Effects of intrahippocampally-injected naloxone and morphine upon behavioural responses to novelty in mice from two selectively-bred lines.

To examine the peptidergic regulation of behavioural responses to novelty, 5-month-old male mice from the inbred selection lines SRH (selected for rearing frequency: high) and SRL (selected for rearing frequency: low) were intrahippocampally micro-injected (0.5 microliter) with either the opiate antagonist naloxone (0.3 microgram), or the opiate agonist morphine (1.0 microgram), or saline vehicle alone, given 15 min prior to individual exposure to 20-min exploration tests in a novel environment. Naloxone exerted opposite effects upon various exploratory acts and locomotor activity in the two strains, that is, it decreased the scores in SRH and augmented them in SRL, while morphine depressed the scores in both. It is suggested that an excess of opioids in SRL, as compared to SRH, is attenuated by this dose of naloxone. In addition to previously obtained evidence of a genotype-dependent cholinergic mechanism in the mouse dorsal hippocampus controlling exploratory responses to novelty, these findings indicate that hippocampal opioid peptides are also involved in the genotype-dependent regulation of exploration.

Behavioural effects of (-)naloxone in mice from four inbred strains.

To study the role of endogenous opioid peptides in the regulation of behavioural responses to novelty, male mice from the inbred strains SRH, SRL, C57BL/6, and DBA/2 were injected IP with either saline alone, or the opiate antagonist naloxone, dissolved in saline, in dosages of 4 or 8 mg/kg. After 10 min, the animals were placed individually for 20 min in a novel environment and some 12 behavioural components were recorded. Naloxone reduced grooming and incipient rearing in all four strains and it reduced sniffing, leaning against the wall, and locomotor activity in some of them. Object-sniffing, object-learning, defecation, freezing, and Straub tail elevation remained unaffected. The results for grooming and locomotor activity are largely in agreement with reports from others. Rather unexpectedly, the drug enhanced rearing responses in all strains. Although in several cases, a genotype-treatment interaction became apparent, the observed strain differences usually persisted and the correlations found between the behavioural components did not alter much. The naloxone-induced reductions in sniffing, leaning, locomotion, and grooming suggest endogenous opioid involvement in the control of behavioural activation in a novel situation. The increases in rearing possibly result from an additional agonist action of naloxone.

A method permitting precise trimming of resin-embedded tissue for ultrathin sectioning in pre-embedding immunoelectronmicroscopy.

Due to the high complexity of the mammalian central nervous system, sampling of immunohistochemically processed brain tissues for electronmicroscopy requires an accurate and reliable technique. For this reason, the flat-embedding method, which allows light microscopical examination of tissue before sampling, is generally employed. Because of the osmification process, however, the tissue is blackish and opaque which hampers light microscopical selection of tissue areas of interest. We have found that tissue translucency is highly improved by an osmification process using an osmium tetroxide-ferrocyanide mixture. We describe a transilluminated chuck that enables visualization of immunostaining in specimens mounted on a trimming instrument, thus allowing for extremely precise sampling of the tissue.

Behavioral responses to novelty and structural variation of the hippocampus in mice. II. Multivariate genetic analysis.

On the basis of results from lesion studies in rodents, covariations are expected to exist between naturally-occurring heritable variations in hippocampal morphology and exploratory behavior elicited by novel surroundings. For this reason, we set up a full diallel cross between five inbred mouse strains and analyzed the behavioral and the hippocampal anatomical variation in male animals from this cross. Employing a bivariate extension of the diallel-cross analysis, estimates were obtained for the phenotypical, environmental, and genetical correlations between the phenotypes studied. A factor analysis performed on the matrix of additive-genetic correlations revealed that variations in the size of the intra- and infrapyramidal mossy fiber terminal fields (iip-MF) are negatively related to open-field exploration and novelty-induced fear. These results indicate that having larger iip-MF projections promotes the collection and processing of information about a novel environment, entailing lower levels of exploration and fear.

Behavioral responses to novelty and structural variation of the hippocampus in mice. I. Quantitative-genetic analysis of behavior in the open-field.

