Christopher J. Heath

The touchscreen operant platform for testing learning and memory in rats and mice

An increasingly popular method of assessing cognitive functions in rodents is the automated touchscreen platform, on which a number of different cognitive tests can be run in a manner very similar to touchscreen methods currently used to test human subjects. This methodology is low stress (using appetitive rather than aversive reinforcement), has high translational potential and lends itself to a high degree of standardization and throughput. Applications include the study of cognition in rodent models of psychiatric and neurodegenerative diseases (e.g., Alzheimers disease, schizophrenia, Huntingtons disease, frontotemporal dementia), as well as the characterization of the role of select brain regions, neurotransmitter systems and genes in rodents. This protocol describes how to perform four touchscreen assays of learning and memory: visual discrimination, object-location paired-associates learning, visuomotor conditional learning and autoshaping. It is accompanied by two further protocols (also published in this issue) that use the touchscreen platform to assess executive function, working memory and pattern separation.

Prenatal and Adolescent Exposure to Tobacco Smoke Modulates the Development of White Matter Microstructure

Prenatal exposure to maternal smoking has been linked to cognitive and auditory processing deficits in offspring. Preclinical studies have demonstrated that exposure to nicotine disrupts neurodevelopment during gestation and adolescence, possibly by disrupting the trophic effects of acetylcholine. Given recent clinical and preclinical work suggesting that neurocircuits that support auditory processing may be particularly vulnerable to developmental disruption by nicotine, we examined white matter microstructure in 67 adolescent smokers and nonsmokers with and without prenatal exposure to maternal smoking. The groups did not differ in age, educational attainment, IQ, years of parent education, or symptoms of inattention. Diffusion tensor anisotropy and anatomical magnetic resonance images were acquired, and auditory attention was assessed, in all subjects. Both prenatal exposure and adolescent exposure to tobacco smoke was associated with increased fractional anisotropy (FA) in anterior cortical white matter. Adolescent smoking was also associated with increased FA of regions of the internal capsule that contain auditory thalamocortical and corticofugal fibers. FA of the posterior limb of the left internal capsule was positively correlated with reaction time during performance of an auditory attention task in smokers but not in nonsmokers. Development of anterior cortical and internal capsule fibers may be particularly vulnerable to disruption in cholinergic signaling induced by nicotine in tobacco smoke. Nicotine-induced disruption of the development of auditory corticofugal fibers may interfere with the ability of these fibers to modulate ascending auditory signals, leading to greater noise and reduced efficiency of neurocircuitry that supports auditory processing.

Nicotine-induced plasticity during development: modulation of the cholinergic system and long-term consequences for circuits involved in attention and sensory processing.

Despite a great deal of progress, more than 10% of pregnant women in the USA smoke. Epidemiological studies have demonstrated correlations between developmental tobacco smoke exposure and sensory processing deficits, as well as a number of neuropsychiatric conditions, including attention deficit hyperactivity disorder. Significantly, data from animal models of developmental nicotine exposure have suggested that the nicotine in tobacco contributes significantly to the effects of developmental smoke exposure. Consequently, we hypothesize that nicotinic acetylcholine receptors (nAChRs) are important for setting and refining the strength of corticothalamic-thalamocortical loops during critical periods of development and that disruption of this process by developmental nicotine exposure can result in long-lasting dysregulation of sensory processing. The ability of nAChR activation to modulate synaptic plasticity is likely to underlie the effects of both endogenous cholinergic signaling and pharmacologically administered nicotine to alter cellular, physiological and behavioral processes during critical periods of development.

Nicotine‐induced phosphorylation of ERK in mouse primary cortical neurons: evidence for involvement of glutamatergic signaling and CaMKII

Extracellular signal‐regulated kinase (ERK) is activated in vivo in a number of brain areas by nicotine and other drugs of abuse. Here we show that nicotine stimulation of cultured mouse cortical neurons leads to a robust induction of ERK phosphorylation that is dependent on nicotine concentration and duration of exposure. Calcium/calmodulin‐dependent protein kinase II activity is necessary for nicotine‐induced ERK phosphorylation and neither cAMP‐dependent protein kinase or protein kinase C appear to be involved. Activity of glutamate receptors, L‐type voltage‐gated calcium channels, and voltage‐gated sodium channels are also required for nicotine‐induced ERK phosphorylation. Nicotine‐induced ERK phosphorylation was inhibited by high concentrations of mecamylamine, however it was not blocked by other broad nicotinic acetylcholine receptor (nAChR) inhibitors (including hexamethonium and chlorisondamine) or nAChR subtype selective inhibitors (such as methyllycaconitine, alpha‐bungarotoxin, dihydro‐beta‐erythroidine, and α‐conotoxin Au1B). In accord with these pharmacological results, nicotine‐induced ERK phosphorylation was normal in primary cultures made from β2 or α7 nAChR subunit knockout mice. The α3/β4 nAChR agonist cytisine did not induce ERK phosphorylation suggesting that α3/β4 nAChRs were not involved in this process. Taken together, these data define a necessary role for glutamatergic signaling and calcium/calmodulin‐dependent protein kinase II in nicotine‐induced ERK phosphorylation in cortical neurons and do not provide evidence for the involvement of classical nAChRs.

