TEACHING CHILDREN WITH AUTISM SPECTRUM DISORDER TO MAND FOR INFORMATION USING “WHICH?”

We examined a procedure consisting of a preference assessment, prompting, contrived conditioned establishing operations, and consequences for correct and incorrect responses for teaching children with autism to mand “which?” We used a modified multiple baseline design across 3 participants. All the children learned to mand “which?” Generalization occurred to the natural environment, to a novel activity, and to a novel container; the results were maintained over time.Key words: mand for information, verbal behavior, verbal operant, whichContrived motivating operations have been used to teach mands for information to children with autism, including the mands “what?” (e.g., Williams, Donley, & Keller, 2000), “where?” (e.g., Betz, Higbee, & Pollard, 2010; Lechago, Carr, Grow, Love, & Almason, 2010), and “who?” (e.g., Endicott & Higbee, 2007; Sundberg, Loeb, Hale, & Eigenheer, 2002). More recently, researchers have examined the effects of contriving establishing operations (CEOs) in four different ways to teach children with autism to acquire the mands “what?” (Marion, Martin, Yu, & Buhler, 2011; Roy-Wsiaki, Marion, Martin, & Yu, 2010) and “where?” (Marion, Martin, Yu, Buhler, & Kerr, in press). Like the mands “what?” and “where?,” the mand “which?” is a mand for information that gives the speaker the ability to gather specific information regarding an item (e.g., “Which book is mine?”). Given the dearth of research that has examined interventions to teach mands for information using “which?,” the purpose of the present study was to extend the work of Marion et al. (2011, in press) by contriving one of four CEOs for teaching the mand “which?” to children with autism, and to assess for generalization to the other CEOs, the natural environment, and over time.     

Cocaine self-administration alters the relative effectiveness of multiple memory systems during extinction

Rats were trained to run a straight-alley maze for an oral cocaine or sucrose vehicle solution reward, followed by either response or latent extinction training procedures that engage neuroanatomically dissociable “habit” and “cognitive” memory systems, respectively. In the response extinction condition, rats performed a runway approach response to an empty fluid well. In the latent extinction condition, rats were placed at the empty fluid well without performing a runway approach response. Rats trained with the sucrose solution displayed normal extinction behavior in both conditions. In contrast, rats trained with the cocaine solution showed normal response extinction but impaired latent extinction. The selective impairment of latent extinction indicates that oral cocaine self-administration alters the relative effectiveness of multiple memory systems during subsequent extinction training.Understanding the psychological and neural mechanisms underlying the acquisition and extinction of drug-seeking behavior has important implications for therapies targeting drug addiction. A better understanding of the neurobiology of extinction can potentially allow for the development of treatments to produce more effective and persistent extinction learning. Dissociable hippocampus-dependent “cognitive” and dorsal striatal-dependent “habit” memory systems are engaged during the initial acquisition of learned behavior (for reviews, see Packard and Knowlton 2002; White and McDonald 2002; Squire 2004). Interestingly, recent evidence indicates that multiple memory systems can also be engaged during the new learning that occurs during behavioral extinction (Gabriele and Packard 2006). For example, the behavior of a rat trained to traverse a straight-alley runway for a food reward can be extinguished using either habit/response or cognitive/latent extinction training procedures. During response extinction, rats are allowed to perform the runway approach response to an empty food cup. In contrast, during latent extinction, rats are placed at the empty food cup without performing the runway approach response. Consistent with evidence indicating a selective role for the hippocampus in cognitive memory, neural inactivation of this brain structure impairs latent extinction and spares response extinction (Gabriele and Packard 2006). Moreover, consistent with evidence that the dorsal striatum selectively mediates habit memory (for review, see Packard and Knowlton 2002), neural inactivation of this brain region impairs response extinction and spares latent extinction (A. Gabriele and M.G. Packard, unpubl.).The transition from initial drug use to eventual addiction may involve, at least in part, the development of compulsive drug-seeking and drug-taking behaviors that are increasingly guided by dorsal striatal-dependent habit learning mechanisms (for reviews, see White 1996; Everitt et al. 2001; Everitt and Robbins 2005; Belin et al. 2008). This hypothesis raises the possibility that once “habit-like” drug-seeking behaviors are firmly acquired, the extinction of such behaviors may be differentially influenced by engaging habit and cognitive memory systems. In the present study, we examined this idea by comparing the relative effectiveness of response and latent extinction training procedures in rats trained to run a straight-alley maze for an oral cocaine reward. Consistent with criteria considered important for demonstrating drug dependence, oral cocaine self-administration produces withdrawal following forced abstinence (Barros and Miczek 1996) and additionally is resistant to reinforcer devaluation (Miles et al. 2003), indicating that this behavior becomes divorced from its consequences in a manner similar to the dorsal striatum-mediated compulsive drug-seeking behavior that may characterize addiction (for reviews, see White 1996; Everitt et al. 2001; Everitt and Robbins 2005; Belin et al. 2008).The apparatus was an elevated (86.4 cm) straight-alley maze with a black Plexiglas floor and clear Plexiglas sides (117.8 cm long, 11.4 cm wide, and 20.3 cm tall). A fluid cup (2.5-cm diameter) was located at the goal end of the maze. The maze was located in a room containing several extra-maze cues.Subjects were 32 adult male Long-Evans rats (275–300 g). Rats were individually housed on a 12:12-h light–dark cycle, with lights on from 8:00 a.m.–8:00 p.m. All animals received food ad libitum.During all behavioral procedures, water bottles were removed from home cages 24 h prior to training, and animals received 15 min/day access to water following each day''s procedures. Training began with 3 d of habituation to the solution to be used during training (cocaine–sucrose [0.1% cocaine HCl/20% sucrose in ddH₂0] or sucrose [20% in ddH₂0] alone). Each habituation day involved presentations of 0.5 mL of the solution in a novel environment consisting of a half-white, half-black box (41.9 cm long, 31.8 cm wide, 35.6 tall) with the fluid cup located in the center of the black side. The number of presentations increased with each habituation day (1, 2, and 4). Each individual presentation had a maximum time of 20 min, and rats were removed when the solution was consumed. Volume consumed and amount of time to consume the solution were recorded for each rat. Each sucrose rat was matched to a cocaine rat to ensure that there were no differences between groups in terms of volume of solution consumed prior to training. For each matched pair, the volume consumed by the rat receiving the cocaine solution during each presentation was measured, and an identical amount was made available to the matched sucrose animal. If, during any given presentation, the cocaine animal did not consume any solution, then the matched sucrose animal received 20 min in the habituation environment with no solution present.Behavioral procedures were similar to those of our previous study using food reward (Gabriele and Packard 2006). During maze training, animals received either the cocaine–sucrose solution or sucrose vehicle solution reward. On days 1–10 of solution-rewarded maze training (six trials per day), rats were placed in the start end and allowed to traverse the maze and drink the available reward solution (0.5 mL). Upon consuming the solution, rats were removed from the maze and placed in an opaque holding box adjacent to the maze for a 30-sec intertrial interval. On each trial, the latency (in seconds) to reach the fluid cup was recorded and used as the measure of task acquisition. If a rat failed to reach the fluid cup within 60 sec, it was removed for the intertrial interval and a latency of 60 sec was recorded.Twenty-four hours following the completion of training (i.e., day 11), rats were assigned to one of two extinction conditions; latent extinction (n = 18, 10 cocaine and eight sucrose) or response extinction (n = 14, seven cocaine and seven sucrose). For both the latent and response conditions, extinction training was administered over 3 d (six trials per day, 30-sec intertrial interval) with no reward solution present. In the latent extinction condition, rats were placed facing the empty fluid cup in the goal end of the maze and were confined for 60 sec by placement of a clear Plexiglas barrier 20 cm from the rear wall of the goal end of the maze. Following confinement, rats were removed from the maze and placed in the holding box for the 30-sec intertrial interval. In the response extinction condition, rats were placed into the start end of the maze as during training and allowed to run to an empty fluid cup at the goal end of the maze. Upon reaching the empty fluid cup and being allowed to discover its emptiness (or after 60 sec if the rat did not reach the reward cup), rats were removed from the maze and placed in the holding box for the 30-sec intertrial interval. Latency to reach the fluid cup was recorded and used as the measure of extinction behavior. On day 3 of extinction, 90 min following the sixth daily extinction trial, all rats were given an additional four extinction “probe” trials in which they were placed in the start end of the maze and latency to reach the empty fluid cup was recorded. These four trials allowed for an assessment of the effectiveness of each extinction procedure.Data from the runway acquisition sessions are presented in Figure 1. A two-way one-repeated-measure ANOVA (Group [cocaine vs. sucrose] × Session) comparing the latencies to reach the fluid cup during acquisition in rats that subsequently received latent extinction revealed a significant effect of Session (F_(9,16) = 61.03, P < 0.001), indicating that latency to reach the fluid cup during acquisition decreased across sessions. However, the absence of a main effect of Group (F_(1,16) = 1.94, n.s.) or interaction between Group and Session (F_(9,16) = 0.53, n.s.) indicates that rats trained to run for cocaine and sucrose acquired the task at similar rates (Fig. 1A). Similar results were observed in rats that subsequently received response extinction (Fig. 1B) in that there was a main effect of Session (F_(9,12) = 13.11, P < 0.001) but no main effect (F_(1,12) = 0.44, n.s.) or interaction (F_(9,12) = 1.50, n.s.) involving drug Group.Open in a separate windowFigure 1.Acquisition of maze runway behavior. (A) Acquisition of maze runway behavior by rats that subsequently received latent extinction. (B) Acquisition of maze runway behavior by rats that subsequently received response extinction. Mean ± SEM of latency (in seconds) to reach the solution cup over training days. For both extinction conditions, there were no group differences in the initial acquisition of runway behavior.The effects of oral cocaine self-administration on latent and response extinction are shown in Figure 2. A two-way ANOVA (Group × Extinction condition) comparing mean runway latencies (collapsed across the four probe trials) for each group revealed a significant main effect of Extinction condition (F_(1,28) = 32.440, P < 0.001), indicating that the response extinction procedures produced greater extinction of the runway response, and a significant interaction effect between Extinction condition and Group (F_(1,28) = 4.813, P < 0.05) but no effect of Group (F_(1,28) = 0.96, n.s.). Simple effects tests showed a significant effect of Group within the latent extinction condition (F_(1,16) = 5.688, P < 0.05) but not the response extinction condition (F_(1,12) = 0.663, n.s.), indicating that oral cocaine self-administration selectively impaired latent but not response extinction. Additionally, a two-way one-repeated-measure ANOVA (Group × Trial) computed on the latencies to reach the fluid cup during response extinction training revealed a main effect of Trial (F_(2,12) = 16.44, P < 0.001), but no significant main effect (F_(1,12) = 2.27, n.s.) or interaction (F_(2,12) = 0.88, n.s.) involving Group, further indicating that oral cocaine did not impair response extinction.Open in a separate windowFigure 2.Effects of oral cocaine self-administration on extinction. The effect of oral cocaine self-administration on runway latent and response extinction. Mean ± SEM latency (in seconds) to reach the fluid cup is shown over the four extinction probe trials. Oral cocaine self-administration impaired latent extinction, but did not impair response extinction.The present experiments investigated the effect of oral cocaine self-administration on response and latent extinction in a straight-alley maze. Following training, rats in the response extinction condition performed the approach response to an empty goal box, whereas rats in the latent extinction condition were placed in the goal box with no reward present. Consistent with previous studies using food reward (e.g., Seward and Levy 1949; Gabriele and Packard 2006), rats rewarded with a sucrose solution were able to extinguish the approach response following both response and latent extinction procedures. In contrast, rats rewarded with a cocaine solution displayed normal response extinction (see also Schoenbaum and Setlow 2005) but impaired latent extinction. The selective impairing effect of oral cocaine self-administration on latent extinction indicates that the drug does not impair processes that contribute to general maze behavior (e.g., motivational, motor, or sensory processes), as any such influence would also likely produce a deficit in response extinction.Previous findings indicate that latent extinction of runway behavior is hippocampus dependent, whereas response extinction is dorsal striatal dependent (Gabriele and Packard 2006; A. Gabriele and M.G. Packard, unpubl.). In view of evidence that the hippocampus and dorsal striatum mediate cognitive and habit learning mechanisms, respectively (for reviews, see Packard and Knowlton 2002; White and McDonald 2002; Squire 2004), the findings suggest that oral cocaine self-administration can affect the relative use of multiple memory systems during extinction learning. The medial prefrontal cortex and basolateral amygdala have been implicated in extinction of several forms of learned behavior, and prior cocaine exposure can impair some forms of extinction learning (Burke et al. 2006; Peters et al. 2008; Quirk and Mueller 2008). However, neural inactivation of medial prefrontal cortex or basolateral amygdala does not affect latent extinction of maze runway behavior (A. Gabriele and M.G. Packard, unpubl.), suggesting that cocaine-induced dysfunction of these structures does not account for the results observed here.One explanation of the cocaine-induced impairment of latent extinction is that the approach response acquired during task acquisition is guided by a supra-normal stimulus-response habit, thereby rendering cognitive learning mechanisms ineffectual during latent extinction training. Consistent with this possibility, drug-seeking behaviors underlying addiction may involve, at least in part, a transition from goal-directed behaviors to habitual behaviors that characterize the function of the dorsal striatal memory system (e.g., Tiffany 1990; White 1996; Packard 1999; Everitt et al. 2001; Porrino et al. 2004; Everitt and Robbins 2005; Belin et al. 2008). Indeed, recent evidence implicates the dorsal striatum in habitual drug-seeking behaviors. For example, intradorsal striatum administration of dopamine antagonists impairs cocaine seeking (Vanderschuren et al. 2005), and inactivation of the dorsal striatum attenuates drug seeking, following both abstinence and extinction (Fuchs et al. 2006; See et al. 2007). Interestingly, disconnection between the ventral and dorsolateral striatum impairs cocaine-seeking behavior (Belin and Everitt 2008), and extended cocaine use enhanced cue-selective firing in the dorsal striatum and reduced cue-selective firing in the ventral striatum in go/no go discrimination learning, indicating an accelerated shift to dorsolateral striatal control (Takahashi et al. 2007). In addition, dopamine release increases in the dorsal striatum of rats following presentation of a response-contingent cue associated with cocaine (Ito et al. 2002). Similar results from fMRI and PET studies of human cocaine addicts showed increased activation in the dorsal striatum (Garavan et al. 2000) and an increase in dopamine release within the dorsal striatum (Volkow et al. 2006) following cue-induced cravings.A second explanation of the cocaine-induced impairment in latent extinction is that drug intake during task acquisition may have affected hippocampal physiology in a manner that negatively impacted the hippocampus-dependent learning that subsequently mediates latent extinction. Consistent with this possibility, chronic cocaine exposure impairs subsequent performance of hippocampus-dependent tasks such as the Morris water maze and the win-shift radial arm maze task (Melnick et al. 2001; Quirk et al. 2001; Mendez et al. 2008). However, it should be noted that the impairments observed in the latter studies were observed following exposure to cocaine doses considerably higher than those used in the present oral self-administration study. Since the current experiments do not explicitly examine the potential neurobiological progression underlying the acquisition of runway responding, further research is necessary to determine whether the cocaine-induced impairment of latent extinction involves the interfering effect of a supra-normal response habit, or a direct impairing effect on hippocampal physiology. It should also be noted that both oral cocaine self-administration and a passive cocaine administration regimen produce results analogous to those presented here, in that they impair “cognitive” representations of rewards (Miles et al. 2003; Schoenbaum and Setlow 2005). However, the relationship between this type of cognitive reward representation (mediated by interactions between basolateral amygdala and orbitofrontal cortex) (Pickens et al. 2003) and cognitive representations in latent extinction mediated by the hippocampus (Gabriele and Packard 2006) is currently unclear.Finally, the selective impairing effect of cocaine self-administration on latent extinction may have implications for understanding the persistent ability of drug-predictive cues and contexts to compel drug-seeking behavior and relapse. Specifically, if the ability to use cognitive learning mechanisms to extinguish drug-seeking behaviors is impaired following the transition from initial to habitual and compulsive drug use, then contextual/relational cues might be expected to maintain greater control over behavior following extinction training. This in turn might suggest that incorporation of response extinction procedures into treatment strategies might provide greater therapeutic efficacy.     

