Disruption of the neuregulin 1 gene in the rat alters HPA axis activity and behavioral responses to environmental stimuli.

Exposure to stress can result in an increased risk for psychiatric disorders, especially among genetically predisposed individuals. Neuregulin 1 (NRG1) is a susceptibility gene for schizophrenia and is also associated with psychotic bipolar disorder. In the rat, the neurons of the hypothalamic paraventricular nucleus show strong expression of Nrg1 mRNA. In patients with schizophrenia, a single nucleotide polymorphism in the 5' region of NRG1 interacts with psychosocial stress to affect reactivity to expressed emotion. However, there is virtually no information on the role of NRG1 in hypothalamic-pituitary-adrenal axis function, and whether the protein is expressed in the paraventricular nucleus is unknown. The present studies utilize a unique line of Nrg1 hypomorphic rats (Nrg1(Tn)) generated by gene trapping with the Sleeping Beauty transposon. We first established that the Nrg1(Tn) rats displayed reduced expression of both the mRNA and protein corresponding to the Type II NRG1 isoform. After confirming, using wild type animals, that Type II NRG1 is expressed in the neurocircuitry involved in regulating hypothalamic-pituitary-adrenal axis responses to environmental stimuli, the Nrg1(Tn) rats were then used to test the hypothesis that altered expression of Type II NRG1 disrupts stress regulation and reactivity. In support of this hypothesis, Nrg1(Tn) rats have disrupted basal and acute stress recovery corticosterone secretion, differential changes in expression of glucocorticoid receptors in the pituitary, paraventricular nucleus and hippocampus, and a failure to habituate to an open field. Together, these findings point to NRG1 as a potential novel regulator of neuroendocrine responses to stress as well as behavioral reactivity.

Regional variability in age-related loss of neurons from the primary visual cortex and medial prefrontal cortex of male and female rats.

During aging, changes in the structure of the cerebral cortex of the rat have been seen, but potential changes in neuron number remain largely unexplored. In the present study, stereological methods were used to examine neuron number in the medial prefrontal cortex and primary visual cortex of young adult (85-90 days of age) and aged (19-22 months old) male and female rats in order to investigate any age-related losses. Possible sex differences in aging were also examined since sexually dimorphic patterns of aging have been seen in other measures. An age-related loss of neurons (18-20%), which was mirrored in volume losses, was found to occur in the primary visual cortex in both sexes in all layers except IV. Males, but not females, also lost neurons (15%) from layer V/VI of the ventral medial prefrontal cortex and showed an overall decrease in volume of this region. In contrast, dorsal medial prefrontal cortex showed no age-related changes. The effects of aging clearly differ among regions of the rat brain and to some degree, between the sexes.

Neuron number decreases in the rat ventral, but not dorsal, medial prefrontal cortex between adolescence and adulthood.

Neuroimaging studies have established that there are losses in the volume of gray matter in certain cortical regions between adolescence and adulthood, with changes in the prefrontal cortex being particularly dramatic. Previous work from our laboratory has demonstrated that cell death can occur as late as the fourth postnatal week in the rat cerebral cortex. The present study examined the possibility that neuronal loss may occur between adolescence and adulthood in the rat prefrontal cortex. Rats of both sexes were examined during adolescence (at day 35) and young adulthood (at day 90). The volume, neuronal number, and glial number of the medial prefrontal cortex (mPFC) were quantified using unbiased stereological techniques. Neurons were lost from the ventral, but not dorsal, mPFC between adolescence and adulthood, suggesting a late wave of apoptosis that was region-specific. This was accompanied by a decrease in the volume of the female ventral mPFC. In contrast to neuron number, the number of glial cells was stable in the ventral mPFC and increased between adolescence and adulthood in the dorsal mPFC. Sex-specific developmental changes in neuron number, glial number, and volume resulted in sex differences in adults that were not seen during adolescence. The loss of neurons at this time may make the peri-adolescent prefrontal cortex particularly susceptible to the influence of environmental factors.

Sexually dimorphic aging of dendritic morphology in CA1 of hippocampus.