Cortico-Thalamic Connectivity is Vulnerable to Nicotine Exposure During Early Postnatal Development through α4/β2/α5 Nicotinic Acetylcholine Receptors

Tobacco smoke exposure during development can result in lasting alterations in sensory processing and attention. This suggests that some constituent of smoke, such as the primary addictive component, nicotine, alters neurodevelopment. Although many effects of developmental nicotine exposure have been identified in humans and animal models, very few mechanistic studies have identified the molecular and anatomical basis for a defined behavioral consequence of developmental exposure. We show in this study that a mouse model of developmental nicotine exposure results in hypersensitive passive avoidance in adulthood. We have used transgenic mice in which β2 subunit containing nicotinic acetylcholine receptors (β2* nAChRs) are expressed exclusively on corticothalamic neurons (β2 tr(CT) mice) to identify the receptor subtypes involved and also to define the circuit level site of action responsible for this persistent, nicotine-induced behavioral phenotype. Further characterization of the native nAChRs expressed in this circuit indicates that both (α4)2(β2)3 and (α4)2(β2)2α5 nAChR subtypes are present in corticothalamic projections. Consistent with a role for (α4)2(β2)2α5 nAChRs in mediating the effect of developmental nicotine exposure on adult passive avoidance behavior, constitutive deletion of the α5 nAChR subunit also alters this behavior. A critical period for this developmental consequence of nicotine exposure was defined by limiting exposure to the early post-natal period. Taken together, these studies identify a novel consequence of developmental nicotine exposure in the mouse, define the nAChR subtypes and neural circuit involved in this behavioral change and delimit the neurodevelopmental period critical for vulnerability to a behavioral alteration that persists into adulthood.

False recognition in a mouse model of Alzheimer's disease: rescue with sensory restriction and memantine

Alzheimer’s disease is commonly regarded as a loss of memory for past events. However, patients with Alzheimer’s disease seem not only to forget events but also to express false confidence in remembering events that have never happened. How and why false recognition occurs in such patients is currently unknown, and treatments targeting this specific mnemonic abnormality have not been attempted. Here, we used a modified object recognition paradigm to show that the tgCRND8 mouse—which overexpresses amyloid β and develops amyloid plaques similar to those in the brains of patients with Alzheimer’s disease—exhibits false recognition. Furthermore, we found that false recognition did not occur when tgCRND8 mice were kept in a dark, quiet chamber during the delay, paralleling previous findings in patients with mild cognitive impairment, which is often considered to be prodromal Alzheimer’s disease. Additionally, false recognition did not occur when mice were treated with the partial N-methyl-d-aspartic acid receptor antagonist memantine. In a subsequent experiment, we found abnormally enhanced N-methyl-d-aspartic acid receptor-dependent long-term depression in these mice, which could be normalized by treatment with memantine. We suggest that Alzheimer’s disease typical amyloid β pathology leads to aberrant synaptic plasticity, thereby making memory representations more susceptible to interfering sensory input, thus increasing the likelihood of false recognition. Parallels between these findings and those from the literature on Alzheimer’s disease and mild cognitive impairment suggest a mechanism underlying false recognition in these patients. The false recognition phenomenon may provide a novel paradigm for the discovery of potential therapies to treat the mnemonic dysfunction characteristic of this disease.

Allelic Variation of Calsyntenin 2 (CLSTN2) Modulates the Impact of Developmental Tobacco Smoke Exposure on Mnemonic Processing in Adolescents