Context representations,context functions,and the parahippocampal–hippocampal system

Psychologists and neurobiologists have a long-standing interest in understanding how the context surrounding the events of our lives is represented and how it influences our behavior. The hippocampal formation emerged very early as a major contributor to how context is represented and functions. There is a large literature examining its contribution that on the surface reveals an array of conflicting outcomes and controversy. This review reveals that these conflicts can be resolved by building Nadel and Willner''s dual-process theory of context representations. Two general conclusions emerge: (1) There are two neural systems that can support context representations and functions—a neocortical system composed primarily of perirhinal and postrhinal cortices and a hippocampal system that includes perirhinal, postrhinal, entorhinal cortices, and the hippocampal formation. (2) These two systems are not equivalent—some context representations and functions are uniquely supported by the hippocampal system. These conclusions are discussed in the context of canonical ideas about the special properties of the hippocampal system that enable it to make unique contributions to memory.Everything we experience happens somewhere. The term “context” is often used to denote this “somewhere.” In the analysis of learning and memory, the context is like the setting for a stage play (Medin and Reynolds 1985). It provides the background for the real action in the drama—the main events. More importantly, as a consequence of learning and memory processes, the context often helps to select appropriate behaviors and determine the explicit and implicit content of our thoughts. Thus, it is not surprising that psychologists have a long-standing empirical and theoretical interest in understanding just what makes up a representation of context and how context representations interact with the main events of our lives to influence our behavior (see Balsam and Tomie 1985).More recently, neurobiologists have increased their interest in the problem of linking context representations and functions to brain systems. The hippocampal formation emerged quite early as a major focal point among many researchers. As a result, there is a substantial literature linking the hippocampal formation and context. However, on the surface, this research yields a dismaying set of conflicting results, with many findings that the hippocampal formation plays a critical role in supporting the influence of context on memory and behavior and many other findings that it does not.The goal of this article is to bring some clarity and order to this state of affairs. I start by providing a working definition of “context” that implicitly underpins its experimental analysis. I then describe several different functions of context that have been studied in the laboratory and are assumed to be theoretically important. I then introduce and build on a dual-process theory of context representations that was put forth several years ago by Lynn Nadel, Jeffrey Willner, and colleague (Nadel and Willner 1980; Nadel et al. 1985) and more recently by my colleagues and me (Rudy and O''Reilly 2001; Rudy et al. 2002). I then apply this framework to a wide range of outcomes from experiments that examined the role of the hippocampal formation in ways in which context influences memory and behavior. Two general conclusions emerge from this exercise. First is that two systems can support context representations and functions: (1) a neocortical system composed primarily of perirhinal and postrhinal cortices and (2) a hippocampal system that includes parahippocampal cortices; perirhinal, postrhinal, and entorhinal cortex; and the hippocampal formation. The second is that these two systems are not equivalent—some context representations and functions are uniquely supported by the hippocampal system. These conclusions are discussed in the context of long-standing ideas about the special properties associated with the hippocampal system that support its unique contributions to memory.     

Sleep spectral power correlates of prospective memory maintenance

Prospective memory involves setting an intention to act that is maintained over time and executed when appropriate. Slow wave sleep (SWS) has been implicated in maintaining prospective memories, although which SWS oscillations most benefit this memory type remains unclear. Here, we investigated SWS spectral power correlates of prospective memory. Healthy young adult participants completed three ongoing tasks in the morning or evening. They were then given the prospective memory instruction to remember to press “Q” when viewing the words “horse” or “table” when repeating the ongoing task after a 12-h delay including overnight, polysomnographically recorded sleep or continued daytime wakefulness. Spectral power analysis was performed on recorded sleep EEG. Two additional groups were tested in the morning or evening only, serving as time-of-day controls. Participants who slept demonstrated superior prospective memory compared with those who remained awake, an effect not attributable to time-of-day of testing. Contrary to prior work, prospective memory was negatively associated with SWS. Furthermore, significant increases in spectral power in the delta-theta frequency range (1.56 Hz–6.84 Hz) during SWS was observed in participants who failed to execute the prospective memory instructions. Although sleep benefits prospective memory maintenance, this benefit may be compromised if SWS is enriched with delta–theta activity.

Prospective memory refers to the maintenance, retrieval, and execution of a previously formed intention (Einstein and McDaniel 1990). Successful prospective memory is essential for a large number of tasks in daily life, such as remembering to attend a doctor''s appointment, to pick up a prescribed medication after that appointment, and to also pick up other needed items (e.g., groceries) while at the drugstore. The above described hypothetical sequence of events integrates previously studied prospective memory variants including time-based (i.e., maintaining a memory to complete an intention at a prespecified time; e.g., Esposito et al. 2015; Occhionero et al. 2017), activity-based (i.e., maintaining a memory to perform an intention before or after a particular activity; e.g., Occhionero et al. 2020), and cue-based (i.e., relying on external cues to prompt a maintained memory for a set intention; e.g., Scullin and McDaniel 2010; Leong et al. 2019b; Scullin et al. 2019).When it is required that memories be maintained across longer periods of time, prospective memory may become less reliable unless sleep occurs (Scullin and McDaniel 2010; Diekelmann et al. 2013a,b; Grundgeiger et al. 2014; Leong et al. 2019a,b; Scullin et al. 2019). Sleep appears to most strongly aid spontaneous retrieval of cue-based prospective memories (Leong et al. 2019a). Several reports have found that slow wave sleep (SWS) supports spontaneous retrieval of cue-based prospective memory intentions (e.g., Diekelmann et al. 2013a; Leong et al. 2019b), although at least one study found an association with rapid eye movement (REM) sleep instead (Scullin et al. 2019). Cue-based prospective memory is hypothesized to be a type of associative memory that binds prospective components (the prospective memory cue) and retrospective components (maintenance of the memory for the prospective memory intention when presented with the cue; Diekelmann et al. 2013b; Leong et al. 2019a).Rodent and human literature, implementing a variety of invasive and noninvasive brain imaging techniques, show that cortical slow oscillations (SOs; <1 Hz) and fast thalamocortical sleep spindles during SWS facilitate associative memory retention (Niknazar et al. 2015; Latchoumane et al. 2017; Helfrich et al. 2018; Mikutta et al. 2019; Muehlroth et al. 2019), whereas faster oscillations, such as those in the theta frequency band (∼4–7 Hz), may inhibit declarative associative memory (Marshall et al. 2011). We therefore hypothesize that prospective memory performance, like other studied associative memory variants, should benefit from oscillations during SWS (Klinzing et al. 2019). However, it remains unknown which SWS microarchitectural features may facilitate or inhibit prospective memory performance.Here, we aimed to first replicate prior findings that prospective memories are better maintained across a 12-h interval including sleep compared with an equivalent interval of wakefulness (e.g., Scullin and McDaniel 2010). We next explored whether sleep-associated memory maintenance might be linked to SWS microarchitectural features. To our knowledge, this is the first experiment to examine whether SWS oscillations differentiate successful from unsuccessful prospective memory performance. Given the role of hippocampal engagement in both associative memory binding (e.g., Yonelinas et al. 2019) and oscillatory coupling during SWS that supports associative memory (Niknazar et al. 2015; Latchoumane et al. 2017; Helfrich et al. 2018; Mikutta et al. 2019; Muehlroth et al. 2019), we hypothesized that prospective memory performance would be supported by SWS and specifically SOs and sleep spindle activity.     

