International Journal of Comparative Psychology

This article describes the integration of neurobiological and molecular genetic approaches in the study of the function of the neuropeptide corticotropin-releasing factor (CRF). CRF is a particularly relevant subject for targeted genetic mutation because of its hypothesized critical role in hormonal and behavioral responses to stressors, its potential role in psychopathology, and the limited neuropharmacological agents available to increase or decrease function in this system. We review the strategy of targeted genetic mutation in neuropharmacology using research directed at understanding the function of CRF and related neuropeptides through molecular genetic manipulation of the levels of the endogenous agonist, its different receptors, and the binding protein. Genetically engineered approaches to functional analysis cross-validate experiments to promote the neurobiological elucidation of complex systems and provide critical information about constituent differences that contribute to individual differences in brain function.

The first generation of genetic mutations in mice, conventional transgenics and knockouts, have provided important new insights into many aspects of brain function, including complex behavior. Moreover, as genetic variants that contribute to the development of mental disorders are found, mice bearing these genes represent novel and powerful animal models of the disorders. However, these mutations are complicated by the fact that they generally occur at early stages of development and in many and perhaps most tissues of the body. This complicates the use of these mice for studies of gene function in adult organisms. The last five years has seen the development of second generation mutations in mice, in which the mutation (either loss or addition of a gene) can be controlled temporally and spatially, that is, the mutation can be induced in adult animals and targeted to a particular brain region and neuronal cell type. Although not yet routine, such inducible and cell-type specific mutations represent powerful new tools to understand the role of a particular gene in complex behavior.

Chronically elevated levels of corticotropin-releasing hormone (CRH) are implicated in human stress-related and affective disorders, including generalized anxiety disorder and major depression. To gain more insight into the relationship between hyperactivity of the CRH system and associated neuroendocrine, autonomic, physiological, and behavioral changes, we have developed a transgenic mouse model of CRH overproduction (CRHOE). In this study, we explored the behavioral consequences of chronic CRH overproduction in mice of the two available transgenic lines (CRH-OE2122 and CRH-OE2123) in paradigms measuring behavioral aspects of stress, anxiety, and depression. These paradigms include tasks based on free exploration of novel environments (unfamiliar homecage and unfamiliar open field), stress-induced hyperthermia, tests associated with anxiety- related behavior (elevated plus maze and light-dark box), and paradigms in which (anti-)depressant-like behaviors can be detected (tail suspension test and forced swim test). The only relatively consistent finding, although not always significant, was reduced locomotor activity in CRH-OE2122 mice, corresponding well with the known effects of CRH on locomotion. Contrary to our predictions, CRH-OE mice did not show an altered response to stress, and a phenotype indicative of increased anxiety and/or depression was not evident in CRH-OE mice.

Our laboratory has discovered that alterations in choline availability to the developing rat fetus lead to long-term changes in spatial and temporal memory function across the lifespan and associated changes in the septo-hippocamal system. The current study was undertaken to determine if performance on an attention task, believed to be relatively independent of septo-hippocampal function, was modified by changes in choline availability prior to birth. A sustained attention task was developed for mice that includes all the features of the 2-choice signal-detection procedure initially applied to rats (McGaughy & Sarter, 1995). Prenatal choline deficiency significantly impaired the ability of adult mice to sustain attention to a brief visual cue throughout a session as evidenced by decreased “Hits” and increased “Omissions” during the second-half of trials. In contrast, prenatal choline supplementation enhanced the ability of mice to detect visual cues but did not alter their ability to maintain attention throughout a session. These data support the view that the effects of alterations in choline availability on brain anatomy, physiology, and function likely extend beyond the septo-hippocampal system that modulates spatial memory. In the case of the sustained attention task, this likely includes cholinergic projections from the basal forebrain to neocortex.

Dopamine terminals in the hippocampus and prefrontal cortex modulate cognitive processes such as spatial learning and working memory. Because dopamine D4 receptors are expressed in these brain areas we have analyzed mutant mice lacking this receptor subtype (D rd4 -/-). Wild-type and D rd4 -/- mice were challenged in two spatial learning paradigms: the Morris water maze and an alternation T-maze. D rd4 -/- mice showed normal place learning ability to find a hidden platform based on spatial extra-maze cues. In addition, Drd4 -/- mice were able to find a new platform location with the same learning plasticity as wild type-mice. Spatial working memory assessed on a T maze showed that Drd4 -/- mice were more efficient than wild-type mice in acquiring the maximum plateau of correct alternation scores. These results provide further evidence that the functional consequence of lacking D4 receptors is more evident in behaviors dependent on the integrity of the prefrontal cortex.

