cardiovascular system and beyond.

A median fin derived from the lateral plate mesoderm and the origin of paired fins.

The development of paired appendages was a key innovation during evolution and facilitated the aquatic to terrestrial transition of vertebrates. Largely derived from the lateral plate mesoderm (LPM), one hypothesis for the evolution of paired fins invokes derivation from unpaired median fins via a pair of lateral fin folds located between pectoral and pelvic fin territories1. Whilst unpaired and paired fins exhibit similar structural and molecular characteristics, no definitive evidence exists for paired lateral fin folds in larvae or adults of any extant or extinct species. As unpaired fin core components are regarded as exclusively derived from paraxial mesoderm, any transition presumes both co-option of a fin developmental programme to the LPM and bilateral duplication2. Here, we identify that the larval zebrafish unpaired pre-anal fin fold (PAFF) is derived from the LPM and thus may represent a developmental intermediate between median and paired fins. We trace the contribution of LPM to the PAFF in both cyclostomes and gnathostomes, supporting the notion that this is an ancient trait of vertebrates. Finally, we observe that the PAFF can be bifurcated by increasing bone morphogenetic protein signalling, generating LPM-derived paired fin folds. Our work provides evidence that lateral fin folds may have existed as embryonic anlage for elaboration to paired fins.

Functional comparison of human ACVR1 and zebrafish Acvr1l FOP-associated variants in embryonic zebrafish.

BACKGROUND: Fibrodysplasia ossificans progressiva (FOP), a rare disease characterized by progressive heterotopic ossification of muscle and connective tissues, is caused by autosomal dominant activating mutations in the type I receptor, ACVR1/ALK2. The classic human FOP variant, ACVR1R206H , shows increased bone morphogenetic protein (BMP) signaling and activation by activins. RESULTS: Here, we performed in vivo functional characterization of human ACVR1R206H and orthologous zebrafish Acvr1lR203H using early embryonic zebrafish dorsoventral patterning as a phenotypic readout for receptor activity. Our results showed that human ACVR1R206H and zebrafish Acvr1lR203H exhibit functional differences in early embryonic zebrafish, and that human ACVR1R206H retained its signaling activity in the absence of a ligand-binding domain (LBD). We also showed, for the first time, that zebrafish Acvr2ba/Acvr2bb receptors are required for human ACVR1R206H signaling in early embryonic zebrafish. CONCLUSIONS: Together, these data provide new insight into ACVR1R206H signaling pathways that may facilitate the design of new and effective therapies for FOP patients.

Neurobehavioral basis of Maier 3-table and other matching-to-place tasks.

The Maier 3-table task comprises three phases conducted each day. During the exploration phase, rats explore the entire apparatus. During the information phase, the rats are placed on one of the three tables where food is found. During the test phase, the animals are placed at the starting point on one of the two remaining tables and must enter the goal table where they previously ate. The acquisition of the Maier 3-table task was slowed down after lesions of the septum, fornix, hippocampus, medial prefrontal cortex, or posterior parietal cortex. Because of its time-consuming nature, the Maier 3-table task has more recently been superseded by appetitive matching-to-place in Y- or T-mazes or the circular water maze, because experimenters skip over the exploration phase. Nevertheless, like the Maier 3-table task, the acquisition of the Y- or T-maze matching-to-place task was retarded after lesions of the medial septum or medial prefrontal cortex, more particularly its prelimbic-infralimbic part. Like the previous task, the water-maze version is sensitive to lesions of the medial septum or retrosplenial cortex. Despite methodological differences between the three procedures, these results indicate common neurobiological bases of matching-to-place learning.

Cocoa Flavanols and the Aging Brain.