A case for methodological overhaul and increased study of executive function in the domestic dog (<Emphasis Type="Italic">Canis lupus familiaris</Emphasis>)

Executive function (EF) allows for self-regulation of behavior including maintaining focus in the face of distraction, inhibiting behavior that is suboptimal or inappropriate in a given context, and updating the contents of working memory. While EF has been studied extensively in humans, it has only recently become a topic of research in the domestic dog. In this paper, I argue for increased study of dog EF by explaining how it might influence the owner–dog bond, human safety, and dog welfare, as well as reviewing the current literature dedicated to EF in dogs. In “EF and its Application to “Man’s Best Friend” section, I briefly describe EF and how it is relevant to dog behavior. In “Previous investigations into EF in dogs” section, I provide a review of the literature pertaining to EF in dogs, specifically tasks used to assess abilities like inhibitory control, cognitive flexibility, and working memory capacity. In “Insights and limitations of previous studies” section, I consider limitations of existing studies that must be addressed in future research. Finally, in “Future directions” section, I propose future directions for meaningful research on EF in dogs.     

Artifact and Essence

An essential property is a property that an object possesses in every possible world in which that object exists. An individual essence is a property (or set of properties) that an object possesses in every world in which that object exists, and that no other object possesses in any possible world. Call the claim that some artifacts possess an individual essence ‘artifactual essentialism’. I will argue that artifactual essentialism is true. In doing so, I will be responding to two recent arguments by Penelope Mackie against artifactual essentialism (Mackie (2006), esp. ch. 3.). In “Individual Essence Properties”, I will rehearse the qualifications that any property must meet if it is to constitute an individual essence, and in “Artifacts and the Recycling Problem” and “Artifacts and the Tolerance Problem”, I will rehearse Mackie’s arguments against artifactual essentialism. In “Artifacts and Weak Unshareability?” and “Artifacts and Strong Unshareability?”, I will show why both of these arguments fail. In “Mona Lisa’s Essence”, I will defend the interesting claim that some artifacts possess an individual essence. In the final section I will entertain some objections to my proposal.     

Exclusion Problems and the Cardinality of Logical Space

Wittgenstein’s atomist picture, as embodied in his Tractatus, is initially very appealing. However, it faces the famous colour-exclusion problem. In this paper, I shall explain when the atomist picture can be defended (in principle) in the face of that problem; and, in the light of this, why the atomist picture should be rejected. I outline the atomist picture in Section 1. In Section 2, I present a very simple necessary and sufficient condition for the tenability (in principle) of the atomist picture. The condition is: logical space is a power of two. In Sections 3 and 4, I outline the colour-exclusion problem, and then show how the cardinality-condition supplies a response to exclusion problems. In Section 5, I explain how this amounts to a distillation of a proposal due to Moss (2012), which goes back to Carruthers (1990: 144–7). And in Section 6, I show how all this vindicates Wittgenstein’s ultimate rejection of the atomist picture. The brief reason is that we have no guarantee that there are any solutions to a given exclusion problem but, if there are any, then there are far too many.     

Individual differences in spatial pattern separation performance associated with healthy aging in humans

Rodent studies have suggested that “pattern separation,” the ability to distinguish among similar experiences, is diminished in a subset of aged rats. We extended these findings to the human using a task designed to assess spatial pattern separation behavior (determining at time of test whether pairs of pictures shown during the study were in the same spatial locations). Using a standardized test of word recall to divide healthy aged adults into impaired and unimpaired groups relative to young performance, we demonstrate that aged impaired adults are biased away from pattern separation and toward pattern completion, consistent with the rodent studies.Memory impairment is a common complaint among aging individuals, yet the variability within the aging population is great in both rats (Gallagher et al. 2006; Robitsek et al. 2008) and humans (Hilborn et al. 2009). A rodent model of aging (Gallagher et al. 2006; Wilson et al. 2006) has demonstrated that ∼50% of healthy rats qualify as cognitively “impaired” by scoring outside the range of the young performance in a standard protocol (Gallagher et al. 1993). The other half, the “unimpaired” rats, perform on par with young adults, demonstrating a natural degree of variability in cognitive aging. In this study, we sought to capitalize on the variability observed in the aging of both rats and humans in a study of spatial pattern separation.One source of variability in memory performance is hypothesized to be tied to changes in the input to the dentate gyrus (DG), which has been shown in the rat to be affected by the aging process. Smith et al. (2000) reported a selective impairment in layer II entorhinal input into the DG and CA3 regions of the hippocampus in rats with cognitive impairment. Similarly, the number of synapses in the outer receiving layer of DG was reduced in autopsied aged brains and correlated with earlier performance on a delayed recall task (Scheff et al. 2006). Finally, in a human imaging study, Small et al. (2002) observed that 60% of their aging sample demonstrated diminished MRI signal in the hippocampal region (including the DG) and also had a greater decline in memory performance. These findings support the notion that changes in the DG associated with aging may affect memory performance.The DG may be particularly important for the computations that underlie pattern separation (Treves and Rolls 1994; McClelland et al. 1995; Norman and O''Reilly 2003). “Pattern separation” refers to the process by which similar inputs are stored as distinct, nonoverlapping representations. In contrast, “pattern completion” refers to the process by which an existing representation can be reinstated by the presentation of a partial or degraded cue. Numerous studies in the rodent have identified the importance of the DG for pattern separation using electrophysiological methods (Leutgeb et al. 2004, 2005, 2007; Leutgeb and Leutgeb 2007), immediate early gene expression (Vazdarjanova and Guzowski 2004), lesions (Lee et al. 2005; Gilbert and Kesner 2006; Goodrich-Hunsaker et al. 2008), and even genetic manipulations (Cravens et al. 2006; Kubik et al. 2007; McHugh et al. 2008). Human neuroimaging has also recently identified activity in the DG (and CA3 regions of the hippocampus) in an object pattern separation task (Kirwan and Stark 2007; Bakker et al. 2008).Given the importance of the DG in pattern separation and its vulnerability to changes that occur with aging, studies have begun to examine pattern separation in older adults. Our laboratory has designed a task to examine object-based pattern separation performance in humans (Kirwan and Stark 2007). In this task, pictures of objects were presented either once or repeatedly throughout the task. Critically, some of the items presented were lures that were similar but not identical to previously shown items. The overlapping features of the lures more heavily engaged pattern separation processes. In young adults, functional magnetic resonance imaging (fMRI) activity in the DG was sensitive to the lures, indicating a role in pattern separation processes in both an explicit (Kirwan and Stark 2007) and implicit (Bakker et al. 2008) version of this task. Toner et al. (2009) used the explicit version of this task to demonstrate that older adults showed a greater tendency to identify lures as “old” (repeated) relative to young adults. These findings were also recently replicated in our laboratory (Yassa et al., in press), with the additional demonstration that older adults exhibit greater fMRI CA3/DG activity for the lures during both encoding and retrieval.Since object-based pattern separation appears to be modulated by the DG in humans, we wondered if these findings could be extended to spatial pattern separation. Rodent studies have demonstrated that the DG has a particular role in spatial pattern separation (Gilbert et al. 2001; Kesner et al. 2004). Specifically, Hunsaker et al. (2008) placed rats with localized DG lesions in an environment with two objects spaced 60 cm apart. When the animals were later placed in the same environment with the same objects now placed 40 cm apart, DG-lesioned animals (unlike control animals) did not re-explore the objects or environment. These data suggest that the DG-lesioned rats were not able to discriminate between the training and test environments. That is, they were impaired in spatial pattern separation. Since converging evidence suggests that one feature of the aging process can be characterized as a DG knockdown, we modified this task design for humans to test spatial pattern separation performance in older adults. While the Hunsaker et al. (2008) task emphasized the distance between the two objects as the source of interference creating a greater need for pattern separation, the paradigm presented here moves an object in any direction, changing both the distance and the angle (i.e., changing more of the spatial relations). We posit that this amount of movement (close, medium, or far) may place similar demands on spatial pattern separation processes as in the rodent task.The present study included 20 young adults (mean age 19.9 yr, range 18–27 yr) and 30 aged adults (mean age 70.4 yr, range 59–80 yr). Aged adults completed a battery of standardized neuropsychological tests, including the Mini-Mental State Exam (Folstein et al. 1975), Rey Auditory–Verbal Learning Task (RAVLT) (Rey 1941), Digit Span, Vocabulary, and Matrices subtests from the Wechsler Adult Intelligence Scale III (Wechsler 1997). The Vocabulary and Matrices scores were entered into a weighted formula along with age, gender, and education to derive estimated IQ scores (Schoenberg et al. 2003). All aged participants scored within the normal age-adjusted ranges on these measures and were cognitively intact. Younger adults also completed the RAVLT and scored within the normal age-adjusted range. These data are presented in Table 

Predictive visual search: Role of environmental regularities in the learning of context cues

Repeatedly searching through invariant spatial arrangements in visual search displays leads to the buildup of memory about these displays (contextual-cueing effect). In the present study, we investigate (1) whether contextual cueing is influenced by global statistical properties of the task and, if so, (2) whether these properties increase the overall strength (asymptotic level) or the temporal development (speed) of learning. Experiment 1a served as baseline against which we tested the effects of increased or decreased proportions of repeated relative to nonrepeated displays (Experiments 1b and 1c, respectively), thus manipulating the global statistical properties of search environments. Importantly, probability variations were achieved by manipulating the number of nonrepeated (baseline) displays so as to equate the total number of repeated displays across experiments. In Experiment 1d, repeated and nonrepeated displays were presented in longer streaks of trials, thus establishing a stable environment of sequences of repeated displays. Our results showed that the buildup of contextual cueing was expedited in the statistically rich Experiments 1b and 1d, relative to the baseline Experiment 1a. Further, contextual cueing was entirely absent when repeated displays occurred in the minority of trials (Experiment 1c). Together, these findings suggest that contextual cueing is modulated by observers’ assumptions about the reliability of search environments.     

