This or not that: select and reject control of relational responding in rats using a blank comparison procedure with odor stimuli.

The blank comparison (BLC) task was developed to assess stimulus relations in discrimination learning; that is, are subjects learning to "select" the correct stimulus (S+) or "reject" the incorrect stimulus (S-) or both? This task has been used to study exclusion learning, mostly in humans and monkeys, and the present study extends the procedure to rats. The BLC task uses an ambiguous stimulus (BLC+/-) that replaces S+ (in the presence of S-) and replaces S- (in the presence of S+). In the current experiment, four rats were trained to remove session-novel scented lids from sand-filled cups in a two-choice, simultaneous presentation procedure called the Odor Span Task (OST) before being trained on the BLC procedure using odors as the discriminative stimuli. The BLC training procedure utilized simple discrimination training (S+ and S-) and added select (S+ and BLC-) and reject (BLC+ and S-) trial types. All rats demonstrated accurate performance in sessions with both select and reject type trials. Next, BLC probe trials were interspersed in standard OST sessions to assess the form of stimulus control in the OST. Rats performed accurately on select type probe trials (similar to baseline OST performance) and also showed above chance accuracy on reject type trials. Thus, we demonstrated that rats could acquire an odor-based version of the BLC task and that both select and exclusion-based (reject) relations were active in the OST. The finding of exclusion in rats under the rigorous BLC task conditions confirms that exclusion-based responding is not limited to humans and non-human primates.

Generalized, cross-modal, and incrementing non-matching-to-sample in rats.

Same/different concept learning has been demonstrated in previous research in rats using matching- and non-matching-to-sample procedures with olfactory stimuli. In Experiment 1, rats were trained on the non-matching-to-sample procedure with either three-dimensional (3D plastic objects; n = 3) or olfactory (household spices, n = 5) stimuli, then tested for transfer to novel stimuli of the same, and then the alternate, modality. While all three rats trained with olfactory stimuli showed generalized non-matching to novel odors, only one rat learned the 3D relation and showed generalized transfer to novel objects. Importantly, in this rat the 3D non-matching relation then immediately transferred to odors. In contrast, rats trained with scents did not show transfer to novel 3D stimuli until after training with one or two 3D stimulus sets. In Experiment 2, four rats were trained on an incrementing non-matching-to-sample task featuring 3D plastic objects as stimuli (3D Span Task). Responses to session-novel stimuli resulted in reinforcement. Only two rats learned the 3D Span Task; one rat performed with high accuracy even with up to 17 session-novel objects in a session. While these findings emphasize the exceptional olfactory discrimination of rats relative to that with 3D/tactile/visual cues, they also show that relational learning can be demonstrated in another modality in this species. Further, the present study provides some evidence of cross-modal transfer of relational responding in rats.

Functional equivalence classes in rats: Repeated olfactory discrimination reversals and delayed stimulus probes.

The simultaneous matching-to-sample procedures that are widely used to study stimulus equivalence in human participants have generally been unsuccessful in animals. However, functional equivalence classes have been demonstrated in pigeons and sea lions using a concurrent repeated reversal discrimination procedure. In this procedure, responding to one set of stimuli is reinforced but responding to a different set is not and the set associated with reinforcement is changed with multiple reversals during the experiment. The experiments reported here were designed to assess whether functional equivalence classes could be demonstrated in rats using similar techniques. Rats were initially trained with two sets of olfactory stimuli (six odors/set). Following many reversals, probe reversal sessions were conducted in which rats were exposed to a subset of the members of each set and, later in the session, the withheld stimuli were introduced. Responding to these delayed probe trials in accord with the reversed contingencies constituted transfer of function. There was some evidence of transfer in Experiment 1, but the effects were relatively weak and variable. Experiment 2 introduced procedural changes and found strong evidence of transfer of function consistent with the formation of functional equivalence classes. These procedures offer a promising strategy to study symbolic behavior in rodents.

Engineering pre-vascularized bone-like tissue from human mesenchymal stem cells through simulating endochondral ossification.