Partly because of its antioxidant and anti-inflammatory properties, cocoa flavanols have been examined in reversing age-related cognitive deficits. Epidemiological studies indicate a relation between flavonoid intake and the prevention of dementia. In confirmation of this relation, several pharmacological studies show the faster speed of responding and better executive performance in flavanol-treated aged or young subjects. The lack of any effect appears in some studies, especially in young subjects, perhaps due to the use of groups with high educational levels and the possibility of a ceiling effect. In several studies, neuropsychological ameliorations were followed by increases in cerebral blood flow. These results are in line with those of animal experimentation since improvements have been found in motor and spatial performances of young and aging mice or rats as well as animal models of Alzheimer's disease and Parkinson's disease. Improvements are also reported in biologic markers of Alzheimer's disease, in particular an increase in soluble Aß and a decrease in tau hyperphosphorylation.

The DST gene in neurobiology.

DST is a gene whose alternative splicing yields epithelial, neuronal, and muscular isoforms. The autosomal recessive Dstdt (dystonia musculorum) spontaneous mouse mutation causes degeneration of spinocerebellar tracts as well as peripheral sensory nerves, dorsal root ganglia, and cranial nerve ganglia. In addition to Dstdt mutants, axonopathy and neurofilament accumulation in perikarya are features of two other murine lines with spontaneous Dst mutations, targeted Dst knockout mice, DstTg4 transgenic mice carrying two deleted Dst exons, DstGt mice with trapped actin-binding domain-containing isoforms, and conditional Schwann cell-specific Dst knockout mice. As a result of nerve damage, Dstdt mutants display dystonia and ataxia, as seen in several genetically modified models and their motor coordination deficits have been quantified along with the spontaneous Dst nonsense mutant, the conditional Schwann cell-specific Dst knockout, the conditional DstGt mutant, and the Dst-b isoform specific Dst mutant. Recent findings in humans have associated DST mutations of the Dst-b isoform with hereditary sensory and autonomic neuropathies type 6 (HSAN-VI). These data should further encourage the development of genetic techniques to treat or prevent ataxic and dystonic symptoms.

Modelling the stand dynamics after a thinning induced partial mortality: A compensatory growth perspective.

Introduction: With increasing forest areas under management, dynamics of managed stands have gained more attention by forest managers and practitioners. Improved understanding on how trees and forest stands would respond to different disturbances is required to predict the dynamics of managed stand.s. Partial mortality commonly occurs in stand development, and different response patterns of trees and stands to partial mortality would govern stand dynamics. Methods: To investigate the possible response patterns using existing knowledge of growth and yield relationships, we developed TreeCG model, standing for Tree's Compensatory Growth, a state-dependent individual tree-based forest growth model that simulates the compensatory growth of trees after experiencing a partial mortality. The mechanism behind the simulation is the redistribution of resources, including nutrients and space, freed from died trees to surviving trees. The developed new algorithm simplified the simulations of annual growth increments of individual trees over a long period of stand development. Results: The model was able to reproduce the forest growth patterns displayed in long-term precommercial thinning experiments. The simulated forest growth displayed the process of compensatory growth from under compensation, to compensation-induced-equality, and to overcompensation over time. Discussion: Our model can simulate stand growth trajectories after different partial harvest regimes at different times and intensities, thus support decisions in best partial harvest strategies. This generic model can be refined with given tree species and specific site conditions to predict stand dynamics after given partial mortality for any jurisdictions under management. The simulation reassembles growth trajectories of natural stands when no thinning is conducted.

Functional Testing of Bone Morphogenetic Protein (BMP) Pathway Variants Identified on Whole-Exome Sequencing in a Patient with Delayed-Onset Fibrodysplasia Ossificans Progressiva (FOP) Using ACVR1R206H -Specific Human Cellular and Zebrafish Models.

