Robert Gerlai

Gene-targeting studies of mammalian behavior: is it the mutation or the background genotype?

Gene targeting to create null mutations in mice is a powerful new tool in biology which will allow the molecular dissection of complex phenotypes such as mammalian brain function, and learning and memory. However, the attempt to interpret the phenotypical changes which arise in null-mutant mice is subject to several caveats. For example, the ability to disrupt a single gene in a targeted manner might lead one to overlook the effects of other genes. Ignoring the genetic background might lead to misinterpretation of results: the present article will demonstrate that the phenotypical abnormalities attributed to the null mutation in several molecular neurobiological studies could simply result from the effects of background genes.

Mice Lacking Metabotropic Glutamate Receptor 5 Show Impaired Learning and Reduced CA1 Long-Term Potentiation (LTP) But Normal CA3 LTP

Class I metabotropic glutamate receptors (mGluRs) have been postulated to play a role in synaptic plasticity. To test the involvement of one member of this class, we have recently generated mutant mice that express no mGluR5 but normal levels of other glutamate receptors. The CNS revealed normal development of gross anatomical features. To examine synaptic functions we measured evoked field EPSPs in the hippocampal slice. Measures of presynaptic function, such as paired pulse facilitation in mutant CA1 neurons, were normal. The response of mutant CA1 neurons to low concentrations of (1S,3R)−1-amino-cyclopentane-1,3-dicarboxylic acid (ACPD) was missing, which suggests that mGluR5 may be the primary high affinity ACPD receptor in these neurons. Long-term potentiation (LTP) in mGluR5 mutants was significantly reduced in the NMDA receptor (NMDAR)-dependent pathways such as the CA1 region and dentate gyrus of the hippocampus, whereas LTP remained intact in the mossy fiber synapses on the CA3 region, an NMDAR-independent pathway. Some of the difference in CA1 LTP could lie at the level of expression, because the reduction of LTP in the mutants was no longer observed 20 min after tetanus in the presence of 2-amino-5-phosphonopentanoate. We propose that mGluR5 plays a key regulatory role in NMDAR-dependent LTP. These mutant mice were also impaired in the acquisition and use of spatial information in both the Morris water maze and contextual information in the fear-conditioning test. This is consistent with the hypothesis that LTP in the CA1 region may underlie spatial learning and memory.

Drinks like a fish: zebra fish (Danio rerio) as a behavior genetic model to study alcohol effects

Zebra fish may be an ideal vertebrate model system for numerous human diseases with which the genetics and biological mechanisms of the disease may be studied. Zebra fish has been successfully used in developmental genetics, and recently, neurobiologists have also started to study this species. A potentially interesting target disease amenable for analysis with zebra fish is drug addiction, e.g. alcoholism. Although genetic tools to manipulate the genome of zebra fish are available, appropriate phenotypical testing methods are often lacking. In this paper, we describe basic behavioral tests to investigate the acute effects of alcohol on zebra fish. These behavioral paradigms will be useful for the genetic and biological analysis of acute and chronic drug effects as well as addiction. In addition to presenting findings for the acute effects of alcohol, we briefly describe our strategy for generating and screening mutants. We hope that our pilot work will facilitate the future development of behavioral tests and the use of zebra fish in the genetic analysis of the biological effects of drugs of abuse.

Enhanced LTP in Mice Deficient in the AMPA Receptor GluR2

AMPA receptors (AMPARs) are not thought to be involved in the induction of long-term potentiation (LTP), but may be involved in its expression via second messenger pathways. However, one subunit of the AMPARs, GluR2, is also known to control Ca2+ influx. To test whether GluR2 plays any role in the induction of LTP, we generated mice that lacked this subunit. In GluR2 mutants, LTP in the CA1 region of hippocampal slices was markedly enhanced (2-fold) and nonsaturating, whereas neuronal excitability and paired-pulse facilitation were normal. The 9-fold increase in Ca2+ permeability, in response to kainate application, suggests one possible mechanism for enhanced LTP. Mutant mice exhibited increased mortality, and those surviving showed reduced exploration and impaired motor coordination. These results suggest an important role for GluR2 in regulating synaptic plasticity and behavior.

VEGF antagonism reduces edema formation and tissue damage after ischemia/reperfusion injury in the mouse brain

VEGF is mitogenic, angiogenic, and a potent mediator of vascular permeability. VEGF causes extravasation of plasma protein in skin bioassays and increases hydraulic conductivity in isolated perfused microvessels. Reduced tissue oxygen tension triggers VEGF expression, and increased protein and mRNA levels for VEGF and its receptors (Flt-1, Flk-1/KDR) occur in the ischemic rat brain. Brain edema, provoked in part by enhanced cerebrovascular permeability, is a major complication in central nervous system pathologies, including head trauma and stroke. The role of VEGF in this pathology has remained elusive because of the lack of a suitable experimental antagonist. We used a novel fusion protein, mFlt(1-3)-IgG, which sequesters murine VEGF, to treat mice exposed to transient cortical ischemia followed by reperfusion. Using high-resolution magnetic resonance imaging, we found a significant reduction in volume of the edematous tissue 1 day after onset of ischemia in mice that received mFlt(1-3)-IgG. 8-12 weeks after treatment, measurements of the resultant infarct size revealed a significant sparing of cortical tissue. Regional cerebral blood flow was unaffected by the administration of mFlt(1-3)-IgG. These results demonstrate that antagonism of VEGF reduces ischemia/reperfusion-related brain edema and injury, implicating VEGF in the pathogenesis of stroke and related disorders.

