Biomedical subjects
Publications and source records attributed to T Steckler.
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Data from several studies suggest that unrestrained secretion of corticotropin-releasing hormone in the CNS produces several signs and symptoms of depression. Recent evidence indicates that blockade of the CRH receptor 1 reduced depression scores in depressed patients. One of the symptoms that occur is depression is impairment in attentional processes. Whether these impairments are due to alterations in the CRH system are so far unknown. In order to investigate whether overproduction of CRH alters attentional process, transgenic mice overproducing CRH were tested on an operant five choice serial reaction time task, a task which taxes sustained and divided attention. Mutants showed impaired autoshaping. During initial discrimination learning, transgenics performed below wildtype level, but with extended training with long stimulus durations, transgenic animals reached similar accuracy levels as wildtype mice. When animals were tested at shortest stimulus duration (0.5s), a mild but significant impairment in accurate responding emerged in transgenics. This was accompanied by longer correct response latencies, while incorrect latencies did not differ between groups, suggesting attentional impairment in CRH transgenics. Because these animals have been reported to also show increased anxiety-related behaviour, animals were treated with the anxiolytic benzodiazepine diazepam. Diazepam failed to affect accuracy, but transgenic mice showed a stronger behavioural disinhibition. This suggests that the attentional impairment seen in CRH overexpressors is independent of alterations in anxiety-like behaviour. These findings may have implications for understanding the pathophysiology of psychiatric disorders such as depression, where it has been suggested that an overactivity of the CRH system accounts for a variety of symptoms, including hyper-arousal and attentional impairment.
Nerve growth factor (NGF) promotes survival and function of basal forebrain cholinergic neurons. We studied NGF and choline acetyltransferase (ChAT) activity after partial quisqualic acid induced lesions of the basal forebrain in 3 and 27 months-old rats, in order to investigate whether NGF-related regeneration is disturbed in old age. 2 weeks post lesion, ChAT activity decreased by 25 to 32% in adult and old rats. 3 months post lesion, the ChAT deficit receded in adult rats, but remained unchanged in old rats. 2 weeks post lesion, NGF levels were reduced by 36 to 44%, but there was no significant difference between adult and old rats. 3 months post lesion, we found increased NGF levels by 44% in the posterior cortex of adult rats. These results indicate that the compensatory NGF increase in the posterior cortex after partial cholinergic lesion of the basal forebrain is slightly impaired in old age.
Corticotropin-releasing hormone (CRH) overproduction and serotonergic dysfunction have both been implicated in a range of psychiatric disorders, such as anxiety and depression, and several studies have shown interactions between these two neurotransmitter systems. In this study, we investigated the effects of CRH challenge on hypothalamo-pituitary-adrenal (HPA) axis activity in female transgenic mice overproducing CRH. Furthermore, the effects of mild stress on HPA axis activity and body temperature were investigated in these mice. Pre- and post-synaptic 5-HT1A receptor function were studied by monitoring body temperature and plasma corticosterone levels after challenge with the 5-HT1A receptor agonist 8-hydroxy-2-(di-n-propyl-amino)-tetralin (8-OH-DPAT). Hypothermia in response to 8-OH-DPAT treatment did not differ between transgenic and wild type mice, indicating unaltered somatodendritic 5-HT1A autoreceptor function in mice overproducing CRH. In wild type mice 8-OH-DPAT increased plasma corticosterone levels, but not in transgenic animals. CRH injection, however, increased corticosterone levels in both groups. These data suggest desensitization of post-synaptic, but not pre-synaptic, 5-HT1A receptors in mice overproducing CRH. These findings resemble those seen in depressed patients following 5-HT1A challenge, which is in accord with the hypothesized role of CRH in the pathogenesis of depression.
Behavioral phenotyping of mutant mice is a new and challenging task for the behavioral neuroscientist. Therefore, standardisation of the experimental conditions is required to permit comparisons between the results of experiments within and between laboratories. Once mutation-induced behavioral changes have been identified, phenotyping of mouse mutants should be performed along a systematic trajectory, which allows for an in-depth characterisation of the mutant under investigation.