LTP in hippocampal area CA1 is induced by burst stimulation over a broad frequency range centered around delta

Long-term potentiation (LTP) is typically studied using either continuous high-frequency stimulation or theta burst stimulation. Previous studies emphasized the physiological relevance of theta frequency; however, synchronized hippocampal activity occurs over a broader frequency range. We therefore tested burst stimulation at intervals from 100 msec to 20 sec (10 Hz to 0.05 Hz). LTP at Schaffer collateral–CA1 synapses was obtained at intervals from 100 msec to 5 sec, with maximal LTP at 350–500 msec (2–3 Hz, delta frequency). In addition, a short-duration potentiation was present over the entire range of burst intervals. We found that N-methyl-d-aspartic acid (NMDA) receptors were more important for LTP induction by burst stimulation, but L-type calcium channels were more important for LTP induction by continuous high-frequency stimulation. NMDA receptors were even more critical for short-duration potentiation than they were for LTP. We also compared repeated burst stimulation with a single primed burst. In contrast to results from repeated burst stimulation, primed burst potentiation was greater when a 200-msec interval (theta frequency) was used, and a 500-msec interval was ineffective. Whole-cell recordings of postsynaptic membrane potential during burst stimulation revealed two factors that may determine the interval dependence of LTP. First, excitatory postsynaptic potentials facilitated across bursts at 500-msec intervals but not 200-msec or 1-sec intervals. Second, synaptic inhibition was suppressed by burst stimulation at intervals between 200 msec and 1 sec. Our data show that CA1 synapses are more broadly tuned for potentiation than previously appreciated.Long-term potentiation (LTP) is used as a model for studying synaptic events during learning and memory (Bliss and Collingridge 1993; Morris 2003; Lynch 2004). At most synapses, LTP is triggered by postsynaptic Ca²⁺ influx through N-methyl-d-aspartic acid (NMDA) glutamate receptors (Collingridge et al. 1983; Harris et al. 1984; Herron et al. 1986) and, under some conditions, through L-type voltage-gated Ca²⁺ channels (Grover and Teyler 1990, 1994; Morgan and Teyler 1999). LTP was discovered in the dentate gyrus (Bliss and Lomo 1973) following several seconds of 10–100 Hz stimulation of the perforant path. Since then, many LTP studies have used similar long, high-frequency stimulation (HFS) protocols, most typically 100 Hz, 1 sec (Bliss and Collingridge 1993). Although effective, HFS does not resemble physiological patterns of activity (Albensi et al. 2007). Patterned stimulation resembling physiological activity, in particular theta burst stimulation, is also effective for LTP induction (Larson et al. 1986; Staubli and Lynch 1987; Capocchi et al. 1992; Nguyen and Kandel 1997). Theta burst stimulation consists of short bursts (4–5 stimuli at 100 Hz) repeated at 5 Hz, which lies within the hippocampal theta frequency range (4–12 Hz) (Bland 1986; Buzsáki 2002). Primed burst stimulation, another form of patterned stimulation, involves delivery of a priming stimulus followed by a single short burst (Larson and Lynch 1986; Rose and Dunwiddie 1986). The temporal requirements for primed burst LTP are quite precise (Diamond et al. 1988; Greenstein et al. 1988; Larson and Lynch 1989): Intervals less than 140 msec or greater than 200 msec are ineffective.The mechanisms underlying theta frequency-dependent LTP have been studied primarily using the primed burst protocol (Larson and Lynch 1986, 1988, 1989; Pacelli et al. 1989; Davies and Collingridge 1996). Activation of GABA_B autoreceptors during the priming stimulus suppresses GABA release during the following burst (Davies et al. 1990; Lambert and Wilson 1994; Olpe et al. 1994), allowing greater postsynaptic depolarization (Larson and Lynch 1986; Pacelli et al. 1989) and more effective NMDA receptor activation (Davies and Collingridge 1996). Consequently, temporal requirements for primed burst potentiation match the time course of GABA_B autoreceptor-mediated suppression of GABA release (Davies et al. 1990; Davies and Collingridge 1993; Mott et al. 1993).Besides theta, hippocampal activity is observed at other frequencies, notably sharp waves (0.01–5 Hz) (Buzsáki 1986, 1989; Suzuki and Smith 1987) and low-frequency oscillations (≤1 Hz) (Wolansky et al. 2006; Moroni et al. 2007). These lower frequencies dominate during slow wave sleep (Buzsáki 1986; Suzuki and Smith 1987; Wolansky et al. 2006; Moroni et al. 2007), and contribute to hippocampal memory processing (Buzsáki 1989; Pennartz et al. 2002). While synchronized population activity over frequencies from <1 Hz to 12 Hz is associated with hippocampal memory function, previous LTP studies have focused on theta. We therefore investigated burst stimulation at frequencies from 0.05 Hz to 10 Hz. We found that CA1 synapses potentiate to some degree over this entire range and that maximal potentiation occurs around delta frequency rather than theta.     

Phenomenological constraints: a problem for radical enactivism

This paper does two things. Firstly, it clarifies the way that phenomenological data is meant to constrain cognitive science according to enactivist thinkers. Secondly, it points to inconsistencies in the ‘Radical Enactivist’ handling of this issue, so as to explicate the commitments that enactivists need to make in order to tackle the explanatory gap. I begin by sketching the basic features of enactivism in sections 1–2, focusing upon enactive accounts of perception. I suggest that enactivist ideas here rely heavily upon the endorsement of a particular explanatory constraint that I call the structural resemblance constraint (SRC), according to which the structure of our phenomenology ought to be mirrored in our cognitive science. Sections 3–5 delineate the nature of, and commitment to, SRC amongst enactivists, showing SRC’s warrant and implications. The paper then turns to Hutto and Myin’s (2013) handling of SRC in sections 6–7, highlighting irregularities within their programme for Radical Enactivism on this issue. Despite seeming to favour SRC, I argue that Radical Enactivism’s purported compatibility with the narrow (brain-bound) supervenience of perceptual experience is in fact inconsistent with SRC, given Hutto and Myin’s phenomenological commitments. I argue that enactivists more broadly ought to resist such a concessionary position if they wish to tackle the explanatory gap, for it is primarily the abidance to SRC that ensures progress is made here. Section 8 then concludes the paper with a series of open questions to enactivists, inviting further justification of the manner in which they apply SRC.     

Perspectivism Versus a Completed Copernican Revolution

I discuss changes of perspective of four kinds in science and about science. Section 2 defends a perspectival nonrealism—something akin to Giere’s perspectival realism but not a realism—against the idea of complete, “Copernican” objectivity. Section 3 contends that there is an inverse relationship between epistemological conservatism and scientific progress. Section 4 casts doubt on strong forms of scientific realism by taking a long-term historical perspective that includes future history. Section 5 defends a partial reversal in the status of so-called context of discovery and context of justification. Section 6 addresses the question of how we can have scientific progress without scientific realism—how progress is possible without the accumulation of representational truth. The overall result (Sect. 7) is a pragmatic instrumentalist perspective on the sciences and how to study them philosophically, one that contains a kernel of realism—instrumental realism.     