Bone morphogenetic protein (BMP) signaling is critical in skeletal development. Overactivation can trigger heterotopic ossification (HO) as in fibrodysplasia ossificans progressiva (FOP), a rare, progressive disease of massive HO formation. A small subset of FOP patients harboring the causative ACVR1R206H mutation show strikingly mild or delayed-onset HO, suggesting that genetic variants in the BMP pathway could act as disease modifiers. Whole-exome sequencing of one such patient identified BMPR1AR443C and ACVR2AV173I as candidate modifiers. Molecular modeling predicted significant structural perturbations. Neither variant decreased BMP signaling in ACVR1R206H HEK 293T cells at baseline or after stimulation with BMP4 or activin A (AA), ligands that activate ACVR1R206H signaling. Overexpression of BMPR1AR443C in a Tg(ACVR1-R206Ha) embryonic zebrafish model, in which overactive BMP signaling yields ventralized embryos, did not alter ventralization severity, while ACVR2AV173I exacerbated ventralization. Co-expression of both variants did not affect dorsoventral patterning. In contrast, BMPR1A knockdown in ACVR1R206H HEK cells decreased ligand-stimulated BMP signaling but did not affect dorsoventral patterning in Tg(ACVR1-R206Ha) zebrafish. ACVR2A knockdown decreased only AA-stimulated signaling in ACVR1R206H HEK cells and had no effect in Tg(ACVR1-R206Ha) zebrafish. Co-knockdown in ACVR1R206H HEK cells decreased basal and ligand-stimulated signaling, and co-knockdown/knockout (bmpr1aa/ab; acvr2aa/ab) decreased Tg(ACVR1-R206Ha) zebrafish ventralization phenotypes. Our functional studies showed that knockdown of wild-type BMPR1A and ACVR2A could attenuate ACVR1R206H signaling, particularly in response to AA, and that ACVR2AV173I unexpectedly increased ACVR1R206H -mediated signaling in zebrafish. These studies describe a useful strategy and platform for functionally interrogating potential genes and genetic variants that may impact the BMP signaling pathway. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Heterogeneity and genomic loci of ubiquitous transgenic Cre reporter lines in zebrafish.

BACKGROUND: The most-common strategy for zebrafish Cre/lox-mediated lineage labeling experiments combines ubiquitously expressed, lox-based Switch reporter transgenes with tissue-specific Cre or 4-OH-Tamoxifen-inducible CreERT2 driver lines. Although numerous Cre driver lines have been produced, only a few broadly expressed Switch reporters exist in zebrafish and their generation by random transgene integration has been challenging due to position-effect sensitivity of the lox-flanked recombination cassettes. Here, we compare commonly used Switch reporter lines for their recombination efficiency and reporter expression pattern during zebrafish development. RESULTS: Using different experimental setups, we show that ubi:Switch and hsp70l:Switch outperform current generations of the two additional Switch reporters actb2:BFP-DsRed and actb2:Stop-DsRed. Our comparisons also document preferential Cre-dependent recombination of ubi:Switch and hsp70l:Switch in distinct zebrafish tissues at early developmental stages. To investigate what genomic features may influence Cre accessibility and lox recombination efficiency in highly functional Switch lines, we mapped these transgenes and charted chromatin dynamics at their integration sites. CONCLUSIONS: Our data documents the heterogeneity among lox-based Switch transgenes toward informing suitable transgene selection for lineage labeling experiments. Our work further proposes that ubi:Switch and hsp70l:Switch define genomic integration sites suitable for universal transgene or switch reporter knock-in in zebrafish.

Detecting Compensatory Growth in Silviculture Trials: Empirical Evidence From Three Case Studies Across Canada.

Compensatory growth (CG) appears common in biology and is defined as accelerated growth after experiencing a period of unfavorable conditions. It usually leads to an increase in biomass that may eventually equal or even surpass that of sites not experiencing disturbance. In forestry, with sufficient time the stand volume lost in a disturbance such as a thinning operation could match or even exceed those from undisturbed sites, respectively called exact and overcompensation. The forest sector could benefit from enhanced productivity and associated ecosystem services such as carbon storage through overcompensation. Therefore, detection of CG in different types of forests becomes important for taking advantage of it in forest management. However, compensatory growth has not been reported widely in forestry, partially due to the paucity of long-term observations and lack of proper indicators. Legacy forest projects can provide a suitable data source, though they may be originally designed for other purposes. Three case studies representing different data structures of silviculture trials are investigated to evaluate if compensatory growth is common in forest stands. Our results showed that compensatory growth occurred in all three cases, and thus suggested that the compensatory growth might indeed be common in forest stands. We found that the relative growth (RG) can serve as a universal indicator to examine stand-level compensatory growth in historical long-term silviculture datasets. When individual tree-based measurements are available, both volume and value-based indicators can be used in detecting compensatory growth, and lumber value-based indicators could be more sensitive in detecting overcompensation.