As a first step towards a multivariate quantitative-genetic analysis of covariations between heritable variation in hippocampal structure and mouse behavior, a univariate analysis of the genetic architecture of behavioral responses to novelty is presented. For several components of exploratory behavior considerable amounts of genetic variation were found and an evolutionary history of stabilizing selection for intermediate levels of exploration was inferred. Comparison of these results with those from a previous study indicated that even a relatively small diallel cross, involving 4-5 inbred strains, may provide useful genetic information on a specific sample of animals. Larger numbers of strains are needed to provide precise estimates of genetic parameters in a population.

Behavioural and neuroanatomical divergence between two sublines of C57BL/6J inbred mice.

Male mice of strains C57BL/6J and DBA/2J differ reproducibly in a number of behaviours displayed in an open-field. In particular, C57BL/6J mice show a higher rearing-up frequency than do DBA/2J animals. Recently, a marked drop occurred in the frequency with which this behaviour is displayed by the C57BL/6J//Nmg (N) subline, that has been separated from the original C57BL/6J line over 62 generations. Comparison of our animals with the C57BL/6J//Kun(K) subline, separated from the Jackson parent over at least 40 generations, showed a significant strain difference for rearing. Since both a positive additive-genetic correlation between rearing and the size of the intra- and infrapyramidal mossy fibre terminal field (iipMF) and a correlated response in the size of the iipMF to selection for rearing have been found previously, we expected to find smaller iipMF in N, as compared with K. After processing for Timm's stain, this predicted difference was indeed found. Skin grafting demonstrated that the two sublines were still completely histocompatible, excluding a possible genetic contamination of N. This provides very strong support for the hypothesis that both the behavioural and the neuroanatomical differences between these sublines are caused by a single spontaneous mutation in the N line and strengthens the idea of a functional relationship between the structural and the behavioural variable.

Behavioural responses to novelty in two inbred mouse strains after intrahippocampal naloxone and morphine.

Male C57BL/6 and DBA/2 mice were injected intrahippocampally with either naloxone (0.5 microgram) or morphine (1.0 microgram), or saline vehicle alone and, after 15 min, some 12 behavioural components carried out in a novel environment were recorded for 20 min. Naloxone reduced exploratory rearing responses, wall-leaning and object-sniffing in strain C57BL/6 and augmented these behaviours in strain DBA/2, while morphine depressed the scores in both. In conjunction with previously obtained evidence that the mouse hippocampus contains a genotype-dependent cholinergic mechanism which regulates responses to novelty, these findings support the hypothesis that hippocampal opioid peptides modulate the cholinergic control of exploration in mice, possibly indirectly through GABAergic pathways. In contrast, locomotor activity, defaecation and tail elevation remained practically unaffected by the two drugs, and grooming showed another kind of genotype-treatment interaction, that is to say, after morphine.

A genetic-correlational study of hippocampal structural variation and variation in exploratory activities of mice.

Our previous work provided evidence that hippocampal opioid peptides form an important neurochemical substrate underlying the gene-dependent exploratory behavior of mice. A prominent hippocampal opioid is dynorphin B, which resides in the mossy fibers exclusively. In order to seek support for causal relationships between dynorphinergic hippocampal mechanisms and exploration, a quantitative-genetic method was chosen. For this purpose, mice from the inbred strains C57BL/6, DBA/2, BLN, and CPB-K were used. Their hippocampal mossy fiber projections were visualized by means of immunohistochemistry, using a highly specific anti-dynorphin B antiserum. The additive-genetic correlations that were estimated suggest pleiotropic gene effects on locomotion, rearing-up, wall-leaning, and several intra- and infrapyramidal mossy fiber (iipMF) variables. Long iipMF, in particular, were found to be associated with high exploratory activity.

A genetic-correlational study of hippocampal neurochemical variation and variation in exploratory activities of mice.

Previously, we have demonstrated that hippocampal mossy fibers, containing the opioid peptide dynorphin B, are functionally connected with the gene-dependent exploratory behavior of mice. In order to seek further evidence of causal relationships between dynorphin B action and exploration, a quantitative-genetic method was chosen. For this purpose, mice from the inbred strains C57BL/6, DBA/2, BLN, and CPB-K were used. By means of radioimmunoassay, the hippocampal level of dynorphin B was monitored in mice that had been exposed to environmental novelty, as compared to naive animals. Clear evidence was obtained that novelty induces the release of hippocampal dynorphin B. Furthermore, low tissue content was found to be causally connected with high exploratory scores.