Prelimbic cortex bdnf knock-down reduces instrumental responding in extinction

Anatomically selective medial prefrontal cortical projections regulate the extinction of stimulus–reinforcement associations, but the mechanisms underlying extinction of an instrumental response for reward are less well-defined and may involve structures that regulate goal-directed action. We show brain-derived neurotrophic factor (bdnf) knock-down in the prelimbic, but not orbitofrontal, cortex accelerates the initial extinction of instrumental responding for food and reduces striatal BDNF protein. When knock-down mice were provided with alternative response options to readily obtain reinforcement, extinction of the previously reinforced response was unaffected, consistent with the hypothesis that the prelimbic cortex promotes instrumental action, particularly when reinforcement is uncertain or unavailable.The rodent medial prefrontal cortex contains cytoarchitectonically distinct subregions that can be differentiated based on efferent and afferent projection patterns, with dorsal regions—including the dorsal prelimbic cortex (PLc)—sharing similar functions that differ from those of the ventromedial prefrontal cortex, which includes the medial orbitofrontal cortex (mOFC) and infralimbic cortex. These dorsal/ventral networks are considered “go” and “stop” systems, respectively, that coincidentally guide behavior (Heidbreder and Groenewegen 2003). For example, the PLc is essential for maintaining instrumental responding for food when reinforcement is uncertain (Corbit and Balleine 2003; Gourley et al. 2008a). By contrast, ventromedial structures are associated with response inhibition, particularly in the context of stimulus–reinforcement associations (Heidbreder and Groenewegen 2003).We explore the hypothesis that the PLc may also promote goal-directed responding in the absence of reinforcement, thereby slowing the extinction of a previously reinforced instrumental response. If this is the case, diminution of the biological factors essential for activity-dependent neuroplasticity and cytoskeletal structure within the PLc might be expected to shift the balance between a dorsal “go” network and ventral “stop” network. The consequence would be a rapid decline in instrumental responding during extinction training. Indeed, we report that such a manipulation—virally knocking down BDNF, which promotes long-term potentiation (Kang and Schuman 1995; Korte et al. 1995, 1996; Patterson et al. 1996) and neuronal outgrowth (McAllister et al. 1995, 1996; Xu et al. 2000a,b; Gorski et al. 2003)—within the PLc facilitates the extinction of instrumental action.In the first experiment, group-housed ≥10 wk-old male mice bred in-house and homozygous for a floxed bdnf gene (Rios et al. 2001) were anaesthetized with 1:1 2-methyl-2-butanol and tribromoethanol (Sigma Aldrich) diluted 40-fold with saline. Mice were infused into the PLc (+2.0AP, −2.8DV, ±0.1ML) with an adeno-associated virus (AAV) expressing enhanced green fluorescent protein (EGFP) ± Cre. With needles (Hamilton Co.) centered at bregma, stereotaxic coordinates were located using Kopf''s digital coordinate system with 1/100-mm resolution (David Kopf Instruments). Viral constructs were infused over 5 min with 0.5 μL/hemisphere; needles were left in place for an additional 4 min. Mice were allowed to recover for at least 2 wk, allowing for viral-mediated gene knock-down (Berton et al. 2006; Graham et al. 2007; Unger et al. 2007). All procedures were Yale University Animal Care and Use Committee approved.Mice were then food-restricted (90-min access/day) and trained to perform an instrumental response (nose poke) for food reinforcement using Med-Associates operant conditioning chambers controlled by Med-Associates software. These 25-min training sessions were conducted daily, and one, two, or three responses on one of three apertures were reinforced with a 20-mg grain-based food pellet (variable ratio 2 schedule of reinforcement; Bioserv). Two-factor (knock-down × session) analysis of variance (ANOVA) with repeated measures (RM) indicated bdnf knock-down did not affect the acquisition of instrumental responding (main effect of infusion and infusion × session interaction Fs < 1) (Fig. 1A).Open in a separate windowFigure 1.PLc bdnf knock-down decreases instrumental responding in extinction. (A) Viral-mediated PLc bdnf knock-down had no effects on the acquisition of an instrumental response for food. Responses made on the active aperture are shown (left). Responding in extinction was, however, diminished during the first extinction session (right). The break in the extinction curve represents the passage of 1 d. (B) A second group of mice was trained to respond for food before viral construct infusion. Responding during reacquisition reminder sessions after recovery was unaffected, but extinction was again immediately facilitated, as indicated by fewer responses made during sessions 1 and 2. Representative EGFP spread is inset. (C) As a control measure, this experiment was replicated in mice initially trained to perform the task, then given a mOFC, rather than PLc, bdnf knock-down. Although reinforced responding during reacquisition was diminished, responding during extinction was unchanged. Representative EGFP spread is inset. (D) In a reversal task, PLc bdnf knock-down mice did not differ in their ability to “reverse” their responding on an aperture on the opposite side of the chamber; response inhibition—extinction of responding on the previously active aperture—under these circumstances was also unchanged. (E) An enlarged EGFP image is shown (taken from inside the white box in C). EGFP radiates laterally from the infusion site, and the medial wall of the PFC can be seen at left. Symbols represent means (+ SEM) per group (*P < 0.05; ^†P = 0.07). Arrows indicate the time of knock-down, relative to testing sessions.Response extinction was then tested with 10 15-min nonreinforced sessions (five sessions/day). Here, responses made on the previously active aperture declined as expected (F_(9,72) = 6.7, P < 0.001). An interaction between group and session for responses on the active aperture was also identified (F_(9,72) = 2.3, P = 0.03). Tukey''s post-hoc tests indicated responses made during session 1 were reduced in knock-down mice (P = 0.002) (Fig. 1A). Responses made during session 2 were reduced at a trend level of significance (P = 0.07), but responding during other sessions did not differ (all Ps > 0.3), suggesting PLc bdnf knock-down facilitated initial response suppression, but not necessarily the consolidation or expression of extinction learning (Rescorla and Heth 1975).Because knock-down could conceivably regulate extinction processes via effects on initial instrumental conditioning, we trained another group of mice to perform the response prior to knock-down. Mice were then matched based on responses made during training, and surgery proceeded. After recovery, mice were given three “reacquisition” sessions identical to training sessions, during which no differences were found for responses made on the reinforced aperture (main effect of group and interaction Fs < 1) (Fig. 1B). When reinforcement was withheld, however, bdnf knock-down mice again made fewer responses relative to control mice during sessions 1 and 2 (interaction F_(9,135) = 2.3, P = 0.02; post-hoc Ps < 0.01) but not later sessions (Fig. 1B). These data further support our conclusion that PLc bdnf knock-down decreases instrumental responding during the early phases of extinction, but do not indicate whether this effect is behaviorally or anatomically specific. In this group, post-mortem EGFP distribution indicated two mice had only unilateral bdnf knock-down; these animals were excluded.To address anatomical specificity, we replicated this experiment with bdnf knocked down in the ventrally situated mOFC. This site was chosen over the infralimbic cortex because we had greater confidence we could achieve anatomically selective knock-down in this larger region. Viral constructs were infused over 3 min with 0.25 μL/hemisphere and needles aimed AP +2.3, DV −3.0, ML ±0.1 and left in place for an additional 4 min. During reacquisition, a main effect of group on responses made on the active aperture indicated mOFC bdnf knock-down, unlike PLc bdnf knock-down, decreased reinforced responding (F_(1,9) = 7.9, P = 0.02; interaction F < 1) (Fig. 1C). No effects of knock-down were, however, detected for responses made during extinction testing (group and interaction Fs < 1) (Fig. 1C). This profile is distinct from PLc bdnf knock-down mice, in which nonreinforced, but not reinforced, responding was affected. In this group, one animal with unilateral bdnf knock-down was excluded.To address behavioral specificity, mice from Figure 1B were retrained until responding for food on the active aperture was reinstated. Then, the location of the active aperture was “reversed,” such that the previously nonreinforced aperture on the opposite side of the chamber wall was reinforced. In other words, mice trained to respond on the right-side aperture were now reinforced for responding on the left-side aperture and vice versa. This “reversal” procedure allowed us to test whether PLc bdnf knock-down facilitates extinction when reinforcement is available upon the acquisition of an alternative response. We used a highly reinforcing variable ratio 2 schedule, and test sessions lasted 45 min.Under these conditions, bdnf knock-down and control mice did not differ, responding on both the previously reinforced and the newly reinforced apertures to the same degree as control mice (main effect of genotype on nonreinforced responding F_(1,14) = 1.9, P = 0.2; reinforced responding F < 1; group × session interaction F < 1) (Fig. 1D). In other words, PLc bdnf knock-down mice showed exaggerated response inhibition in the absence of reinforcement, but not when a competing response to obtain food reinforcement was available. Main effects of session on responses made on the active and inactive apertures indicated mice acquired the “reversal” (F_(3,45) = 15.2, P < 0.001; F_(3,45) = 5.7, P = 0.002, respectively).In a final behavioral experiment, male group-housed C57BL/6J mice (Charles River Laboratories, Kingston, New York), also ≥10 wk of age at the start of the experiment, were trained and infused with BDNF to evaluate whether acute PLc BDNF infusion produced the opposite effects of gene knock-down: slowed extinction. Human recombinant BDNF (Chemicon) dissolved in sterile saline in a concentration of 0.4 μg/μL (Gourley et al. 2008b) was used, with 0.2 μL/site at AP +2.0, DV −2.5, ML ±0.1 (Gourley et al. 2008a) infused over 2 min with needles left in place for 2 min after infusion.Several studies indicate BDNF has behavioral effects for several days after infusion into the striatum (Horger et al. 1999), ventral tegmental area (Lu et al. 2004), hippocampus (Shirayama et al. 2002; Gourley et al. 2008b), and prefrontal cortex (Berglind et al. 2007, 2009). Therefore, we utilized a single-infusion protocol: Food restriction resumed on day 5 after surgery, at which point mice appeared active. Testing resumed on day 7, at which point mice were subjected to three nonreinforced test sessions. bdnf knock-down mice were affected during the first and second sessions only, so this protocol would be expected to capture the window during which BDNF had effects, if any. These mice showed the typical reduction of responding across sessions (F_(2,14) = 8.6, P = 0.004) (Fig. 2). It is worth noting that responding in control mice was lower than in previous experiments; this is likely due to the more limited recovery and food restriction time after surgery. Nonetheless, we found no effect of BDNF on responding (F < 1; infusion × session interaction F_(2,14) = 1.4, P = 0.3).Open in a separate windowFigure 2.Effects of PLc BDNF microinfusion. Mice were initially trained to perform the nose poke response for food. Responses on the active aperture during training are shown at left. Mice were then infused with BDNF; subsequent instrumental responding during extinction was unaffected. (Inset) Adrenal glands were extracted and weighed after the last extinction session as a measure expected to be sensitive to PLc manipulations. Here, BDNF decreased gland weights (represented as the weight of both glands normalized to total body weight). Symbols represent means (+ SEM) per group, *P < 0.05.To verify a physiological response to PLc BDNF infusions (despite a lack of behavioral effect), we rapidly euthanized mice after the last session and extracted and weighed the adrenal glands, which secrete the hormone, corticosterone. Corticosterone secretion is sensitive to medial prefrontal cortex lesions (Diorio et al. 1993; Rangel et al. 2003) and noradrenergic depletion (Radley et al. 2008), and adrenal weights correlate with PLc BDNF expression levels (Gourley et al. 2008a). As expected, BDNF-infused mice had lighter adrenal glands (t₍₁₀₎ = 4.2, P = 0.002) (Fig. 2), indicating effects of BDNF infusion were detectable on this measure, though not on response diminution per se.Local bdnf knock-down could conceivably act in part by retarding anterograde BDNF transport to, or BDNF synthesis in, major PLc projections sites (Sobreviela et al. 1996; Altar et al. 1997; Conner et al. 1997; Kokaia et al. 1998). BDNF in those projection regions—the dorsal and ventral striatum and multiple hypothalamic subregions (Öngür and Price 2000)—as well as in the PLc itself, was therefore quantified by enzyme-linked immunosorbent assay (ELISA; Promega) in knock-down, control, and BDNF-infused mice.Brains were rapidly harvested from extinguished mice in Figures 1A and ​and2,2, and frozen and sliced into 1-mm-thick coronal sections. Brain regions were dissected bilaterally or with a single midline extraction by tissue punch (1.2-mm diameter). Tissue was then sonicated in lysis buffer (200 μL: 137 mM NaCl, 20 mM tris-Hcl [pH 8], 1% igepal, 10% glycerol, 1:100 Phosphatase Inhibitor Cocktails 1 and 2; Sigma) and stored at −80°C. ELISAs were conducted using 65 μL/sample/well and in accordance with manufacturer''s instructions. BDNF concentrations were normalized to each sample''s total protein concentration, as determined by Bradford colorimetric protein assay (Pierce). BDNF was analyzed by ANOVA or ANOVA-on-Ranks for non-normally distributed PLc values.In the PLc, BDNF was diminished in bdnf knock-down mice as expected (H_(2,18) = 0.2, P = 0.006, post-hoc Ps < 0.05), but BDNF expression in BDNF-infused mice did not differ from the control group (P > 0.05) (Fig. 3A). BDNF in the hypothalamus (F_(2,19) = 2.6, P = 0.1) and nucleus accumbens (F < 1) was not affected. By contrast, dorsal (primarily dorsomedial) striatal BDNF expression differed between groups (F_(2,20) = 5.4, P = 0.01), with knock-down mice expressing less BDNF than the BDNF-infused group (P = 0.01). BDNF in knock-down mice did not, however, significantly differ from control mice (P = 0.09).Open in a separate windowFigure 3.Quantification of BDNF in the PLc, dentate gyrus, and downstream projection sites. (A) BDNF was quantified in the PLc and major projection sites after viral-mediated gene knock-down or acute microinfusion. BDNF was diminished in the PLc of knock-down mice as expected. BDNF was also reduced in the dorsal striatum (dstri) of these animals, while other regions were unaffected by this manipulation. NAC refers to the nucleus accumbens. (B) To confirm the effects of acute BDNF infusion could be detected under some circumstances, tissue from mice infused with BDNF into the dentate gyrus (dentate) was also analyzed. Under these circumstances, elevated BDNF was detected in the hypothalamus. *P < 0.05 relative to control and BDNF-infused groups; ^§P < 0.05 relative to BDNF-infused mice; and P = 0.09 relative to control mice.For additional analyses, we conducted ELISAs on tissue from drug-naïve mice that had had a BDNF infusion of the same volume and concentration in the dorsal hippocampus, rather than PLc. As here, these animals had been subsequently tested in an instrumental conditioning task and were sacrificed 7 d after infusion (for behavioral reports, see Fig. 4 in Gourley et al. 2008b). Like the PLc, the hippocampus projects to the striatum and hypothalamus (Groenewegen et al. 1987; Kishi et al. 2000). In this instance of acute hippocampal infusion, BDNF expression was increased in hypothalamic samples (infusion × brain region interaction F_(3,27) = 3.5, P = 0.03, post-hoc P = 0.009), consistent with previous findings (Sobreviela et al. 1996). Other regions were not affected (Ps > 0.6) (Fig. 3B).Taken together, these data indicate long-term distal effects of acute BDNF infusion are detectable when BDNF is infused into the dorsal hippocampus, though not necessarily PLc. Our data do not preclude the possibility, however, that acute PLc BDNF infusion has long-term consequences for BDNF-regulated intracellular signaling cascades in these downstream sites. For example, extracellular-signal regulated kinase 1/2 phosphorylation in the nucleus accumbens is enhanced by single BDNF infusions aimed at the anterior cingulate/PLc border (Berglind et al. 2007).To summarize, we provide evidence for decreased responding in instrumentally trained mice with PLc-selective bdnf knock-down tested in extinction. Recall of extinction learning did not appear to be affected, as group differences were restricted to test sessions 1 and 2. Time of instrumental training was not a factor, as mice trained to respond for food both before and after knock-down showed a characteristically rapid decline in responding when reinforcement was withheld.Testing mice in a spatial “reversal” task, in which mice learn simultaneously to inhibit responding on one operant and respond instead on a previously nonreinforced operant, eliminated differences in nonreinforced responding between groups. In other words, in the presence of positive reinforcement, knock-down mice did not show exaggerated response inhibition. This behavioral pattern is consistent with the PLc''s role in maintaining goal-directed action particularly under low-reinforcement conditions (Corbit and Balleine 2003; Gourley et al. 2008a). If PLc bdnf played a more general role in extinction learning, one would expect PLc bdnf knock-down mice to show rapid response diminution regardless of whether reinforcement was readily available or not, but our reversal experiment clearly illustrated this was not the case.BDNF ELISA indicated the gene knock-down protocol utilized here results in an ∼48% reduction in BDNF within the PLc and a modest reduction in the downstream dorsal striatum, providing direct evidence for effects of bdnf knock-down on PLc projection neurons (though local interneurons would also be expected to have been infected). Such effects on striatal BDNF expression may be selective to chronic manipulations, as our acute infusion protocol had no consequences for expression in downstream regions, despite actions on a peripheral measure (adrenal gland weight) and evidence of downstream effects after hippocampal infusion.While we report bdnf knock-down rapidly decreased responding early in extinction, we found that acute BDNF infusion had no effects. How might we reconcile these findings? First, it is possible that prefrontal BDNF overexpression must be chronic to have behavioral effects in this task. Second, supraphysiological BDNF-induced structural destabilization and neuronal remodeling (Horch et al. 1999; Horch and Katz 2002) or activation of cortical interneurons (Rutherford et al. 1998) may have counteracted any effects on extinction. Cortical interneuron activation in particular—a process thought to stabilize cortical activity to maintain homeostasis in local circuits—could conceivably negate any effects of BDNF infusion on prefrontal projection neurons (cf., Turrigiano and Nelson 2004; see also Berglind et al. 2007). Last, while single prefrontal BDNF infusions have been reported to suppress cue-induced drug-seeking behavior (Berglind et al. 2007, 2009), such effects may be more acute and/or selectively mediated by Pavlovian, rather than instrumental, processes.Traditionally, extinction research has focused on Pavlovian fear extinction, in which the infralimbic cortex, and not PLc, is considered the major regulatory site (Quirk and Mueller 2008). Our findings suggest the PLc may, however, be indirectly involved in instrumental extinction, as bdnf knock-down facilitated rapid response diminution in the absence of reinforcement, but not when a competing response was reinforced. These findings are consistent with the idea that under normal circumstances, the PLc invigorates responding by maintaining sensitivity to reinforcement previously available upon completion of a particular instrumental action (Corbit and Balleine 2003) or previously associated with a Pavlovian cue (Vidal-Gonzalez et al. 2006). Future studies will address whether PLc BDNF is indeed critical to the maintenance of action–outcome behavior, since the mechanisms of goal-directedness are not well-characterized. This is despite the possibility that their identification may aid in therapeutically facilitating goal-directed action when response extinction is an unproductive behavioral choice.     