Currently, most in vitro engineered bone tissues do not contain viable blood vessel systems, so the vascularization depends on post-implantation angiogenesis from the host, which is often insufficient for repairing large bone defects. In this study, we aimed to create pre-vascularized bone-like tissue from human bone marrow-derived mesenchymal stem cells (HBMSCs) within the self-generated extracellular matrix by simulating the developmental endochondral ossification. Afterward, a three-dimensional (3D) culture of human umbilical vein endothelial cells (HUVECs)/HBMSCs was introduced to cover bone-like constructs surface for vascularization. Lastly, the pre-vascularized bone-like tissues were subcutaneously implanted into mice and the quality of newly formed blood vessels and bones were later assessed. We particularly examined whether the pre-existing HUVECs/HBMSCs vascular networks within the implants were able to integrate with the host's blood vessels and facilitate bone formation. Our results showed that this developmentally informed procedure resulted in a robust osteogenic differentiation of HBMSCs. Moreover, the bone-like constructs markedly promoted HUVEC/HBMSCs network formation in vitro. After 28 days of implantation in mice, the experimental group, in which bone-like constructs were pre-vascularized with HUVEC/HBMSCs networks, exhibited significantly more functional blood vessels than the control group that contained HUVEC and HBMSC single cells. Interestingly, increased levels of bone formation and absorption markers were also observed in the pre-vascularized bone-like constructs. Taken together, these findings demonstrated the potential of pre-vascularized bone-like constructs in repairing bone defects.

Incrementing non-matching- but not matching-to-sample is rapidly learned in an automated version of the odor span task.

The odor span task (OST) is frequently used to assess memory capacity in rodents. Odor stimuli are presented in a large arena and choices of session-novel odors produce food reward. The procedure can be described as an incrementing non-matching-to-sample contingency because on each trial one new stimulus is presented along with one or more previously presented (non-reinforced) comparison odors. An automated version of this task has recently been developed in which odors are presented with an olfactometer in an operant chamber using a successive conditional discrimination procedure. The present study compared the acquisition of matching- vs. non-matching-to-sample versions of the task with six rats tested under each procedure. All six rats trained on the non-matching variation showed rapid acquisition of the discrimination with high rates of responding to odor stimuli when they were session-novel and low rates of responding to subsequent presentations of those odors. However, only three of the six rats trained on the matching variation met acquisition criteria, and two of the three that did acquire the task required extensive training to do so. These results support findings from the OST that rats can differentiate between stimuli that are session-novel and those previously encountered, but also that a matching contingency is more difficult to learn than a non-matching arrangement. These findings parallel differences observed between acquisition of simple matching- and non-matching-to-sample tasks, but accounts such as novelty preference or the oddity preference effect may not be sufficient to explain the present results.

Repeated reversals of concurrent olfactory discriminations in rats: A search for functional equivalence.

Equivalence class formation has been difficult to demonstrate in nonhumans, but one method that has been successful is a simple discrimination procedure in which contingencies associated with two sets of arbitrary discriminative stimuli are repeatedly reversed. Pigeons and sea lions shift responding after encountering the newly-reversed contingency with only a few set members, showing evidence of functional equivalence. We used this strategy to determine whether similar findings would occur in rats using olfactory stimuli. Rats were trained to nose-poke in the presence of six stimuli arbitrarily designated as members of the positive set; responses to the six members of the negative set were not reinforced. When discriminative performance was established, contingencies associated with each set were reversed and re-reversed each time subjects met a performance criterion. All subjects successfully acquired the concurrent simple discriminations and were exposed to between 12 and 60 reversals, but none showed clear evidence of functional class formation until a final procedure in which the stimulus sets that had been in place were arbitrarily rearranged. Acquisition with these new stimulus sets was impaired, showing that class membership generated by the original stimulus sets interfered with learning the new ones, thus providing evidence of functional equivalence.

