Discrimination Learning In Mice Research Articles

An automated homecage system for multiwhisker detection and discrimination learning in mice.

Automated, homecage behavioral training for rodents has many advantages: it is low stress, requires little interaction with the experimenter, and can be easily manipulated to adapt to different experimental conditions. We have developed an inexpensive, Arduino-based, homecage training apparatus for sensory association training in freely-moving mice using multiwhisker air current stimulation coupled to a water reward. Animals learn this task readily, within 1–2 days of training, and performance progressively improves with training. We examined the parameters that regulate task acquisition using different stimulus intensities, directions, and reward valence. Learning was assessed by comparing anticipatory licking for the stimulus compared to the no-stimulus (blank) trials. At high stimulus intensities (>9 psi), animals showed markedly less participation in the task. Conversely, very weak air current intensities (1–2 psi) were not sufficient to generate rapid learning behavior. At intermediate stimulus intensities (5–6 psi), a majority of mice learned that the multiwhisker stimulus predicted the water reward after 24–48 hrs of training. Both exposure to isoflurane and lack of whiskers decreased animals’ ability to learn the task. Following training at an intermediate stimulus intensity, mice were able to transfer learning behavior when exposed to a lower stimulus intensity, an indicator of perceptual learning. Mice learned to discriminate between two directions of stimulation rapidly and accurately, even when the angular distance between the stimuli was <15 degrees. Switching the reward to a more desirable reward, aspartame, had little effect on learning trajectory. Our results show that a tactile association task in an automated homecage environment can be monitored by anticipatory licking to reveal rapid and progressive behavioral change. These Arduino-based, automated mouse cages enable high-throughput training that facilitate analysis of large numbers of genetically modified mice with targeted manipulations of neural activity.

Improved visual discrimination learning in mice with partial 5-HT2B gene deletion

The neurotransmitter serotonin (5-hydroxytryptamine, 5-HT) has been linked to multiple aspects of cognition. For example, in rodents, discrimination and reversal learning are altered by experimentally induced changes in brain serotonin levels, and reduced expression of the 5-HT2B receptor subtype in mice and humans is associated with decreased serotonergic tone and increased behavioral impulsivity. Serotonin modulates cognitive flexibility as well as fear and anxiety, but the specific contributions of 5-HT2B receptors to these behaviors is unknown. The current study assessed mice with partial Htr2b deletion for performance on a touchscreen-based pairwise visual discrimination and reversal learning task followed by a test of cued fear learning. Male Htr2b heterozygous mice (+/-) and littermate controls (+/+) were trained to discriminate between two visual stimuli presented on a touch-sensitive screen, one which predicted delivery of a 14-mg food pellet and the other which was not rewarded. Once discrimination performance criterion was attained, the stimulus-reward contingencies were reversed. Htr2b +/- mice were faster to reach discrimination criterion than +/+ controls, and made fewer errors. Htr2b +/- mice were also slower to make responses and collect rewards. Conversely, measures of reversal learning were not different between genotypes. Pavlovian cued fear conditioning was also normal in Htr2b +/-mice. These data demonstrate a selective improvement in touchscreen-based discrimination learning in mice with partial deletion of the 5-HT2B receptor, and provide further insight into the role of the 5-HT2B receptor in cognition.

Genetic inactivation of synaptosomal-associated protein 25 (SNAP-25) in adult hippocampal neural progenitors impairs pattern discrimination learning but not survival or structural maturation of newborn dentate granule cells.

Adult neurogenesis is necessary for proper cognition and behavior, however, the mechanisms that underlie the integration and maturation of newborn neurons into the pre-existing hippocampal circuit are not entirely known. In this study, we sought to determine the role of action potential (AP)-dependent synaptic transmission by adult-generated dentate granule cells (DGCs) in their survival and function within the existing circuitry. We used a triple transgenic mouse (NestinCreERT2 :Snap25fl/fl : tdTomato) to inducibly inactivate AP-dependent synaptic transmission within adult hippocampal progenitors and their progeny. Behavioral testing in a hippocampal-dependent A/B contextual fear-discrimination task revealed impaired discrimination learning in mice harboring SNAP-25-deficient adult-generated dentate granule cells (DGCs). Despite poor performance on this neurogenesis-dependent task, the production and survival of newborn DGCs was quantitatively unaltered in tamoxifen-treated NestinCreERT2 :Snap25fl/fl : tdTomato SNAP compared to tamoxifen-treated NestinCreERT2 :Snap25wt/wt : tdTomato control mice. Although SNAP-25-deficient adult DGCs displayed a small but statistically significant enhancement in proximal dendritic branching, their overall dendritic length and distal branching complexity was unchanged. SNAP-25-deficient newborn DGCs also displayed robust efferent mossy fiber output to CA3, with normal linear density of large mossy fiber terminals (LMTs). These studies suggest that AP-dependent neurotransmitter release by newborn DGCs is not essential for their survival or rudimentary structural maturation within the adult hippocampus.