Distinct roles for dorsal CA3 and CA1 in memory for sequential nonspatial events

Previous studies have suggested that dorsal hippocampal areas CA3 and CA1 are both involved in representing sequences of events that compose unique episodes. However, it is uncertain whether the contribution of CA3 is restricted to spatial information, and it is unclear whether CA1 encodes order per se or contributes by an active maintenance of memories of sequential events. Here, we developed a new behavioral task that examines memory for the order of sequential nonspatial events presented as trial-unique odor pairings. When the interval between odors within a studied pair was brief (3 sec), bilateral dorsal CA3 lesions severely disrupted memory for their order, whereas dorsal CA1 lesions did not affect performance. However, when the inter-item interval was extended to 10 sec, CA1 lesions, as well as CA3 lesions, severely disrupted performance. These findings suggest that the role of CA3 in sequence memory is not limited to spatial information, but rather appears to be a fundamental property of CA3 function. In contrast, CA1 becomes involved when memories for events must be held or sequenced over long intervals. Thus, CA3 and CA1 are both involved in memory for sequential nonspatial events that compose unique experiences, and these areas play different roles that are distinguished by the duration of time that must be bridged between key events.Episodic memory involves the ability to encode and retrieve the order of events in individual experiences (Tulving 1983). Recent evidence in both animals and humans indicates that the hippocampus plays a critical role in this capacity. In animals, damage to the hippocampus impairs memory for the order of associated elements that compose an episode (Fortin et al. 2002; Kesner et al. 2002), and hippocampal neuronal activity reflects processing of the order of events in both spatial (Dragoi and Buzsáki 2006; Foster and Wilson 2007) and nonspatial episodes (Manns et al. 2007). In humans, hippocampal activation has also been related to memory for the order of elements (Kumaran and Maguire 2006; Lehn et al. 2009; Ross et al. 2009).Within the hippocampal circuitry, contributions of the CA3 and CA1 fields are probably most extensively studied, but this work has not yet clarified the distinct roles of these areas in sequence memory. Computational models suggest that the recurrent connections of CA3 cells operate as an attractor network that computes associations between elements (Norman and O''Reilly 2003; Rolls 2007) and is suitable for representing sequences of events in episodic memories (Jensen and Lisman 1996; Levy 1996; Lisman 1999). Studies on the effects of selective damage within the hippocampus have shown that CA3 is critical for remembering sequences of spatial locations (Hunsaker et al. 2008a), but not sequences of nonspatial events (Hoge and Kesner 2007). It is, therefore, uncertain whether CA3 is critical for sequence memory per se, rather than other aspects of spatial processing. Other observations suggest that CA1 may be involved in memory for the order of both spatial (Hunsaker et al. 2008a) and nonspatial stimuli (Hoge and Kesner 2007; Manns et al. 2007). However, it is not clear whether the contribution of CA1 involves integrating sequential elements of a memory or instead participates by active maintenance of event memories that underlies bridging sequential events in an episode (Kesner et al. 2005).To shed light on these issues, we compared the effects of selective damage to CA3 and CA1 on memory for the order of nonspatial events that occurred in unique episodes. We designed a task, based on the delayed-nonmatching-to-sample test, wherein subjects were required to remember the order of two sequentially presented stimuli in trial–unique-paired associations (Fig. 1).Open in a separate windowFigure 1.Test of memory for the order of stimuli in trial-unique odor pairs. At study, animals were presented with 10 odor-paired associates and odors in a pair were presented one at a time. At test, animals were presented with the same 10 odor pairs and were required to distinguish pairs where the odors within a pair were presented in the same order as during study (“old”) from pairs where the odors were presented in the reverse order (“new”). Old and new order test pairs were presented in a pseudorandom order. The first odor in each test pair acted as a cue to the ordering of the odors within a test pair; the animal was required to place its nose over the cup, but no digging response was required or rewarded. When the second cup was presented, the animal could dig to retrieve a reward if the order was new. If the order was old, the animal was required to approach an empty cup in the back of the home cage to obtain reward.     

Memory deficits are associated with impaired ability to modulate neuronal excitability in middle-aged mice