Graphene oxide-functionalized nanocomposites promote osteogenesis of human mesenchymal stem cells via enhancement of BMP-SMAD1/5 signaling pathway.

Biomaterials that can harness the intrinsic osteogenic potential of stem cells offer a promising strategy to accelerate bone regeneration and repair. Previously, we had used methacrylated gelatin (GelMA)-based scaffolds to achieve bone formation from human mesenchymal stem cells (hMSCs). In this study, we aimed to further enhance hMSC osteogenesis by incorporating graphene oxide (GO)-based nanosheets into GelMA. In vitro results showed high viability and metabolic activities in hMSCs encapsulated in the newly developed nanocomposites. Incorporation of GO markedly increased mineralization within hMSC-laden constructs, which was further increased by replacing GO with silica-coated graphene oxide (SiGO). Mechanistic analysis revealed that the nanosheet enhanced the production, retention, and biological activity of endogenous bone morphogenetic proteins (BMPs), resulting in robust osteogenesis in the absence of exogenous osteoinductive growth factors. Specifically, the osteoinductive effect of the nanosheets was abolished by inhibiting the BMP signaling pathway with LDN-193189 treatment. The bone formation potential of the technology was further tested in vivo using a mouse subcutaneous implantation model, where hMSCs-laden GO/GelMA and SiGO/GelMA samples resulted in bone volumes 108 and 385 times larger, respectively, than the GelMA control group. Taken together, these results demonstrate the biological activity and mechanism of action of GO-based nanosheets in augmenting the osteogenic capability of hMSCs, and highlights the potential of leveraging nanomaterials such as GO and SiGO for bone tissue engineering applications.

Successive incrementing non-matching-to-samples in rats: An automated version of the odor span task.

The odor span task is a procedure frequently used to study remembering of multiple stimuli in rodents. A large arena is used and odor stimuli are presented using scented cups. Selection of each odor is reinforced when first presented, but not on subsequent presentations; correct selections depend on remembering which stimuli were previously presented. The use of an arena setting with manual stimulus presentation makes the odor span task labor-intensive and limits experimental control; thus, an automated version of the task would be of value. The present study used an operant chamber equipped with an olfactometer and trained rats using successive conditional discrimination procedures under an incrementing non-matching-to-samples contingency. High rates of responding developed to odor stimuli when they were session-novel with low rates of responding to subsequent presentations of that odor. Additional experiments assessed variations of the procedure to determine the role of the frequency of odor presentation and the retention interval separating sample and comparison. Discrimination was impaired with long retention intervals suggesting the importance of this variable. These findings confirmed that rats differentiate between stimuli that are session-novel and those previously encountered and support the use of an automated procedure as an alternative to the odor span task.

Effects of set size on identity and oddity abstract-concept learning in rats.

Match (MTS) and non-match-to-sample (NMTS) procedures are used to assess concepts of identity and oddity across species and are measured by transfer performance to novel stimuli. The number of exemplars used in training (set size) has been shown to affect learning with evidence of larger set sizes promoting concept learning in several species. The present study explored the effects of set size and procedure on concept learning in rats using olfactory stimuli. Concept learning was assessed for 20 rats via transfer tests consisting of novel stimuli after rats were initially trained to either MTS or NMTS with two or ten stimuli as exemplars. No difference was found in acquisition or transfer between MTS and NMTS, but rats trained with ten stimuli performed better on novel transfer tests than rats trained with two. When set size was expanded for rats originally trained with two stimuli and rats were re-tested with ten novel stimuli, performance showed full transfer demonstrating that training with multiple exemplars facilitates concept learning.

Effects of NMDA antagonist dizocilpine (MK-801) are modulated by the number of distractor stimuli in the rodent odor span task of working memory.