Behavioural phenotyping of mouse mutants is not a goal in itself but serves to characterise the behavioural effects of naturally occurring or experimentally induced mutations. Genetically engineered mouse mutants are valuable tools to elucidate the genetic control of behaviour and the interaction between genetic and environmental factors. However, a prerequisite for their use is the ability to assess different elements of behaviour. To this end, a battery of tests, which should be flexible enough to meet the needs of a particular study, should be used to characterise the behavioural phenotype. Detailed and extensive information about the effects of gene mutations is crucial for model building and model evaluation. Model building is an iterative process, switching between experimental data and theory formation. In order to facilitate this process and to allow comparison of results within and between laboratories, the standardisation of breeding, housing, and testing conditions is essential. The development and standardisation of sensitive, valid behavioural tests which are suited to phenotype mouse mutants is both a responsibility and a challenge to investigators of mouse behaviour.
Several factors account for murine cognitive abilities, and manipulation of genes which would act at the effector molecules involved in stimulus processing, reward-related properties and/or motor output can easily confound behavioural data obtained from mouse mutants responding on cognitive tasks. Therefore, tests may be needed which allow a better dissociation between true cognitive processes (accuracy) and other factors that may alter performance (motor or motivational bias). Part of this can be achieved by using methods which enable parametric variation of task difficulty. Part of it can also be achieved by using data analysis that allows a distinction between accuracy and bias, such as the mathematical methods of signal detection theory (SDT). SDT formally addresses the possibility that a given gene product or lack thereof affects performance by affecting motivation rather than cognition. It proposes that performance in a task depends on two factors, that is the sensitivity (or accuracy) of the neural systems mediating a cognitive process and the subject's motivational state, the latter of which can be represented as bias. SDT analysis can be easily applied to murine data. This overview will discuss the advances and limitations of the various SDT measures and illustrate the value of this type of analysis for understanding cognitive performance of 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 microg) 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 microg 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 microg 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.
With the increasing demand on phenotyping of mouse mutants there is a clear need to develop novel paradigms for testing mice. Mice are able to learn a non-matching to position rule to high accuracy in a variety of maze paradigms, but an operant version of this task is desirable. In the present study, mice of the C57BL/6 and DBA/2 strains were trained and tested on an operant delayed non-matching to position (DNMTP) paradigm. Data were analysed according to the methods of signal detection theory (SDT), which allows conclusions as to whether strain differences in DNMTP performance are more related to changes in accuracy or in motivational factors. Mice can learn to respond on an operant DNMTP paradigm with high accuracy, and accurate performance depends on the duration of the delay-period, i.e. forgetting curves can be generated. Comparison between the two strains of mice revealed that DBA/2 mice learned faster than C57BL/6 mice to associate the lever press with food during initial shaping, but no further strain differences were observed in accurate responding during later stages of the experiment. However, differences in biased responding and, in particular, responsivity were observed between the two strains. Muscarinic blockade with scopolamine (0.1--1.0 mg/kg) failed to affect accuracy in the two strains, but altered responsivity. This task should be of great value for a more in-depth analysis of cognitive function in mutant mice as it allows a better dissociation between mnemonic and non-mnemonic factors. In particular, such paradigm may be of interest for testing conditional mutants, which allow time-sensitive induction or inhibition of gene expression, i.e. where animals can be trained while non-impaired to stable baseline and then tested once the gene is activated or inhibited.
Corticotropin-releasing hormone (CRH) interacts with noradrenergic, dopaminergic and cholinergic systems of the brain, and these interactions are thought to be of relevance for the stress response, anxiety-related behavior, and cognitive function. CRH mediates its central effects through two high-affinity membrane receptors, CRH receptor subtypes 1 and 2. It is however unclear at present whether cholinergic or catecholaminergic cells express these receptors themselves or whether the effects of CRH are indirectly mediated through interaction with other neurotransmitter systems. Therefore, this study investigated whether choline acetyltransferase immunoreactive neurons of the murine basal forebrain and brainstem nuclei, and tyrosine hydroxylase immunoreactive neurons located within the locus coeruleus, ventral tegmental area and substantia nigra co-express CRH receptor 1, employing a double-immunocytochemical procedure. Using an antibody against the C-terminus of the CRH type 1 receptor (CRH-R1), CRH-R1-like immunoreactivity was found in all cholinergic basal forebrain nuclei except the nucleus basalis magnocellularis. In particular, the diagonal band of Broca (vertical and horizontal limbs) showed a high degree of co-localization of CRH-R1 immunoreactivity and choline acetyltransferase immunoreactivity (both limbs >90%). A less intense immunoreactivity but still high rate of co-localization was detected in the cholinergic neurons of the medial septum (80%), while lowest co-localization was observed in choline acetyltransferase immunoreactive neurons of the substantia innominata (58%). An intermediate degree of co-localization (75%) was seen in the brainstem pedunculopontine tegmental nucleus, while the other major brainstem cholinergic nucleus, the laterodorsal tegmental nucleus, showed an even higher degree of choline acetyltransferase immunoreactivity-positive cells also immunoreactive for CRH-R1 (92%). All catecholaminergic structures studied displayed a pattern of CRH-R1 immunoreactivity strongly overlapping the pattern of tyrosine hydroxylase immunoreactivity. The intensity of the CRH-R1 signal was relatively low within the ventral tegmental area and the substantia nigra pars compacta, while the CRH-R1 signal was very intense and detected in almost all of the neurons of the locus coeruleus. These results clearly demonstrate that the cholinergic and catecholaminergic systems provide direct anatomical substrates for CRH action through the CRH-R1. These findings are of particular relevance for understanding the action of recently developed CRH-R1 antagonistic drugs which may offer a new therapeutic approach to treat stress-related disorders such as anxiety and depression and their concomitant alterations in arousal and cognitive functions.