Normal aging disrupts hippocampal neuroplasticity and learning and memory. Aging deficits were exposed in a subset (30%) of middle-aged mice that performed below criterion on a hippocampal-dependent contextual fear conditioning task. Basal neuronal excitability was comparable in middle-aged and young mice, but learning-related modulation of the post-burst afterhyperpolarization (AHP)—a general mechanism engaged during learning—was impaired in CA1 neurons from middle-aged weak learners. Thus, modulation of neuronal excitability is critical for retention of context fear in middle-aged mice. Disruption of AHP plasticity may contribute to contextual fear deficits in middle-aged mice—a model of age-associated cognitive decline (AACD).Plasticity of intrinsic neuronal excitability increases the overall storage capacity of neurons and therefore likely plays a critical role in learning and memory (Zhang and Linden 2003). Increased neuronal excitability via reductions of the post-burst afterhyperpolarization (AHP) is hypothesized as a general mechanism underlying learning and memory tasks (Disterhoft et al. 1986; Disterhoft and Oh 2006). The AHP serves to limit subsequent firing in response to excitation (Madison and Nicoll 1984; Lancaster and Adams 1986; Storm 1990; Sah and Bekkers 1996). Generally speaking, the size of the AHP is inversely related to neuronal excitability, and the measurement of the AHP is routinely used as an index of neuronal excitability.Our laboratory and others have shown that AHP reductions are observed in hippocampal neurons from animals that learn hippocampal-dependent tasks including trace eyeblink conditioning in rabbit and rat (de Jonge et al. 1990; Moyer Jr et al. 1996, 2000; Kuo 2004) and spatial water maze in rat and mouse (Oh et al. 2003; Tombaugh et al. 2005; Ohno et al. 2006b). Learning-related reductions in the AHP have also been observed in cortical neurons following odor discrimination (Saar et al. 1998) and extinction learning (Santini et al. 2008). In vitro, activity-dependent plasticity of the AHP is induced using physiologically relevant stimuli (Kaczorowski et al. 2007). Because the AHP serves to limit subsequent firing, learning-related reductions in the AHP are poised to facilitate mechanisms crucial for information storage, such as long-term potentiation (LTP), synaptic integration (Sah and Bekkers 1996), metaplasticity (Le Ray et al. 2004), and spike-timing dependent plasticity (STDP) (Le Ray et al. 2004).Hippocampal neurons from naïve aged rodents and rabbits show a decrement in basal excitability evidenced by a robust enhancement of the AHP (Landfield and Pitler 1984; Moyer Jr et al. 1992, 2000; Oh et al. 1999; Kumar and Foster 2002, 2004; Power et al. 2002; Hemond and Jaffe 2005; Murphy et al. 2006b; Gant and Thibault 2008). Enhancement of the AHP in hippocampal neurons in aged animals correlates with impaired performance on learning paradigms that depend on a functional hippocampus, such as trace eyeblink and spatial water maze (Moyer Jr et al. 2000; Tombaugh et al. 2005; Murphy et al. 2006a). Pharmaceuticals aimed at reducing the AHP and increasing basal excitability (Moyer Jr et al. 1992; Moyer Jr and Disterhoft 1994) have been successful at restoring performance of aged rats on trace eyeblink conditioning (Deyo et al. 1989; Straube et al. 1990; Kowalska and Disterhoft 1994). Interestingly, AHPs from neurons recorded from aged learners are indistinguishable from young learners; both are reduced compared to that of aged weak-learners (Moyer Jr et al. 2000; Tombaugh et al. 2005). These data suggest that mechanisms that permit learning-related modulation of the AHP are also critical determinants of learning abilities in an aged population. To date, age-related impairments in hippocampal-dependent tasks and biophysical alterations in hippocampal neurons have largely focused on studies that compare animals at extreme ends of the aging spectrum.In an effort to better understand physiological changes that underlie the onset of early cognitive decline, the development of rodent models of “normal” age-associated cognitive decline (AACD), as well as mild cognitive impairment (MCI), is critical (Pepeu 2004). Therefore, we set out to characterize the development of age-related deficits indicative of hippocampal dysfunction in middle-aged C57Bl6/SJL mice and to examine the biophysical changes in hippocampal neurons that accompany such deficits.Recently, age-related deficits in contextual fear memory following trace fear conditioning were reported in a subset of middle-aged rats (Moyer Jr and Brown 2006). Because the dorsal hippocampus is critical for trace and contextual fear conditioning in mice and rats (McEchron et al. 1998; Chowdhury et al. 2005; Misane et al. 2005), trace fear conditioning is an ideal paradigm for exploring cellular mechanisms that underlie early-age-related cognitive decline.Here we investigate the effects of “early” aging on trace fear conditioning by comparing performance outcomes of young (2 mo, n = 7; and 4 mo, n = 8) and middle-aged (8 mo, n = 22) male C57/SJL F1 hybrid mice. Mice were trained and tested singly, and the experimenter was blind to the training and retention status of the mice. All animal procedures were approved by the Northwestern University Animal Care and Use Committee. Preliminary data were reported previously (Kaczorowski 2006).To assess hippocampal function with aging, young and middle-aged mice were trained on a trace fear conditioning task followed by retention tests of the auditory conditioned stimulus (CS) and contextual CS memory. The basic protocol for trace fear conditioning has been described previously (Ohno et al. 2006a). Mice were trained in a Plexiglas conditioning chamber with a stainless-steel floor grid used for shock delivery. After the baseline period (150 sec), mice received four pairings of the CS (tone; 15 sec, 3 kHz, 75 dB) and US (shock; 1 sec, 0.7 mA). The CS and unconditioned stimulus (US) were separated by a 30-sec empty trace interval. The intertrial interval was set at 210 ± 10 sec. The training chamber was wiped with 95% ethyl alcohol, illuminated with a 10-W bulb in an otherwise dark room, and provided with 65-dB white noise to make it distinct. During training on trace fear conditioning, no effect of age was observed on measures of baseline freezing (F_(2,34) = 2.0, P = 0.15), the expression of freezing during tone (F_(2,34) = 0.6, P = 0.6), or post-shock freezing (F_(2,34) = 0.2, P = 0.8), suggesting that middle-aged and young mice do not differ in measures of anxiolysis or expression of behavioral freezing (measured index of fear) (Fig. 1A).Open in a separate windowFigure 1.Onset of early aging deficits in 8-mo-old middle-aged mice. (A) Baseline (BL) freezing and auditory CS freezing during trace fear conditioning was similar between young (2 mo and 4 mo) and middle-aged (8 mo) mice. (B1) Mean baseline freezing and retention of the auditory CS memory (tones 1–4) were comparable in young (2 mo and 4 mo) and middle-aged (8 mo) mice. (B2) Middle-aged mice showed a significant decrease in freezing compared to young (2 mo and 4 mo) mice when exposed to the original context chamber where they had been trained 1 d earlier; (*) P < 0.05.Retention of the auditory CS:US memory was tested 24 h later in a novel context that differed in its location, size, scent, lighting, background noise, and flooring (bedding) compared to the training chamber. Data from three mice (one young, two middle-aged) were excluded because of video malfunction. Following a 150-sec baseline, mice received four presentations of the tone CS in the absence of footshock. Neither baseline freezing (F_(2,31) = 2.6, P = 0.1) nor conditional freezing in response to the tone CS (F_(2,31) = 1.6, P = 0.2) differed between young and middle-aged mice (Fig. 1B1). Thus, retention of the auditory CS following trace fear conditioning was intact in middle-aged compared to young mice. Although deficits in retention of auditory trace fear have been reported in aged mice and rats (Blank et al. 2003; McEchron et al. 2004; Villarreal et al. 2004), the results herein agree with report of intact trace fear memory in middle-aged rats (Moyer Jr and Brown 2006).One hour after this testing, retention of the contextual fear memory was assessed by placing mice in the original context (in the absence of the tone and footshock) and measuring freezing for 10 min. A subtle but significant difference in freezing was observed as a function of age (F_(2,31) = 4.3, P = 0.02; Fig. 1B ​2).2). A student''s post-hoc t-test revealed that mean freezing (collapsed across 10 min) of middle-aged mice was reduced compared to 2-mo (P < 0.05) and 4-mo (P < 0.05) young mice. Contextual fear memory deficits have been similarly reported in aged mice (Fukushima et al. 2008). Studies that failed to observe contextual fear deficits in aged (>18 mo) mice may result from a floor effect because young mice showed weak conditioning to the context (∼30% freezing) (Feiro and Gould 2005; Gould and Feiro 2005) or employment of delay (Corcoran et al. 2002; Feiro and Gould 2005; Gould and Feiro 2005) compared to trace procedures (Moyer Jr and Brown 2006). Although differences in experimental parameters are plausible, heterogeneity in the performance of aged mice may make detection of age-related impairments difficult owing to increased variability.Open in a separate windowFigure 2.Selective deficits on retention of contextual fear in middle-aged weak-learner mice. (A,B) Summary plot and histogram show young mice (100%, n = 6) at 2 mo of age showed robust recall of contextual fear memory (range 75%–99%) with mean and standard deviation (SD) of 91% ± 10%, whereas retention of middle-aged mice (n = 21) varied to a much greater extent (range 21%–95%; M, SD = 74% ± 19%). Distribution of middle-aged mice relative to their mean percent freezing shows two distinct populations. Middle-aged mice with freezing levels less than 3 SD from the mean freezing in young wild-type (WT) mice (61%, dashed line) were characterized as having weak contextual fear memory (weak learners) and those with freezing levels ≥62% as having strong contextual fear memory (learners). (C) Baseline (BL) freezing, expression of post-shock freezing and freezing during retention tests for auditory CS and trace CS memories, were comparable in both weak-learners and learners. (D) Selective deficits in retention of contextual fear memories were observed in middle-aged weak learners as compared to middle-aged learners; (*) P < 0.05.Previous studies in the rat report heterogeneity in spatial water maze and contextual fear conditioning in middle-aged and/or aged rats compared to young animals (Fischer et al. 1992; Wyss et al. 2000; Moyer Jr and Brown 2006). Therefore, we determined if middle-aged impairments of context fear (Fig. 2A) were driven by a subset of impaired mice. The degree of age-related impairment in each middle-aged mouse was determined by comparison to a reference group of young mice tested concurrently (shown in Fig. 1). The behavioral criterion for retention of contextual fear in middle-aged mice was set at 61%, which was 3 standard deviations (SD) below the mean freezing in young mice (mean and SD, 91% ± 10%; Fig. 1). A bimodal distribution of freezing of middle-aged mice was observed (Fig. 2B), where 70% of middle-aged mice performed above criterion and were labeled learners (n = 14), and 30% of middle-aged mice performed below criterion and were labeled as weak learners (n = 6). Comparison on measures of baseline freezing (F_(1,18) = 1.8, P = 0.2) and expression of post-shock freezing (F_(1,18) = 2.1, P = 0.2) revealed no differences between the groups during auditory trace fear training (Fig. 2C). Similarly, no differences in baseline freezing (F_(1,18) = 0.03, P = 0.9) or acquisition/recall of conditioned auditory trace fear (tone, F_(1,18) = 0.08, P = 0.8; trace, F_(1,18) = 2.4, P = 0.1) were observed 24 h later during retention tests. Thus, deficits ascribed to middle-aged weak learners were limited to contextual processing/retention, where middle-aged weak learners responded to the contextual CS with significantly lower levels of freezing compared to middle-aged learners (F_(1,18) = 47, P = 0.001; Fig. 2D). To summarize, we found that onset of cognitive decline in the C56Bl6/SJL mice was first apparent in a subset of middle-aged mice. Middle-aged weak learners showed a mild but specific deficit in hippocampal-dependent contextual learning/memory (spatial learning) but not hippocampal-dependent auditory trace learning/memory (temporal learning), assessed following trace fear conditioning.Given that contextual fear deficits occurred in a subset of middle-age mice, we were able to directly assess age-related alterations in excitability and AHP plasticity in CA1 neurons as they relate to learning abilities (learners vs. weak learners). Within 1 h of cessation of behavioral tests, middle-aged learners and weak learners were decapitated under deep halothane anesthesia and their brains quickly removed and placed into ice-cold artificial cerebral spinal fluid (aCSF): 125 mM NaCl, 25 mM glucose, 25 mM NaHCO₃, 2.5 mM KCl, 1.25 mM NaH₂PO₄, 2 mM CaCl₂, 1 MgCl₂ (pH 7.5, bubbled with 95%O₂/5%CO₂). Naïve mice were removed from their home cage and underwent identical decapitation procedures. Slices (300 μm) of the dorsal hippocampus and adjacent cortex were made using a Leica vibratome. The slices were first incubated for 30 min at 34°C in bubbled aCSF, and held at room temperature in bubbled aCSF for 1–4 h before use. Recording electrodes prepared from thin-walled capillary glass were filled with potassium methylsulfate-based internal solution and had a resistance of 5–6 MΩ.Whole-cell current-clamp recordings were performed on CA1 hippocampal pyramidal neurons of middle-aged learners (n = 36, 14 mice) and weak-learners (n = 15, 6 mice), as well as middle-aged naïve mice (n = 35 cells, 18 mice). Neuronal excitability was compared by measuring the post-burst AHP generated by 25 action potentials at 50 Hz (Fig. 3A), a stimulus shown to reliably evoke an AHP of sizable—but not maximal—amplitude from hippocampal neurons of mice (Ohno et al. 2006b). A significant difference in the peak amplitude of the AHP from learners, weak learners, and naïve mice was observed (F_(2,83) = 5, P < 0.01). Because the peak AHP and sAHP amplitudes did not differ between neurons from weak-learners and naïve mice (Fig. 3B). No differences in membrane resistance (F_(2,83) = 1.6, P = 0.2) or action potential properties, elicited using a brief (2 msec) near threshold current step (pA), were observed (Open in a separate window^aP < 0.05 compared to Weak L.^bP < 0.05 compared to Naïve.^cP < 0.05 compared to Pooled.Open in a separate windowFigure 3.Learning-related AHP plasticity is impaired in middle-aged weak-learner mice. (A) Representative traces showing the sAHP is reduced in neurons from (black) middle-aged learners compared to (blue) weak-learner mice and (gray) naïve mice. (Inset) The medium AHP (mAHP) of neurons from (black) learner mice was decreased compared to (blue) weak-learner mice and (gray) naïve mice. (B) No differences in the AHP from naïve and weak learners were observed; therefore, their data were pooled, and mean AHP was plotted by time on a log scale. (Inset) The mean amplitude of the peak AHP (1 msec) and sAHP (600 msec) was significantly reduced in neurons from learners compared to AHPs from weak learners and naïve labeled control; (*)P < 0.05.The results presented here are important in two respects. First, we demonstrate that the successful acquisition and recall of trace fear conditioning results in a significant reduction in the AHP in CA1 hippocampal neurons from the mouse. Our data are similar to previous reports showing learning-related reductions of the AHP in hippocampal neurons following training on hippocampal-dependent tasks (Disterhoft and Oh 2007) and thus strengthen the case for neuronal excitability change as a general mechanism underlying hippocampal-dependent learning. Second, we demonstrate that the onset of age-related cognitive decline in the C56Bl6/SJL mouse (termed “weak learners”) first manifests as a specific deficit in spatial associative learning in a subset of middle-age mice. These data, combined with a previous report from middle-aged rats (Moyer Jr and Brown 2006), suggest that initiation of age-related hippocampal dysfunction results in specific spatial—as opposed to temporal—deficits in associative learning and memory during middle age. By combining trace fear conditioning with whole-cell patch-clamp recordings in middle-aged mice, we revealed that “early” age-related impairments in spatial associative learning—like those in the aged hippocampus (Tombaugh et al. 2005)—result in part from an impairment of AHP plasticity of hippocampal neurons. Because AHP reductions are poised to facilitate mechanisms crucial for information storage, it is interesting that trace fear conditioning facilitates the long-term potentiation (LTP) of field excitatory postsynaptic potentials in the CA1 region of the rat hippocampus (Song et al. 2008).Generally speaking, both LTP and activation of AHP currents (I_AHP and sI_AHP) are sensitive to changes in intracellular Ca²⁺ (Storm 1990; Sah 1996; Malenka and Nicoll 1999). Thus, dysregulation of Ca²⁺ homeostasis in the hippocampus of middle-aged rats via enhancement of Ca²⁺-induced Ca²⁺ release (CICR) is an important finding (Gant et al. 2006). Age-related enhancement of Ca²⁺-dependent AHPs has been shown to raise the threshold for induction of LTP (Kumar and Foster 2004). These data support our hypothesis that impairments in contextual fear reported herein, as well as deficits in spatial water maze reported in middle-aged rats (Frick et al. 1995; Markowska 1999; Kadish et al. 2009), result from dysfunction of AHP plasticity.Studies in middle-aged mice have important implications for the treatment of “normal” age-associated cognitive decline (AACD), as well as mild cognitive impairment (MCI) (Pepeu 2004). Further studies aim to examine alterations in cholinergic function in our middle-aged mouse model, as the cholinergic agonist carbachol suppressed the AHP in neurons from naïve middle-aged mice (Supplemental Fig. 1). Activation of cholinergic receptors shape neuronal excitability and synaptic throughput (Tai et al. 2006) through multiple Ca²⁺-dependent processes (Gahwiler and Brown 1987; Tai et al. 2006). Restoration of cholinergic function has been shown to rescue deficits on hippocampal-dependent tasks in aged rodent and mouse models of Alzheimer''s disease (AD) (Disterhoft and Oh 2006), as well as in human AD patients (Cummings et al. 1998; Morris et al. 1998; Pettigrew et al. 1998), and therefore is a potential target aimed at the rescue of early age-related cognitive decline.     