The rodent odor span task (OST) uses an incrementing non-matching to sample procedure in which a series of odors is presented and selection of the session-novel odor is reinforced. An OST is frequently used to test the effects of neurobiological variables on memory capacity as the number of odors to remember increases during the course of the session. In this regard, one important finding has been that NMDA receptor antagonists selectively impair OST performance at doses that spare accuracy on control tasks. However, in many versions of the odor span task the number of stimuli to remember is confounded with the number of distractor odors presented to the rat on each trial. The present study compared the effects of the NMDA antagonist dizocilpine when the number of choices was held constant at two (one novel odor-S+ and one previously presented distractor odor-S-) and when the number of choice stimuli was permitted to increase up to 10 (one S+ and 9 S-). Dizocilpine impaired OST accuracy at doses that had no effect on a reference memory control task in both 2-choice and 10-choice conditions; however, the dose-response function was shifted to the left in the 10-choice tests. The impairments produced by dizocilpine were exacerbated as the memory load increased in both 2- and 10-choice conditions. These findings support the hypothesis that NMDA antagonism reduces the number of stimuli that rats can remember accurately, but the interaction between the effective DZP dose and the number of distractors shows that drug effects on OST performances may involve attentional factors in addition to memory capacity. The findings also demonstrate that variations in number of OST distractors can be used to alter sensitivity of the task.

Generalized identity in a successive matching-to-sample procedure in rats: Effects of number of exemplars and a masking stimulus.

Two experiments examined the emergence of generalized identity matching in rats using a successive discrimination procedure with olfactory stimuli. Trials consisted of the presentation of two odors separated by a 1-s interstimulus interval. Responses during the second odor presentation were reinforced only if the two odors were identical. In Experiment 1, rats were trained with two odors and then exposed to sessions that included unreinforced probe trials with novel odors. There was evidence of higher response rates on matching probe trials in some rats, but matching did not approach baseline levels. Additional training with four exemplars produced transfer to novel odors that was equivalent to baseline levels. Experiment 2 tested the possibility that detection of stimulus change, rather than generalized identity, was responsible for the transfer seen in Experiment 1. Thus, a masking odor was inserted during the 1-s interstimulus interval so that stimulus change occurred on all trials. Although response rates on probe trials were lower than baseline rates, above chance transfer to novel stimuli was still observed in four of the five animals tested. These findings support the hypothesis that transfer of matching to novel odors in this successive matching-to-sample paradigm is based on a generalized identity relation.

Abstraction, Multiple Exemplar Training and the Search for Derived Stimulus Relations in Animals.

Symmetry and other derived stimulus relations are readily demonstrated in humans in a variety of experimental preparations. Comparable emergent relations are more difficult to obtain in other animal species and seem to require certain specialized conditions of training and testing. This article examines some of these conditions with an emphasis on what animal research may be able to tell us about the nature and origins of derived stimulus relations. We focus on two areas that seem most promising: 1) research generated by Urcuioli's (2008) theory of the conditions necessary to produce symmetry in pigeons, and 2) research that explores the effects of multiple exemplar training on emergent relations. Urcuioli's theory has successfully predicted emergent relations in pigeons by taking into account their apparent difficulty in abstracting the nominal training stimulus from other stimulus properties such as location and temporal position. Further, whereas multiple exemplar training in non-humans has not consistently yielded arbitrarily-applicable relational responding, there is a growing body of literature showing that it does result in abstracted same-different responding. Our review suggests that although emergent stimulus relations demonstrated in non-humans at present have not yet shown the flexibility or generativity apparent in humans, the research strategies reviewed here provide techniques that may permit the analysis of the origins of derived relational responding.

Successive odor matching- and non-matching-to-sample in rats: A reversal design.

There is a growing body of research on matching- and non-matching-to-sample (MTS, NMTS) relations with rats using olfactory stimuli; however, the specific characteristics of this relational control are unclear. In the current study we examine MTS and NMTS in rats with an automated olfactometer using a successive (go, no-go) procedure. Ten rats were trained to either match- or non-match-to-sample with common scents (apple, cinnamon, etc.) as olfactory stimuli. After matching or non-matching training with four odorants, rats were tested for transfer twice with four new odorants on each test. Most rats trained on MTS showed immediate transfer to new stimuli, and most rats trained on NMTS showed full transfer by the second set of new odors. After meeting criterion on the second transfer test, the contingencies were reversed with four new odor stimuli such that subjects trained on matching were shifted to non-matching and vice versa. Following these reversed contingencies, the effects of the original training persisted for many trials with new odorants. These data extend previous studies on same-different concept formation in rats, showing strong generalization requiring few exemplars. The critical role of olfactory stimuli is discussed.