Distribution of dynorphin B and methionine-enkephalin in the mouse hippocampus: influence of genotype.

Immunohistochemical techniques were used to localize dynorphin B and methionine-enkephalin in the mouse hippocampus. Methionine-enkephalin-like immunoreactivity was found within the somata of interneurons distributed mainly in and around the CA1 stratum pyramidale and stratum granulosum as well as in the mossy fibers. Dynorphin B appeared to be confined to the mossy fiber pathway. In addition, we observed a difference between the inbred mouse strains DBA/2 and C57BL/6 with regard to the areas of the dynorphinergic mossy fiber projections: the intra- and infrapyramidal terminal fields were larger in the latter group.

A genotype-dependent hippocampal dynorphinergic mechanism controls mouse exploration.

Following microinjections with two dilutions of anti-dynorphin B antiserum into the hippocampal CA3 region, adult male mice from the inbred strains DBA/2 and C57BL/6 were individually tested for various exploratory behaviors in a novel environment and compared to preimmune serum control animals. Treatment augmented vertically-oriented exploratory acts in strain DBA/2 and reduced the scores in strain C57BL/6 so that strain differences originally present between the controls were reversed or eliminated after antiserum. These opposite effects indicate that a hippocampal dynorphinergic mechanism is involved in the regulation of novelty-induced behavior in mice and that its modulatory function depends on the genotype. It is concluded that DBA/2 animals exposed to novelty, as compared to C57BL/6, are characterized by an over-release of hippocampal dynorphin B which is neutralized in part by small amounts of antibody.

Y-chromosomal influences on renal and adrenal measures in mice.

From a four-way cross between unrelated inbred strains of mice, a random-breeding line was developed that segregated at two coat-color loci and carried Y chromosomes from different sources. Adult males were used for measurements of body weight, kidney weight, renal papilla length, and adrenal gland weight. Clear evidence was obtained of Y-linked influences on papilla length and adrenal weight, whereas direct effects of Autosome 9 were indicated with regard to adrenal weight only. Epistatic interaction of the Y chromosome with Autosome 4 was found for kidney weight and a 4-9 interaction appeared for papilla length. The direct effects of Y-chromosomal variation are interpreted in terms of a functional relationship between adrenal action and the development of Henle's loops and, in connection with previous findings, discussed in terms of adrenal functioning in the regulation of hippocampally-mediated behaviors.

Genetic control of hippocampal cholinergic and dynorphinergic mechanisms regulating novelty-induced exploratory behavior in house mice.

Neurobehavioral genetics endeavors to trace the pathways from genetic and environmental determinants to neuroanatomical and neurophysiological systems and, thence, to behavior. Exploiting genetic variation as a tool, the behavioral sequelae of manipulating these neuronal systems by drugs and antisera are analyzed. Apart from research in rats, this paper deals mainly with the genetically-influenced regulation in mice of exploratory behaviors that are adaptive in novel surroundings and are hippocampally-mediated. Special attention is paid to neuropeptidergic, GABAergic, and cholinergic synaptic functions in the mouse hippocampus. The behaviorally different inbred mouse strains C57BL/6 and DBA/2 show opposite reactions (reductions and increases, respectively, in exploration rates) to peripheral and intrahippocampal injections with agents that interfere with peptidergic, cholinergic, and GABAergic neurotransmission. These findings can be explained by an interdependent over-release of opioids, arrested GABA release, and excess acetylcholine in the hippocampal neuronal network of DBA/2 mice, as compared to C57BL/6 mice where these systems are functionally well balanced. Very similar results have been obtained with the lines SRH and SRL, derived from C57BL/6 and DBA/2, and genetically selected for rearing behavior. Most probably, the opioids act to disinhibit exploratory responses. An additional genetic approach is mentioned, in which four inbred mouse strains and one derived heterogeneous stock are used for estimating genetic correlations between structural properties of the hippocampal mossy fibers and levels of hippocampal dynorphin B, on the one hand, and frequencies of exploratory responses to environmental novelty, on the other.

Direct genetic and maternal influences on behavior and growth in two inbred mouse strains.