High Resolution Quantitative Synaptic Proteome Profiling of Mouse Brain Regions After Auditory Discrimination Learning

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory discrimination learning in mice is described. In this learning paradigm, mice are trained in a shuttle box Go/NoGo-task to discriminate between rising and falling frequency-modulated tones in order to avoid a mild electric foot-shock. The protocol involves the enrichment of synaptosomes from four brain areas, namely the auditory cortex, frontal cortex, hippocampus, and striatum, at different stages of training. Synaptic protein expression patterns obtained from trained mice are compared to naïve controls using a proteomic approach. To achieve sufficient analytical depth, samples are fractionated in three different ways prior to mass spectrometry, namely 1D SDS-PAGE/in-gel digestion, in-solution digestion and phospho-peptide enrichment. High-resolution proteomic analysis on a mass spectrometer and label-free quantification are used to examine synaptic protein profiles in phospho-peptide-depleted and phospho-peptide-enriched fractions of synaptosomal protein samples. A commercial software package is utilized to reveal proteins and phospho-peptides with significantly regulated relative synaptic abundance levels (trained/naïve controls). Common and differential regulation modes for the synaptic proteome in the investigated brain regions of mice after training were observed. Subsequently, meta-analyses utilizing several databases are employed to identify underlying cellular functions and biological pathways.

High Resolution Quantitative Synaptic Proteome Profiling of Mouse Brain Regions After Auditory Discrimination Learning.

The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory discrimination learning in mice is described. In this learning paradigm, mice are trained in a shuttle box Go/NoGo-task to discriminate between rising and falling frequency-modulated tones in order to avoid a mild electric foot-shock. The protocol involves the enrichment of synaptosomes from four brain areas, namely the auditory cortex, frontal cortex, hippocampus, and striatum, at different stages of training. Synaptic protein expression patterns obtained from trained mice are compared to naïve controls using a proteomic approach. To achieve sufficient analytical depth, samples are fractionated in three different ways prior to mass spectrometry, namely 1D SDS-PAGE/in-gel digestion, in-solution digestion and phospho-peptide enrichment. High-resolution proteomic analysis on a mass spectrometer and label-free quantification are used to examine synaptic protein profiles in phospho-peptide-depleted and phospho-peptide-enriched fractions of synaptosomal protein samples. A commercial software package is utilized to reveal proteins and phospho-peptides with significantly regulated relative synaptic abundance levels (trained/naïve controls). Common and differential regulation modes for the synaptic proteome in the investigated brain regions of mice after training were observed. Subsequently, meta-analyses utilizing several databases are employed to identify underlying cellular functions and biological pathways.

Proteome rearrangements after auditory learning: high-resolution profiling of synapse-enriched protein fractions from mouse brain.

Learning and memory processes are accompanied by rearrangements of synaptic protein networks. While various studies have demonstrated the regulation of individual synaptic proteins during these processes, much less is known about the complex regulation of synaptic proteomes. Recently, we reported that auditory discrimination learning in mice is associated with a relative down‐regulation of proteins involved in the structural organization of synapses in various brain regions. Aiming at the identification of biological processes and signaling pathways involved in auditory memory formation, here, a label‐free quantification approach was utilized to identify regulated synaptic junctional proteins and phosphoproteins in the auditory cortex, frontal cortex, hippocampus, and striatum of mice 24 h after the learning experiment. Twenty proteins, including postsynaptic scaffolds, actin‐remodeling proteins, and RNA‐binding proteins, were regulated in at least three brain regions pointing to common, cross‐regional mechanisms. Most of the detected synaptic proteome changes were, however, restricted to individual brain regions. For example, several members of the Septin family of cytoskeletal proteins were up‐regulated only in the hippocampus, while Septin‐9 was down‐regulated in the hippocampus, the frontal cortex, and the striatum. Meta analyses utilizing several databases were employed to identify underlying cellular functions and biological pathways. Data are available via ProteomeExchange with identifier PXD003089. How does the protein composition of synapses change in different brain areas upon auditory learning? We unravel discrete proteome changes in mouse auditory cortex, frontal cortex, hippocampus, and striatum functionally implicated in the learning process. We identify not only common but also area‐specific biological pathways and cellular processes modulated 24 h after training, indicating individual contributions of the regions to memory processing.