Probiotic effects on anxiety-like behavior in animal models.

Gut microbiota have been shown to be useful in treating gastrointestinal diseases, cancer, obesity, infections, and, more recently, neuropsychiatric conditions such as degenerative diseases and depression. There has also been recent expansion in testing probiotics and prebiotics on anxiety-like behaviors in animals. Current results indicate that probiotic substances of the Lactobacillus and Bifidobacterium type are effective in reducing anxiety-like behaviors in mice or rats evaluated in the elevated plus-maze, the open-field, the light-dark box, and conditioned defensive burying. Probiotics are also effective in reducing serum or plasma corticosterone levels after acute stress. It is hypothesized that probiotics cause anxiolytic-like effects via vagal influences on caudal solitary nucleus, periaqueductal gray, central nucleus of the amygdala, and bed nucleus of the stria terminalis. Further experimentation is needed to trace the neurochemical anatomy underlying anxiolytic-like behaviors of gut microbiata exerting effects via vagal or nonvagal pathways.

The Hole-Board Test in Mutant Mice.

First described by Boissier and Simon in (Ther Recreat J 17:1225-1232, 1962), the hole-board has become a recognized test of anxiety and spatial memory. Benzodiazepines acting at the GABAA-BZD site increase hole-pokes in rats and mice, indicating a loss in behavioral inhibition concordant with the behavior of mutant mice deficient in the GABA transporter. Hole-poking also depends on arousal mechanisms dependent on dopaminergic transmission, as indicated by drug and null mutant studies. In addition, the behavior is modified in natural and null mutants affecting the cerebellum as well as null mutants affecting neuropeptides, growth factors, cell adhesion, and inflammation. Further research is required to determine convergences between genetic and pharmacological effects.

Intrahepatic Injection of Sodium Pentobarbital as an Alternative to Intraperitoneal Injection for the Euthanasia of Rats (Rattus norvegicus).

The most commonly accepted method of rat euthanasia in North America is intraperitoneal injection of sodium pentobarbital (PB). However, misinjection can occur, and intraperitoneal PB may cause pain and distress. The objective of this study was to test an alternative method of euthanasia: intrahepatic injection of PB. A pilot study was conducted to develop a method of intrahepatic injections (evaluated using CT scans and test injections), followed by a full study comparing intraperitoneal (n = 14) and intrahepatic PB injections (n = 66) in adult rats. Full study outcomes were: 1) time from injection to loss of right- ing reflex (LORR), 2) time from injection to cessation of heartbeat (CHB), 3) number of failed euthanasia attempts, and 4) confirmation of successful intrahepatic injection or misinjection via necropsy. All injections were performed by a veterinary student. CT revealed that intrahepatic injections were feasible. Times (median [range]) to LORR and CHB were faster after successful intrahepatic injections (LORR, 3 s [1 to 5 s]; CHB, 8 s [2 to 242 s]) than after intraperitoneal injections (LORR, 89.5 s [73 to 110 s], CHB: 284.5 s [237 to 423 s]). The misinjection rate was higher with intrahepatic injections (59%) than with intraperitoneal injections (29%), but intrahepatic misinjection still resulted in fast and successful euthanasia (LORR, 29 s [1 to 96 s]; CHB, 216 s [12 to 330 s]), with the injectate distributed between the intraperitoneal and intrahepatic locations. The number of failed euthanasia attempts with intrahepatic injections was low (n = 2). Intrahepatic injections show potential as an alternative to intraperitoneal injections for rat euthanasia.