Glucocorticoids enhance the locomotion-stimulating and the rewarding properties of stimulant drugs. Amphetamine-induced conditioned activity was investigated in B6C3F1 (controls) and antisense transgenic mice. The latter expresses a neurofilament-promotor-driven antisense RNA complementary to a fragment of cDNA that codes for the mouse glucocorticoid receptor. This gene expression leads to approximately a 50% reduction in glucocorticoid receptor mRNA in the brain. Transgenic mice showed an increased novelty response when tested in an open field, in terms of both distance traveled and number of rearings. Moreover, they displayed enhanced amphetamine-induced conditioned activity. Behavioral sensitization was observed in controls, whereas behavioral tolerance developed in transgenic mice. These data support the concept of an enhanced stress response in these transgenic mice, rather than a general downregulation of the stress response because of impaired glucocorticoid receptor function.
Knowledge of the genetic and molecular events underlying the neuroendocrine and behavioural sequelae of the response to stress has advanced rapidly over recent years. The response of an individual to a stressful experience is a polygenic trait, but also involves non-genetic sources of variance. Using a combination of top-down (quantitative trait locus [QTL] and microarray analysis) and bottom-up (gene targeting, transgenesis, antisense technology and random mutagenesis) strategies, we are beginning to dissect the molecular players in the mediation of the stress response. Given the wealth of the data obtained from mouse mutants, this review will primarily focus on the contributions made by transgenesis and knockout studies, but the relative contribution of QTL studies and microarray studies will also be briefly addressed. From these studies it is evident that several neuroendocrine and behavioural alterations induced by stress can be modelled in mouse mutants with alterations in hypothalamic-pituitary-adrenal axis activity or other, extrahypothalamic, neurotransmitter systems known to be involved in the stress response. The relative contribution of these models to understanding the stress response and their limitations will be discussed.
Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptor subtype 1 (PAC1) have been suggested to play a role in the modulation of learning and memory. However, behavioral evidence for altered mnemonic function due to altered PAC1 activity is missing. Therefore, the role of PAC1 in learning and memory was studied in mouse mutants lacking this receptor (PAC1 knock-out mice), tested in water maze two-choice spatial discrimination, one-trial contextual and cued fear conditioning, and multiple-session contextual discrimination. Water maze spatial discrimination was unaffected in PAC1 mutants, while a mild deficit was observed in multiple session contextual discrimination in PAC1 knock-out mice. Furthermore, PAC1 knock-out mice were able to learn the association between context and shock in one-trial contextual conditioning, but showed faster return to baseline than wild-type mice. Thus, the effects of PAC1 knock-out on modulating performance in these tasks were subtle and suggest that PAC1 only plays a limited role in learning and memory.
Differences in locomotor activity, exploratory activity and anxiety-like behaviour of C57BL/6ChR,C57BL/6J, Swiss Webster/J and A/J strain were investigated in an anxiety battery. The battery consisted of paradigms studying spontaneous behaviour after a mild stressor, tasks of innate anxiety (light-dark box, elevated plus maze, novel object exploration), response to a conflict situation (Vogel conflict), conditioned fear and response to inescapable swim stress. Locomotor activity was studied in an open field and compared with locomotion in the other tests. Exploratory behaviour was studied in a 16-hole board task. The data confirm previous studies suggesting that A/J mice are a relatively anxious strain. Also, the data indicated that locomotor activity was independent of the paradigm employed, while the rank order of strain-dependent effects on anxiety-related behaviour changed as a function of the task under study. Our data provide further support for the notion that choice of strain is essential in studies of anxiety-related behaviour. Influence of strain should be considered in pharmacological and lesion studies, as well as in studies with mutant mice. In addition, the data indicate that different anxiety paradigms tax different aspects of anxiety, suggesting that a battery of different tests should be used in studies of anxiety-related behaviour.