Mice carrying a threonine286 to alanine mutation in subunit α of Ca2+/CaM-dependent protein kinase II (α-CaMKIIT286A) have been reported to show deficits in hippocampal long-term potentiation as well as spatial learning deficits similar to those induced in mice with hippocampal lesions. In this series, we sought to extend this analysis by investigating the performance of α-CaMKII mutants on other, potentially hippocampal, learning tasks. Experiment 1 examined fear conditioning, Experiment 2 examined the acquisition of free-operant instrumental conditioning and its dependence upon the action-outcome association, and Experiment 3 assessed interval timing using an instrument version of the peak procedure.We found evidence of a deficit in contextual fear conditioning in Experiment 1 and in peak timing in Experiment 3, but neither a deficit in fear conditioning to a discrete tone CS nor in the acquisition of instrumental conditioning in Experiment 2. Furthermore, all of the mice in Experiment 2 showed a normal instrumental outcome devaluation effect, suggesting that encoding of the action-outcome association was unaffected by this point mutation. It appears, therefore, that although learning in CaMKII mutants is affected on specific tasks, they do not have a general learning deficit, and that the influence of this mutation, and therefore, of α-CaMKII, is both localized anatomically and relatively specialized functionally.

The Morris water maze (MWM) has been widely used as a diagnostic tool to detect alterations in hippocampal-dependent learning induced by pharmacological or genetic manipulations in rats and mice. However, with the frequent use of the paradigm some questions have arisen regarding the complex nature of the effects of environmental and biological factors that influence behavioral performance of rodents in this task. One of the most contentious issues is whether MWM can consistently detect genetic differences independent of environmental, i.e., laboratory and experimental conditions. In the present paper, we demonstrate that changes in environmental factors, due to holding mice in large vs. small home cages, can lead to significant and robust spatial learning alterations in one inbred strain (C57BL/6) while having minimal or no effects on another (129/SVEV). The detected genotype-environment interaction underscores the need for experimenters to diligently control and document all possible environmental factors in order to make their results comparable across multiple test environments and laboratories.

Behavioral and pharmacological testing in mice has been revamped following the development of new tools for the manipulation of genetic information. We present the results from the peak procedure, an operant test that assesses the capacity to perceive, remember, and act upon temporal information. We studied the basic timing abilities in two different strains of mice, the C57Bl/6J and C3H/HeJ, and their response to psychoactive substances. Scopolamine and high doses of d-amphetamine disrupted performance by increasing response variability. The effect of d-amphetamine was particularly clear in C3H mice. Whereas scopolamine did not seem to affect the location of the response, the effect of a low dose of d-amphetamine, a leftward shift, was consistent with the hypothesis that it accelerates the internal time keeping mechanism. Physostigmine alone improved performance by reducing variability between trials without affecting the response location. Pretreatment with physostigmine partially blocked the deleterious effects of scopolamine. Methylphenidate did not have major effects on timing behavior in C57 but in the highest dose shifted the response of C3H mice to the left. The higher sensitivity of the C3H strain to the effects of d-amphetamine and methylphenidate support its value as an animal model of attention deficit disorder. The performance of mice in this temporal task was comparable to that observed in rats and pigeons, and seemed exquisitely sensitive to pharmacological manipulation.

Mice can be shown to process temporal information as if they use an internal stopwatch that can be run, stopped, and reset on command and whose speed of “ticking” is adjustable. In addition, interval-timing behavior can be separated into clock, memory, and decision stages of information processing such that one stage can be modified without changing the others. In order to demonstrate the efficacy of interval-timing procedures in the evaluation of behavioral phenotypes, proline transporter (PROT) deficient mice (+/+, +/-, and -/-) were assessed for motor control (Rotarod beam), spatial memory (Morris water-maze), and temporal generalization (peak-interval procedure) competency. The findings demonstrate that interval-timing procedures can be profitably integrated into a behavioral battery and used to selectively diagnose the psychological abnormalities associated with transgenic, knock-out, and knock-down mouse models of human diseases.

The memory mechanism carries information forward in time. Screening for genetic distortions in this information (screening for changes in the content of memory) is likely to do a better job of distinguishing between genetic effects on memory mechanisms and genetic effects on performance mechanisms than screening for changes in the strength of learned behavior. The peak procedure is the most commonly used screen for duration memory. Mice give peak data strikingly similar to data from the rat and the pigeon. The data, however, favor a model in which the decision criterion for starting the response (putting the head into the hole) and stopping the response are independently determined. For reasons we explain, this prevents the estimation of scalar memory error and memory variability from simple peak data.

The hypothesis that the standard acoustic startle response (ASR) paradigm contains the elements of interval timing was tested. Acoustic startle stimuli were presented at a 10-s interstimulus interval (ISI) for 100 trials leading to habituation of the ASR. The ISI was then changed to either a shorter (5-s) or a longer (15-s) duration using a betweensubjects design. Dishabituation of the ASR was used to measure the degree of temporal generalization for the interval-timing process. The ASR showed dishabituation at both shorter and longer ISI values on the first trial following the change in ISI. The dishabituation resulting from the change in ISI was temporary and the ASR rapidly returned to levels of response habituation showing rate sensitivity to the frequency of stimulus presentation. Interval timing may be a standard feature of this habituation paradigm, it serves to anticipate the time of occurrence of the subsequent stimulus and to prepare the startle response, and provides a computational dimension lacking in the habituation process per se.