BACKGROUND Exposure to nicotine in tobacco smoke during development has been linked to subsequent deficits in attention and memory. The present study tested for evidence that genetic variation may contribute to individual differences in vulnerability to the effects of developmental exposure to tobacco smoke on memory and medial temporal lobe function in adolescents. METHODS Verbal and visuospatial memory were assessed and functional magnetic resonance imaging (fMRI) data were acquired in 101 adolescents systematically characterized for prenatal and adolescent exposure to tobacco smoke, while they performed an encoding and recognition memory task. The impact of allelic variation at loci within CLSTN2 (encoding synaptic protein calsyntenin 2) and KIBRA, shown previously to modulate early and delayed recall of words, on the dependent measures was examined. RESULTS KIBRA genotype did not exert significant main or interacting effects with prenatal or adolescent exposure to tobacco smoke on verbal or visuospatial memory. Previous observations of a beneficial effect of the CLSTN2 C allele on verbal recall were replicated. Adolescent exposure to tobacco smoke reversed this beneficial effect and was associated with increased activation of parahippocampal gyrus during early and delayed recognition in CLTSN2 C allele carriers. While the CLSTN2 C allele conferred enhanced functional connectivity between brain regions subserving accurate verbal recognition, adolescent exposure to tobacco smoke reversed this effect. CONCLUSIONS These findings extend previous work demonstrating that calsyntenins play an essential role in learning and indicate that this role is modulated both by CLSTN2 genotype and, during adolescent development, by exposure to tobacco smoke.

An epigenetic mechanism mediates developmental nicotine effects on neuronal structure and behavior

Developmental nicotine exposure causes persistent changes in cortical neuron morphology and in behavior. We used microarray screening to identify master transcriptional or epigenetic regulators mediating these effects of nicotine and discovered increases in Ash2l mRNA, encoding a component of a histone methyltransferase complex. We therefore examined genome-wide changes in trimethylation of histone H3 on Lys4 (H3K4me3), a mark induced by the Ash2l complex associated with increased gene transcription. A large proportion of regulated promoter sites were involved in synapse maintenance. We found that Mef2c interacts with Ash2l and mediates changes in H3K4me3. Knockdown of Ash2l or Mef2c abolished nicotine-mediated alterations of dendritic complexity in vitro and in vivo, and attenuated nicotine-dependent changes in passive avoidance behavior. In contrast, overexpression mimicked nicotine-mediated alterations of neuronal structure and passive avoidance behavior. These studies identify Ash2l as a target induced by nicotinic stimulation that couples developmental nicotine exposure to changes in brain epigenetic marks, neuronal structure and behavior.

Oral nicotine consumption does not affect maternal care or early development in mice but results in modest hyperactivity in adolescence

Nicotine exposure during development can alter behavior in adulthood in mice. One route of nicotine administration that can mimic some of the dynamics of human smoking is administration of the drug to pregnant and nursing mice through the drinking water. It is critical to determine if nicotine administration has an impact on maternal behavior as such changes could lead to persistent behavioral alterations in the offspring, independent of the neuropharmacological effects of the drug. While a number of studies have detected nicotine exposure-induced changes, the effects of nicotine administration through the drinking water on maternal behavior in mice have not been examined comprehensively. In the current study we have compared maternal behaviors of C57BL/6J mice exposed to nicotine in the drinking water to behaviors of animals exposed to saccharin (vehicle) in the drinking water for the first 7days after birth of their litters and find no significant between-group differences in any behaviors measured except passive nursing. We have also assessed the effects of nicotine administration through the drinking water on postnatal weight gain of the pups and find no significant differences between groups. Open-field locomotor activity differences between exposed and unexposed offspring in adolescence were also assessed, with transient hyperactivity detected in nicotine-exposed mice. These data suggest that behavioral differences identified between animals exposed to nicotine through maternal drinking water administration are primarily due to the neuropharmacological effects of the drug and not due to effects of exposure on maternal behavior.

The NEWMEDS rodent touchscreen test battery for cognition relevant to schizophrenia

RationaleThe NEWMEDS initiative (Novel Methods leading to New Medications in Depression and Schizophrenia, http://www.newmeds-europe.com) is a large industrial-academic collaborative project aimed at developing new methods for drug discovery for schizophrenia. As part of this project, Work package 2 (WP02) has developed and validated a comprehensive battery of novel touchscreen tasks for rats and mice for assessing cognitive domains relevant to schizophrenia.ObjectivesThis article provides a review of the touchscreen battery of tasks for rats and mice for assessing cognitive domains relevant to schizophrenia and highlights validation data presented in several primary articles in this issue and elsewhere.MethodsThe battery consists of the five-choice serial reaction time task and a novel rodent continuous performance task for measuring attention, a three-stimulus visual reversal and the serial visual reversal task for measuring cognitive flexibility, novel non-matching to sample-based tasks for measuring spatial working memory and paired-associates learning for measuring long-term memory.ResultsThe rodent (i.e. both rats and mice) touchscreen operant chamber and battery has high translational value across species due to its emphasis on construct as well as face validity. In addition, it offers cognitive profiling of models of diseases with cognitive symptoms (not limited to schizophrenia) through a battery approach, whereby multiple cognitive constructs can be measured using the same apparatus, enabling comparisons of performance across tasks.ConclusionThis battery of tests constitutes an extensive tool package for both model characterisation and pre-clinical drug discovery.