Mice with a genetic disruption (knockout) of the multiple drug resistance (Mdr1a) gene were used to examine the effect of the absence of the drug-transporting P-glycoprotein at the blood-brain barrier on the uptake of amitriptyline (AMI) and fluoxetine (FLU) and their metabolites into the brain. One hour after intraperitoneal injection of AMI or FLU, knockout (-/-) and wild-type (+/+) mice were sacrificed and drug concentrations of brain, kidney, liver, testis, and plasma were measured. The plasma concentrations of the AMI metabolites and the brain:spleen ratios of AMI, nortriptyline (NOR), 10-OH-AMI and 10-OH-NOR were significantly higher in the -/- mice, demonstrating that AMI and its metabolites are substrates of the P-glycoprotein and that mdr1a activity at the level of the blood-brain barrier reduces the penetration of these substances into the brain. In contrast, tissue distributions of FLU and its metabolites among the various tissues tested were indistinguishable between groups. The herein reported differences in brain penetration of antidepressant drugs depending on the presence of the mdr1a gene may offer an explanation for differences in the treatment response at a given plasma concentration. Moreover, individual differences in mdr1 gene activity may account for variable response patterns at different episodes and development of therapy resistance.
In the post-genome era, the mouse will have a major role as a model system for functional genome analysis. This requires a large number of mutants similar to the collections available from other model organisms such as Drosophila melanogaster and Caenorhabditis elegans. Here we report on a systematic, genome-wide, mutagenesis screen in mice. As part of the German Human Genome Project, we have undertaken a large-scale ENU-mutagenesis screen for dominant mutations and a limited screen for recessive mutations. In screening over 14,000 mice for a large number of clinically relevant parameters, we recovered 182 mouse mutants for a variety of phenotypes. In addition, 247 variant mouse mutants are currently in genetic confirmation testing and will result in additional new mutant lines. This mutagenesis screen, along with the screen described in the accompanying paper, leads to a significant increase in the number of mouse models available to the scientific community. Our mutant lines are freely accessible to non-commercial users (for information, see http://www.gsf.de/ieg/groups/enu-mouse.html).
Dehydroepiandrosteone (DHEA) and its sulfate ester (DHEAS) are the major secretory products of the human adrenal glands and serve as precursors for both androgenic and estrogenic steroids. DHEA/S concentrations are particularly high in the brain, and DHEA/S and related steroids can be synthesized de novo in brain glial cells. Therefore, the term 'neurosteroids' has been coined for these compounds. This review summarizes findings in neurosteroid physiology on a cellular and molecular level, and outlines current concepts of how these compounds modulate physiological functions of the brain. Today, despite promising preclinical and human data the present clinical studies provide only weak evidence, if any, in favour of a DHEA replacement therapy.
Electrophysiological and behavioural experiments were performed in transgenic mice expressing a dominant-negative form of cAMP response element-binding protein (CREBA133) in the limbic system. In control littermate in vitro slice preparation, tetanizing the lateral amygdala-basolateral amygdala (BLA) pathway with a single train (100 Hz for 1 s) produced short-term potentiation (STP) in the BLA. Five trains (10-s interstimulus interval) induced long-term potentiation (LTP), which was completely blocked by the N-methyl-D-aspartate (NMDA) receptor antagonist D(-)-2-amino-5-phosphonopentanoic acid (AP5; 50 microM). When GABAergic (gamma-aminobutyric acid) inhibition was blocked by picrotoxin (10 microM), LTP became more pronounced. Low-frequency stimulation (1 Hz for 15 min) induced either long-term depression (LTD) or depotentiation. LTD remained unaffected by AP5 (50 microM) or by the L- and T-type Ca2+-channel blockers nifedipine (20 microM) and Ni2+ (50 microM), but was prevented by picrotoxin (10 microM), indicating a GABAergic link in the expression of LTD in the BLA. When conditioned fear was tested, a mild impairment was seen in one of three transgenic lines only. Although high levels of mRNA encoding CREBA133 lead to downregulation of endogenous CREB, expression of LTP and depotentiation were unaltered in BLA of these transgenic animals. These results could suggest that residual CREB activity was still present or that CREB per se is dispensable. Alternatively, other CREB-like proteins were able to compensate for impaired CREB function.