Role of Muscarinic Acetylcholine Receptors in Serial Feature-Positive Discrimination Task during Eyeblink Conditioning in Mice.

We investigated the role of muscarinic acetylcholine receptors (mAChRs) in eyeblink serial feature-positive discrimination learning in mice using the mAChR antagonist. A 2-s light cue was delivered 5 or 6 s before the presentation of a 350-ms tone paired with a 100-ms periorbital electrical shock (cued trial) but not before the tone-alone presentation (non-cued trial). Mice received 30 cued and 30 non-cued trials each day in a random order. We found that saline-injected control mice were successfully discriminating between cued and non-cued trials within a few days of conditioning. The mice responded more frequently to the tone in cued trials than in non-cued trials. Analysis of conditioned response (CR) dynamics revealed that the CR onset latency was shorter in cued trials than in non-cued trials, despite the CR peak amplitude not differing significantly between the two conditions. In contrast, scopolamine-injected mice developed an equal number of CRs with similar temporal patterns irrespective of the presence of the cue during the 7 days of conditioning, indicating in a failure to acquire conditional discrimination. In addition, the scopolamine administration to the control mice after they had successfully acquired discrimination did not impair the conditional discrimination and expression of pre-acquired CR. These results suggest that mAChRs may play a pivotal role in memory formation in the conditional brain state associated with the feature cue; however they are unlikely to be involved in the development of discrimination after conditional memory had formed in the serial feature-positive discrimination task during eyeblink conditioning.

Automated Training Systems in Real and Virtual Worlds for Tactile Discrimination Learning in Mice

Somatic sensations are thought to be represented by the neural activity in the primary somatosensory cortex (S1), which can then be used for higher brain functions, such as decision-making, and memory. However, it is still unclear how neural activity represents these sensations and contributes to those functions. In a previous study, we showed that the intensity of limb stimulation in awake rodents could be coded by dendritic activity of layer 5 pyramidal neurons in S1(1). This implies that somatosensation of tactile stimuli can also be used as a primary mode of input for higher brain areas. To understand causal relationship between S1 activity and higher brain functions, we developed a new experimental system in which mice discriminated texture in a modified Y-maze. With this maze, mice were successfully trained to discriminate a specific tactile stimulus on the floor of the maze at an area just before the branch point. On the basis of these results, we designed a task in virtual world situations. This system also can present tactile stimulation while mice are running within the maze. Now, we are using this system for electrophysiological and optogenetic experiments, and trying to gain new knowledge in the field of neurobehavioral research.

Pavlovian contextual and instrumental biconditional discrimination learning in mice

Genetically-modified animal models are a powerful tool for investigating the link between neurological and behavioral changes and for the development of therapeutic interventions. Executive function deficits are symptomatic of many human clinical disorders but few tasks exist for studying executive functions in mice. To address this need, we describe procedures for establishing Pavlovian contextual and instrumental biconditional discriminations (BCDs) in C57BL/6J mice. In the first experiment, contextual cues disambiguated when two short duration stimulus targets would be followed by food pellets. In the second experiment, discrete visual cues signaled when lever press or nose poke responses would be continuously reinforced with food pellets. Mice learned both BCDs as evidenced by differential responding in each cue during training and, more critically, during extinction testing. The implications of these findings for using BCD tasks to analyze the neural substrates of executive processing in animal models are discussed.

Olfactory discrimination learning in mice lacking the fragile X mental retardation protein

An automated training system was used to compare the behavior of knockout (KO) mice lacking the fragile X mental retardation protein with that of wild-type (WT) mice (C57Bl/6 strain) in the acquisition and retention of olfactory discriminations. KO and WT mice did not differ in the acquisition of a four-stage nose poke shaping procedure. In two separate experiments, mutant mice required substantially more training to acquire a series of novel olfactory discrimination problems than did control mice. The KO mice required significantly more sessions to reach criterion performance, made significantly more errors during training, and more often failed to acquire discriminations. Both KO and WT mice showed similar error patterns when learning novel discriminations and both groups showed evidence of more rapid learning of later discriminations in the problem series. Both groups showed significant long-term memory two or four weeks after training but WT and KO mice did not differ in this regard. A group of well-trained mice were given training on novel odors in sessions limited to 20–80 trials. Memory of these problems at two day delays did not differ between WT and KO mice. Tests using ethyl acetate demonstrated that WT and KO mice had similar odor detection thresholds.