Behavioral pharmacology of the odor span task: Effects of flunitrazepam, ketamine, methamphetamine and methylphenidate.

The Odor Span Task is an incrementing non-matching-to-sample procedure that permits the study of behavior under the control of multiple stimuli. Rats are exposed to a series of odor stimuli and selection of new stimuli is reinforced. Successful performance thus requires remembering which stimuli have previously been presented during a given session. This procedure has been frequently used in neurobiological studies as a rodent model of working memory; however, only a few studies have examined the effects of drugs on performance in this task. The present experiments explored the behavioral pharmacology of a modified version of the Odor Span Task by determining the effects of stimulant drugs methylphenidate and methamphetamine, NMDA antagonist ketamine, and positive GABAA modulator flunitrazepam. All four drugs produced dose-dependent impairment of performances on the Odor Span Task, but for methylphenidate and methamphetamine, these occurred only at doses that had similar effects on performance of a simple odor discrimination. Generally, these disruptions were based on omission of responding at the effective doses. The effects of ketamine and flunitrazepam were more selective in some rats. That is, some rats tested under flunitrazepam and ketamine showed decreases in accuracy on the Odor Span Task at doses that did not affect simple discrimination performance. These selective effects indicate disruption of within-session stimulus control. Overall, these findings support the potential of the Odor Span Task as a baseline for the behavioral pharmacological analysis of remembering.

Variability in orthopedic surgeon treatment preferences for nondisplaced scaphoid fractures: A cross-sectional survey.

PURPOSE: The absence of a best practice treatment standard contributes to clinical variation in medicine. Often in the absence of evidence, a standard of care is developed and treatment protocols are implemented. The purpose of this study was to examine whether the standard of care for the treatment of nondisplaced scaphoid fractures is uniform among orthopedic surgeons. METHODS: A survey of orthopedic surgeons actively practicing in the US or abroad was conducted to elicit preferred treatment strategies for nondisplaced scaphoid fractures. The surgeons were recruited at orthopedic conferences, clinical visits, and via email. The survey included demographic questions along with a short clinical vignette. The option for fracture management included surgical versus nonsurgical treatment. For those who chose nonsurgical treatment, type/duration of immobilization was recorded. Cost analysis was performed to estimate direct and indirect costs of various treatment options. RESULTS: A total of 494 orthopedic surgeons completed the survey. The preference for surgical treatment was preferred in 13% of respondents. Hand/upper extremity specialists were significantly more likely to operate compared with generalists (p = 0.0002). Surgeons younger than forty-five were nearly twice as likely to choose surgery (p = 0.01). There was no clear consensus on duration of immobilization as 30% of surgeons chose 6 weeks, 33% selected 8 weeks, and 27% opted for 12 weeks. Total cost of surgery was 49% greater than that of nonoperative treatment. With each additional week of immobilization for nonoperative treatment, the total costs of surgical treatment near that of nonoperative treatment. CONCLUSION: There exist clear trends in how specific demographic groups choose to treat the nondisplaced scaphoid fracture. Whether these trends are the result of generational gaps or additional subspecialty training remains difficult to determine, but there is need to pursue a more consistent approach that benefits the patients and the health care system as a whole.

Lack of Agreement on Distal Radius Fracture Treatment.