Newborn male DBA/2 and C57BL/6 mice were either crossfostered, fostered within strains, or, after handling, left with their own dams and compared for nine behavioral compenents observed in single animals at an adult age and for body weight measured at 4 weeks and 3 months. Many strain differences between the unfostered control groups emerged and these remained largely unchanged in comparisons between the infostered and between the outfostered groups. In neither infostered group were significant influences of fostering per se found. Some variation due to foster strain was detected mainly in the DBA outfostered group, namely, for 4-week body weight, rearing frequency, and locomotor activity, but no resemblance to the alien strain was brought about by this. Effects of litter occurred rarely. Unlike the C57BL strain, the DBAs showed a clear pattern of interdependence among exploratory acts. In spite of problems that may be attached to the crossfostering method, it was concluded that postnatal genotype-dependent maternal influences were rather unimportant as a source of variation in behavior and growth as measured between these two strains, relative to direct genetic effects and, possibly, prenatal factors.

Zinc-induced peripheral anosmia and behavioral responses to novelty in mice: a quantitative-genetic analysis.

Adult male mice were made anosmic by intranasal flushing with a 5% zinc sulfate solution. Twelve behavioral variables were measured in treated as well as saline-irrigated control animals placed in a novel environment. The genetic underpinnings and the genotype-treatment interactions with regard to these behaviors were analyzed in a classical Mendelian cross between the inbred strains C57BL/6 and DBA/2 and in a full 4 X 4 diallel cross, replicated five times, between these strains and strains C3H/St an CPB-K. Based on the hypothesis of an evolutionary history of directional selection for a well-balanced information-processing system, one might expect directional dominance for decrease in exploration after anosmization. Although decreases were found for several behavioral phenotypes, only few and relatively unimportant genotype-treatment interactions were present. This absence of any kind of genetic variation for behavioral change after anosmization points to an extremely strong directional selection which has eliminated all less favorable alleles. The findings support the hypothesis of directional selection for an efficient olfactory information-processing system.

The genetic architecture of behavioural responses to novelty in mice.

The genetic architectures of 12 behavioural variables measured in adult male mice placed in a novel environment were analysed in a replicated 4 X 4 diallel cross. The results were combined with those obtained in a classical cross involving two of the four strains. Based on the hypothesis of an evolutionary history of stabilising selection for mouse exploratory behaviour, we expected additive genetic effects and ambidirectional dominance. Such genetic architectures were actually found for those exploratory behaviours where epistatic effects were of minor importance. Similar findings emerged for some non-exploratory phenotypes. All behaviours analysed appeared to be polygenically controlled.

Role of hippocampal Met-enkephalin in the genotype-dependent regulation of exploratory behavior in mice.

Intrahippocampal microinjections with anti-Met-enkephalin antiserum enhanced novelty-induced vertically oriented exploratory acts and horizontal locomotor activity in inbred mouse strain DBA/2 and reduced these behaviors in C57BL/6 so that strain differences originally present between the normal serum controls were eliminated after antiserum treatment. These opposite effects suggest that hippocampal Met-enkephalin participates in the genotype-dependent control of mouse exploration.

A comparison between the full diallel cross and the simplified triple-test cross.

The simplified triple-test cross (sTTC) is a mating design that, because of its economic use of the experimental material as compared with other designs, seems very attractive. In theory, its power is almost equal to that of more elaborate designs such as the diallel cross. To evaluate the merits of both designs in a genetic analysis of mouse behavior, the results of a previous replicated 4×4 diallel cross (Crusio and van Abeelen 1986) were reanalyzed as a sTTC. We found that, at least with the fairly low number of strains employed, the sTTC analysis is clearly inferior to the diallel cross. This finding, in combination with some theoretical considerations, leads to the conclusion that the sTTC design is not a very useful one for such studies.

Ontogeny of behavior in two inbred lines of selected mice.

The development over 11 ages of 14 reflex responses, 26 behavioral components, jumpiness, and left-right discrimination learning was studied in 2 strains of mice genetically selected for high (SRH) and low (SRL) rearing response frequency. Significant age-dependent differences between the strains were found mainly, but not exclusively, in the behavioral components, e.g., rearing, jumping, scratching, defecating. Two-week-old pups of both strains showed good acquisition and retention in learning tests without shock, but the "jumpy" behavior disturbed performance to a certain degree in the SRH strain. Although less active initially, SRH mice were ahead at later developmental stages. Whether these differences are determined by the gene controlling rearing frequency or by other genes remains undecided.