Impaired discrimination learning in mice lacking the NMDA receptor NR2A subunit

N-Methyl-D-aspartate receptors (NMDARs) mediate certain forms of synaptic plasticity and learning. We used a touchscreen system to assess NR2A subunit knockout mice (KO) for (1) pairwise visual discrimination and reversal learning and (2) acquisition and extinction of an instrumental response requiring no pairwise discrimination. NR2A KO mice exhibited significantly retarded discrimination learning. Performance on reversal was impaired in NR2A KO mice during the learning phase of the task; with no evidence of heightened perseverative responses. Acquisition and extinction of an instrumental behavior requiring no pairwise discrimination was normal in NR2A KO mice. The present findings demonstrate a significant and selective deficit in discrimination learning following loss of NR2A.

The parsing of food reward

what is it that is habit-forming about food? Sweet foods and fatty foods are habit-forming and sweet-fatty foods perhaps more than doubly so. Many kitchen and industrial experiments have gone into the determination of the amounts of sugar and butter to optimize the palatability of a given meal or

Holeboard discrimination learning in mice

We have adapted to mice a holeboard-learning task, which allows simultaneous assessment of spatial working and reference-memory performance. The holeboard apparatus consists of an open-field chamber with a 16-hole floor insert. Across trials, animals have to learn that the same four holes of 16 are always baited. Here, we show that C57BL/6 mice readily acquire this task within 4 days when submitted to six trials per day or within 8 days when submitted to only four trials per day. We also show that C57BL/6, Swiss-Webster, CD-1 and DBA/2 mice acquire this task similarly, despite the fact that some differences could be observed in measures of exploratory activity during habituation and training. Moreover, the muscarinic antagonist scopolamine disrupts learning at doses of 0.1 and 1.0 mg/kg, although the highest dose appeared to have side-effects. Lastly, we found that amyloid precursor protein transgenic mice have a selective disruption in their working-memory performance only during reversal training (i.e. after a change in the configuration of the baited holes). Overall, our data indicate that this spatial learning task is well adapted to mice and will be useful to characterize spatial memory in various genetic or pharmacological mouse models.

A simple and reliable test of olfactory learning and memory in mice.

The present paper describes a quick and efficient method for assessing olfactory discrimination learning in mice. In training mice received trials in which one odor (CS+) was paired with sugar and another odor (CS-) was paired with no sugar. When the mice were subsequently placed in a chamber with CS+ odor at one end and CS- odor at the other, they spent more time digging in CS+ than in CS- odor. In Experiment 2 mice trained with this procedure and tested after 60 days also spent more time digging in CS+ than CS- in the test phase, indicating that this olfactory discrimination task is effective for assessing long-term memory. In addition to the outbred strain of CD1 mice used in Experiments 1 and 2, C57Bl/6NCr/BR and DBA/2NCr/BR mice used in Experiment 3 also acquired this learned odor discrimination. Moreover, Experiment 4 showed that DBA animals were capable of acquiring this odor discrimination after receiving only two training trials (one exposure each to CS+ and CS-) per day for 4 days.

Interaction between the cholinergic system and CRH in the modulation of spatial discrimination learning in mice

Both cholinergic and CRH systems have been linked to cognitive processes such as learning and memory, and neuroanatomical as well as neurochemical evidence suggests important interactions between these two systems. Moreover, recent reports of pro-mnestic effects of CRH open the possibility that CRH could have beneficial effects in animals with cholinergic dysfunction. In a first experiment, spatial discrimination of C57BL/6 mice treated with various doses of scopolamine (0.5–2.0 mg/kg IP) was tested in a two-choice water maze task. Scopolamine, but not methylscopolamine, impaired accuracy and decreased responsivity. In contrast, similar doses of the nicotinic antagonist mecamylamine had no effect on choice accuracy but altered responsivity, as indicated by increased errors of omission and a reduction in swim speed during early experimental stages. ICV CRH (0.5–1.0 μg) also failed to significantly affect accuracy, but a strong tendency was observed to impair percentage correct responses. Measures of responsivity, such as errors of omission, choice latency and distance traveled, and of thigmotaxis were not significantly affected by CRH. However, initial swim speed was reduced by the peptide. Combined treatment with scopolamine (0.5 mg/kg IP) and CRH (0.5 μg ICV) had only mild, and primarily independent, effects, but overall suggested that concomitant blockade of muscarinic receptors and activation of the CRH system would rather act synergistically to disrupt spatial discrimination learning. Synergistic effects were also observed when animals receiving a combination of mecamylamine (2.0 mg/kg IP) and CRH (0.5 μg ICV) were tested, both in terms of responsivity and thigmotaxis, and there was limited evidence that part of these effects were potentiating. Thus, the cholinergic and CRH systems interact in the modulation of learning, but CRH, contrary to prediction, worsens the impairment caused by cholinergic blockade.