INTRODUCTION: Variation in clinical practice resulting from the absence of evidence-based treatment protocols has negative implications on both the cost and the quality of medical care. The objective of this study was to assess whether a standard of care for the treatment of extra-articular nondisplaced distal radius fracture has developed despite the lack of a conclusive recommendation from the American Academy of Orthopaedic Surgeons. METHODS: A case-vignette survey was conducted. Treatment type and duration of casting selections were analyzed. The cost implications of responses were assessed. Participants were practicing orthopedists primarily in the mid-Atlantic region of the United States. Orthopedists (n = 494) were recruited via E-mail and at the American Academy of Orthopaedic Surgeons Annual Meeting held in Chicago in March 2013. Inclusion criteria required that participants be graduates of an accredited medical school and be practicing orthopedists at the time of survey distribution. The main outcome measure was surgical or nonsurgical intervention. RESULTS: Nonsurgical treatment was selected by 60% of respondents, with surgery preferred by 37%. Duration of casting responses varied from 2 to 12 weeks. Among nonsurgical responses, 69% indicated 6 weeks as their preferred duration of casting (95% confidence interval, 64.9-73.1%). Surgery imposes a 76% greater total cost to society than nonsurgical treatments. CONCLUSIONS: Our findings suggest the absence of a consensus strategy for the treatment of extra-articular nondisplaced distal radius fractures. Implications of variance in treatment on cost and quality support the need for established, evidence-based guidelines or further clinical trials to assist in the management of this common fracture.

Emergent identity but not symmetry following successive olfactory discrimination training in rats.

The search for symmetry in nonhuman subjects has been successful in recent studies in pigeons (e.g., Urcuioli, 2008). The key to these successes has been the use of successive discrimination procedures and combined training on identity, as well as arbitrary, baseline relations. The present study was an effort to extend the findings and theoretical analysis developed by Urcuioli and his colleagues to rats using olfactory rather than visual stimuli. Experiment 1 was a systematic replication of Urcuioli's (2008) demonstration of symmetry in pigeons. Rats were exposed to unreinforced symmetry probes following training with two arbitrary and four identity conditional discriminations. Response rates on symmetry probe trials were low and provided little evidence for emergent symmetry in any of the seven rats tested. In Experiment 2, a separate group of six rats was trained on four identity relations and was then exposed to probe trials with four novel odor stimuli. Response rates were high on identity probe trials, and low on nonmatching probe trials. The similar patterns of responding on baseline and probe trials that were shown by most rats provided a demonstration of generalized identity matching. These findings suggest that the development of stimulus control topographies in rats with olfactory stimuli may differ from those that emerge in pigeons with visual stimuli. Urcuioli's (2008) theory has been highly successful in predicting conditions necessary for stimulus class formation in pigeons, but may not be sufficient to fully understand determinants of emergent behaviors in other nonhuman species.

What-Where-When Memory in the Rodent Odor Span Task.

While the Odor Span Task (OST) was developed to assess working memory in rodents, it appears that odor ("What") and time since an odor was last reinforced ("When") jointly control responding in the OST. The OST uses an incrementing non-match to sample procedure such that the number of stimuli to remember increases during the session; the rodent is trained to remember stimuli within a session but not between sessions. We used a variation of the OST to add a "Where" dimension to the task to examine whether rodents could learn to respond to scents based on contextual cues as well. In Experiment 1, 6 rats well-trained on the OST procedure were exposed to four target scents in a holding cage before the OST session began [What-Where-When (WWW) condition]. When these target scents appeared in the OST, rats treated them as novel scents despite their being previously encountered that day; WWW responding was comparable to baseline (BL) responding. Controls were implemented to account for relative familiarity: frequency of target presentation and time since the target odor was presented. On both types of control probes, rats typically responded to target scents less than during WWW or BL conditions, took longer to make a response, and visited more comparison stimuli. In Experiment 2, the study was replicated adding reinforcement delivery for responding to pre-session presentation of target stimuli. Subjects were the same 6 rats plus 2 additional rats also well-trained on the OST. Results were similar to those from Experiment 1. These data indicate that the variables controlling performance on the OST task include What stimulus is presented, Where (i.e., in which location) it was presented, and When it was presented. Thus, the OST-probe methodology may provide a useful vehicle for the study of episodic-like memory processes in non-humans.