New behavioral protocols to extend our knowledge of rodent object recognition memory

Animals often show an innate preference for novelty. This preference facilitates spontaneous exploration tasks of novelty discrimination (recognition memory). In response to limitations with standard spontaneous object recognition procedures for rodents, a new task (“bow-tie maze”) was devised. This task combines features of delayed nonmatching-to-sample with spontaneous exploration. The present study explored aspects of object recognition in the bow-tie maze not amenable to standard procedures. Two rat strains (Lister Hooded, Dark Agouti) displayed very reliable object recognition in both the light and dark, with the Lister Hooded strain showing superior performance (Experiment 1). These findings reveal the potential contribution of tactile and odor cues in object recognition. As the bow-tie maze task permits multiple trials within a session, it was possible to derive forgetting curves both within-session and between-sessions (Experiment 1). In Experiment 2, rats with hippocampal or fornix lesions performed at normal levels on the basic version of the recognition task, contrasting with the marked deficits previously seen after perirhinal cortex lesions. Next, the training protocol was adapted (Experiment 3), and this modified version was used successfully with mice (Experiment 4). The overall findings demonstrate the efficacy of this new behavioral task and advance our understanding of object recognition.Understanding the neural basis of recognition memory, the ability to discriminate whether a stimulus is novel or familiar, is heavily reliant on animal research. Here, advances have been closely tied to the introduction of new behavioral tests. The preeminent example concerns one-trial tests of recognition memory for monkeys using delayed nonmatching-to-sample (Mishkin and Delacour 1975). These tasks reward the natural preference that monkeys have for selecting novel items and permit multiple recognition trials within a single session. These features make the task relatively easy to train and then maximize findings from small group sizes. Although rat tasks closely based on delayed nonmatching-to-sample have been devised (Aggleton 1985; Mumby et al. 1990; Steckler et al. 1998; Prusky et al. 2004), they are very rarely employed as they are difficult to train and performance levels are unreliable.Almost all studies of rodent recognition memory now employ the spontaneous object recognition test and its direct variants (Ennaceur and Delacour 1988; Dix and Aggleton 1999; Winters et al. 2008). These tasks again take advantage of an innate preference for novel items, but this preference is displayed by spending more time exploring novel than familiar stimuli. In the standard version of the task, a rodent (rat or mouse) is placed in an arena containing two identical objects and then freely allowed to explore these objects for several minutes (“sample” phase). After a delay, the rodent is placed back in the arena (“test” phase), which now contains one familiar object (a copy of the sample phase objects) and a novel object. Recognition is signified by greater exploration of the novel object. Because the task measures spontaneous behavior, it requires minimal pretraining, but for the same reason it is prone to considerable variance. Unlike delayed nonmatching-to-sample, each trial (sample plus test phase) takes many minutes, and so only one recognition trial is normally given per session. Advantages are that proactive interference between objects is minimized, and the one-trial design lends itself to episodic-like tests of memory (Dere et al. 2005; Good et al. 2007). Disadvantages include the fact that data accumulation, with appropriate counterbalancing, is slow.To address these limitations, a new object recognition test using a “bow-tie maze” (Fig. 1A) has been developed for rats (Albasser et al. 2010). This test combines features of delayed nonmatching-to-sample with spontaneous object preference: It permits multiple trials per session, but the measure of recognition comes from the preferential exploration of novelty. The rat is first placed in one end of a bow-tie–shaped maze that contains a single object (object A; Fig. 1B). After a minute, the rat is allowed to run to the other end of the maze where there are two dissimilar objects (A and B; Fig. 1B). Object A is familiar as it is identical to the object previously explored, while object B is novel. Consequently, a rat will typically prefer to explore object B. On the next trial, a minute later, the rat shuttles back to the initial start point, but this time encounters objects B and C. Object B is now familiar, while object C is novel. The next trial, 1 min later, is between object C (now familiar) and object D (novel), and so on. A food reward placed under every object promotes shuttling back and forth within the maze, and encourages interaction with the objects. Unlike delayed nonmatching-to-sample, the food reward is not contingent on first selecting the novel object.Open in a separate windowFigure 1.(A) Schematic of the bow-tie maze. A sliding door separates the two ends of the maze in which two objects are placed. (B) General procedure showing the presentation order of the objects in the standard object recognition task. All objects are rewarded (+). (Arrow) Rat movements. (Black print) Novel objects, (gray print) familiar objects.The present study used the bow-tie maze to explore recognition memory on four fronts. In Experiment 1, recognition in the light and recognition in the dark were compared to help determine the cues available to detect object familiarity. It is known that rats can perform recognition tasks when solely reliant on visual cues (Aggleton 1985; Bartko et al. 2007; Winters and Reid 2010), but object recognition based on other modalities remains largely unexplored (Winters and Reid 2010). Potential cues for recognition include odor differences and tactile information. It is known that rats can discriminate novel from familiar olfactory cues (Otto and Eichenbaum 1992; Kesner et al. 2002; Fortin et al. 2004; Wolff et al. 2006), while tactile (e.g., vibrissae) cues can be used to distinguish surfaces (e.g., Birrell and Brown 2000). The bow-tie maze is ideal for studying object recognition in the dark as the rats are rewarded for visiting items in set locations (to receive food rewards), and so should readily approach the objects. In contrast, running the standard spontaneous object recognition test (in an arena) in the dark would be problematic as it is not clear how the rats would first appreciate the presence of the to-be-discriminated objects.An additional goal of Experiment 1 was to determine how readily the bow-tie maze could be used to compile within-session and between-session forgetting curves. A limitation with the standard spontaneous object task is that with only one trial per session it can be very time consuming to create forgetting curves, while within-session forgetting curves for individual animals are not feasible. These shortcomings create limitations when examining manipulations thought to affect memory. The possibility of deriving within-session forgetting curves is particularly appealing as: (1) it minimized the impact of those factors that introduce variance when performance is compared across sessions, and (2) the animal need not be removed from the maze, which could additionally disrupt performance, e.g., by increasing stress. A further component of Experiment 1 manipulated object memory strength by presenting objects either once (“single”) or six times (“repeated”). Recognition was tested after a 3-h delay with the twin goals of determining whether repeated presentation would aid performance and whether these performance levels would be sufficiently above chance so that they could be used to examine factors involved in longer term memory.At the same time, Experiment 1 provided the opportunity to compare two rat strains, Dark Agouti (DA) and Lister Hooded (LH). Previous studies suggest that the Dark Agouti strain might be particularly good at visual recognition tasks (Aggleton 1996), though others have argued that this strain has aberrant behavioral properties, including higher anxiety and higher levels of inappropriate nonspatial behaviors in spatial learning tasks (Mechan et al. 2002; Harker and Whishaw 2004; but see Aggleton and Vann 2004).Experiment 2 examined the ability of rats with either hippocampal or fornix lesions to perform object recognition in the bow-tie maze. There is a longstanding debate over the impact of hippocampal damage on recognition memory, with mixed findings coming from spontaneous object recognition tests (Clark et al. 2000; Mumby 2001; Gaskin et al. 2003; Winters et al. 2008). The bow-tie maze should prove informative as numerous trials can be run to assess the impact of selective brain lesions. Although in this initial study only short retention delays were examined, these same delays and conditions are highly sensitive to perirhinal cortex lesions (Aggleton et al. 2010; Horne et al. 2010), a brain region regarded as vital for recognition memory (Brown and Aggleton 2001). In Experiment 3 the training protocol changed so that objects did not cover food rewards. Rather, a single food reward was always placed between the test objects. This modification was examined because: (1) it would preclude any exploration scores that were simply derived from attempts to move the test objects in order to uncover the food reward, and (2) it would introduce a task variant that might be amenable to small rodents not able to move objects. Accordingly, Experiment 4 examined the performance of mice (strain C57Bl/6) on a test of object recognition based on the modified version of the bow-tie maze from Experiment 3.