Zebrafish as an emerging model for studying complex brain disorders

The zebrafish (Danio rerio) is rapidly becoming a popular model organism in pharmacogenetics and neuropharmacology. Both larval and adult zebrafish are currently used to increase our understanding of brain function, dysfunction, and their genetic and pharmacological modulation. Here we review the developing utility of zebrafish in the analysis of complex brain disorders (including, e.g., depression, autism, psychoses, drug abuse, and cognitive deficits), also covering zebrafish applications towards the goal of modeling major human neuropsychiatric and drug-induced syndromes. We argue that zebrafish models of complex brain disorders and drug-induced conditions are a rapidly emerging critical field in translational neuroscience and pharmacology research.

Alarm substance induced behavioral responses in zebrafish (Danio rerio)

Zebrafish (zebra danio) are becoming increasingly popular in behavioral neuroscience and behavior genetics. This small vertebrate may be utilized in modeling human brain disorders. One of the major neuropsychiatric conditions still not well understood is abnormally increased fear and anxiety. Zebrafish may be an appropriate organism with which these human diseases can be modeled and their biological mechanisms investigated. Predator induced anxiety paradigms have been suggested as useful methods in translational research. Shoaling fish, such as zebrafish, are known to respond to alarm substances with antipredatory or alarm reactions. However, these responses are not well characterized in zebrafish. In the current paper, we investigate the behavioral responses of zebrafish elicited by its alarm substance. Using observation-based as well as video-tracking aided behavior quantification methods we demonstrate significant alarm substance-induced behavioral changes that are independent of the presence of a predatory fish stimulus. The results suggest that, once refined, the use of alarm substance with zebrafish will allow the development of high throughput behavioral paradigms for drug and mutation screening aimed at the analysis of the biological mechanisms of fear in vertebrates.

The social zebrafish: behavioral responses to conspecific, heterospecific, and computer animated fish.

Zebrafish has been in the forefront of developmental biology and genetics, but only recently has interest in their behavior increased. Zebrafish are small and prolific, which lends this species to high throughput screening applications. A typical feature of zebrafish is its propensity to aggregate in groups, a behavior known as shoaling. Thus, zebrafish has been proposed as a possible model organism appropriate for the analysis of the genetics of vertebrate social behavior. However, shoaling behavior is not well characterized in zebrafish. Here, using a recently developed software application, we first investigate how zebrafish respond to conspecific and heterospecific fish species that differ in coloration and/or shoaling tendencies. We found that zebrafish shoaled with their own species but not with two heterospecific species, one of which was a shoaling the other a non-shoaling species. In addition, we have started the analysis of visual stimuli that zebrafish may utilize to determine whether to shoal with a fish or not. We systematically modified the color, the location, the pattern, and the body shape of computer animated zebrafish images and presented them to experimental zebrafish. The subjects responded differentially to some of these stimuli showing preference for yellow and avoidance of elongated zebrafish images. Our results suggest that computerized stimulus presentation and automated behavioral quantification of zebrafish responses are feasible, which in turn implies that high throughput forward genetic mutation or drug screening will be possible in the analysis of social behavior with this model organism.

Behavioral tests of hippocampal function: simple paradigms complex problems

Behavioral tests have become important tools for the analysis of functional effects of induced mutations in transgenic mice. However, depending on the type of mutation and several experimental parameters, false positive or negative findings may be obtained. Given the fact that molecular neurobiologists now make increasing use of behavioral paradigms in their research, it is imperative to revisit such problems. In this review three tests, T-maze spontaneous alternation task (T-CAT), Context dependent fear conditioning (CDFC), and Morris water maze (MWM) sensitive to hippocampal function, serve as illustrative examples for the potential problems. Spontaneous alternation tests are sometimes flawed because the handling procedure makes the test dependent on fear rather than exploratory behavior leading to altered alternation rates independent of hippocampal function. CDFC can provide misleading results because the context test, assumed to be a configural task dependent on the hippocampus, may have a significant elemental, i.e. cued, component. MWM may pose problems if its visible platform task is disproportionately easier for the subjects to solve than the hidden platform task, if the order of administration of visible and hidden platform tasks is not counterbalanced, or if inappropriate parameters are measured. Without attempting to be exhaustive, this review discusses such experimental problems and gives examples on how to avoid them.

Zebra fish: an uncharted behavior genetic model.

The zebra fish has been a preferred subject of genetic analysis. It produces a large number of offspring that can be kept in small aquaria, it can be easily mutagenized using chemical mutagens (e.g., ethyl nitrosourea [ENU]), and high-resolution genetic maps exist that aid identification of novel genes. Libraries containing large numbers of mutant fish have been generated, and the genetic mechanisms of the development of zebra fish, whose embryo is transparent, have been extensively studied. Given the extensive homology of its genome with that of other vertebrate species including our own and given the available genetic tools, zebra fish has become a popular model organism. Despite this popularity, however, surprisingly little is known about its behavior. It is argued that behavioral analysis is a powerful tool with which the function of the brain may be studied, and the zebra fish will represent an excellent subject of such analysis. The present paper is a proof of concept study that uses pharmacological manipulation (exposure to alcohol) to show that the zebra fish is amenable to the behavioral genetic analysis of aggression and thus may allow us to reveal molecular mechanisms of this behavioral phenomenon relevant to vertebrates.