Magnetic field effects on spatial discrimination learning in mice

A magnetic sense has been demonstrated in several species, including mice. It has been suggested that the basis of this sense, and its role in navigation, rests on the existence of ferromagnetic materials in the brain. Several researchers have shown changes in navigation during exposure to relatively weak magnetic fields, which presumably interact with magnetite in the brain. In this study, mice were exposed to the powerful magnetic field of an MRI scanner and their left-right discrimination learning was compared with a control group. Results indicate a significant decrement in such learning ability immediately after exposure. Suggestions for future research are presented.

Effects of intrahippocampal injections of somatostatin and cysteamine on spatial discrimination learning in mice

The involvement of hippocampal somatostatin (SS-14) in spatial discrimination learning in mice was evaluated by bilaterally administering either SS-14 or its depletor, cysteamine, into the hippocampus through chronically implanted guide cannulae. Two separate experiments were performed using an eight-arm radial maze. In Experiment 1, mice were trained for 4 days on a discrimination task in which only three arms (A, B, and C) were used. In each daily session, subjects were each presented in 10 trials with the pair AB and the pair BC, with arm B consistently baited over trials (e.g., A − B + and B + C −). Immediately after completion of the fourth daily session with these regular trials, they were given 10 additional probe trials with free access to all three arms (e.g., A − B + C −). In Experiment 2, animals had free access to all eight arms. They were trained to discriminate the three arms that were consistently baited over trials (8 trials per day for 7 days). Intrahippocampal injections were given each day, 30 min before testing. The results of both experiments showed that, compared with saline, SS-14 administration (0.2 µg/0.2 µl per hippocampus) resulted in faster acquisition rates. In contrast, animals treated with cysteamine (20 µg) were dramatically impaired. In Experiment 1, however, the SS-14 treatment group was significantly impaired relative to both the saline and the intact control groups when testing was shifted from regular to probe trials. The impaired ability of somatostatin-treated mice to adapt to probe trials, together with their improved performance on both types of spatial discrimination task, suggests that this peptide produced a change in the strategy the animals used to solve these tasks. Notwithstanding, the present findings seem difficult to reconcile with current theories about hippocampal function.

A computer-controlled Y-maze for the analysis of vibrissotactile discrimination learning in mice

An automatized, computer-controlled Y-maze is described in which mice are trained to discriminate between two vibrissotactile, and/or visual stimuli, other modalities or combinations of modalities being testable as well. Movements of the mouse are recorded by photocells and monitored on a computer screen. Forward passage of the mouse is ensured by movable gates, and, if necessary, by brief air blows. Wrong choices are punished by air blows as well. The controller is a Hewlett-Packard Series 80 microcomputer with a 16 channel parallel input/output interface; programs are in BASIC. The program analyzes start latency, decision and homing time, side preference, inspections and ultimate choice, as well as choice strategies based on discrimination, left/right habits and short-term memory. Thus we can determine the nature of discrimination errors, and establish individual behavioral profiles of the animals. Results are both printed alphanumerically and plotted. The apparatus may be used for studying sensory physiology as well as cerebral lateralization, and drug or gene effects on memory and learning.

The contribution of the rewarded and the non-rewarded stimulus in visual discrimination learning in mice

The relative contribution of the rewarded and non-rewarded stimulus in the learning of a visual discrimination task in various species is unclear. A number of different parameters have been suggested as influencing the importance of the one over the other, among them the type of reinforcement. Our results show that with both positive reinforcement (using water as reward after a correct choice, and no reinforcement after an incorrect choice) and negative reinforcement (electroshock punishment after an incorrect choice) mice use the position of the incorrect stimulus as a cue for the discrimination. Mice trained by positive reinforcement without the added negative reinforcement of blocking the door on which the incorrect stimulus is projected, do not learn a brightness discrimination task. This suggested that the added negative reinforcement of the closed door had a strong influence on the learning behaviour in our experiments.

A simple decoder arrays to a for linking photocell microcomputer

Inputs from photocell arrays (such as those incorporated in mazes) to a microcomputer must be decoded. For up to 31 channels, this can be conveniently done by using cascaded 8-to-3 line priority encoders (74LS148) and five lines on a parallel data bus. The arrangement is expandable. It is currently used in an automatized Y-maze for vibrisso-tactile discrimination learning in mice.