During aging, rats of both sexes experience a decline in performance on hippocampal-dependent tasks. Investigations into the neuroanatomical correlates of this functional decline have been conducted almost exclusively in male subjects. In the present study, dendritic spine density in stratum radiatum and complexity of the entire apical dendritic tree were quantified using Golgi-Cox-stained tissue in young (3-5 months) and aged (19-22 months) rats of both sexes. Because both cognitive decline and hippocampal morphology may be influenced by ovarian hormonal state, young adult females were examined during either proestrus or estrus, and aged females were examined in one of two reproductively senescent states: persistent estrus or persistent diestrus. A sex difference in dendritic branching of CA1 pyramidal cells was found among young adults. However, this difference disappeared during aging, due to a reduction in branching with age for males but not for females. Spine density was not influenced by age or sex, nor did ovarian hormone status influence either measure. These results are consistent with our previous findings in the rat medial prefrontal cortex and primary motor cortex and with the human literature, which indicate that age-related atrophy of cognitive brain regions is more severe for males than females.

Sex differences in mouse cortical thickness are independent of the complement of sex chromosomes.

Although the morphology of the cerebral cortex is known to be sexually dimorphic in several species, to date this difference has not been investigated in mice. The present study is the first to report that the mouse cerebral cortex is thicker in males than in females. We further asked if this sex difference is the result of gonadal hormones, or alternatively is induced by a direct effect of genes encoded on the sex chromosomes. The traditional view of mammalian neural sexual differentiation is that androgens or their metabolites act during early development to masculinize the brain, whereas a feminine brain develops in the relative absence of sex steroids. We used mice in which the testis determination gene Sry was inherited independently from the rest of the Y chromosome to produce XX animals that possessed either ovaries or testes, and XY animals that possessed either testes or ovaries. Thus, the design allowed assessment of the role of sex chromosome genes, independent of gonadal hormones, in the ontogeny of sex differences in the mouse cerebral cortex. When a sex difference was present, mice possessing testes were invariably masculine in the morphology of the cerebral cortex, independent of the complement of their sex chromosomes (XX vs. XY), and mice with ovaries always displayed the feminine phenotype. These data suggest that sex differences in cortical thickness are under the control of gonadal steroids and not sex chromosomal complement. However, it is unclear whether it is the presence of testicular secretions or the absence of ovarian hormones that is responsible for the thicker male cerebral cortex.

Ovarian hormone replacement to aged ovariectomized female rats benefits acquisition of the morris water maze.

Ovarian steroids have been suggested to aid in preserving cognitive functioning during aging in both humans and other animals. Spatial memory relies heavily on the hippocampus, a structure that is sensitive to the influence of both ovarian hormones and aging. The present study investigated the outcome of ovarian hormone replacement during aging on performance in a spatial version of the Morris water maze. Female rats were ovariectomized at 14 months of age and received one of three types of replacement prior to testing at 16 months: acute estrogen replacement (2 days), chronic estrogen replacement (28 days), or chronic replacement of both estrogen and progesterone (28 days). Control animals, which did not receive replacement hormones, displayed significant overnight forgetting during acquisition of the task. Ovarian hormone replacement, both acute and chronic, prevented forgetting. Previous studies have demonstrated that high levels of ovarian hormones are detrimental to performance of young adult female rats on this task (Warren and Juraska, 1997; Chesler and Juraska, 2000). The current study found an opposite effect during aging: ovarian hormone replacement was beneficial. This suggests that animal models of menopause, aimed at exploring the protective effects of hormone replacement therapy on cognition during human female aging, require the use of aged female animals.

Migration patterns of sympathetic preganglionic neurons in embryonic rat spinal cord.

The displacement of immature neurons from their place of origin in the germinal epithelium toward their adult positions in the nervous system appears to involve migratory pathways or guides. While the importance of radial glial fibers in this process has long been recognized, data from recent investigations have suggested that other mechanisms might also play a role in directing the movement of young neurons. We have labeled autonomic preganglionic cells by microinjections of horseradish peroxidase (HRP) into the sympathetic chain ganglia of embryonic rats in order to study the migration and differentiation of these spinal cord neurons. Our results, in conjunction with previous observations, suggest that the migration pattern of preganglionic neurons can be divided into three distinct phases. In the first phase, the autonomic motor neurons arise in the ventral ventricular zone and migrate radially into the ventral horn of the developing spinal cord, where, together with somatic motor neurons, they form a single, primitive motor column (Phelps P. E., Barber R. P., and Vaughn J. E. (1991). J. Comp. Neurol. 307:77-86). During the second phase, the autonomic motor neurons separate from the somatic motor neurons and are displaced dorsally toward the intermediate spinal cord. When the preganglionic neurons reach the intermediolateral (IML) region, they become progressively more multipolar, and many of them undergo a change in alignment, from a dorsoventral to a mediolateral orientation. In the third phase of autonomic motor neuron development, some of these cells are displaced medially, and occupy sites between the IML and central canal. The primary and tertiary movements of the preganglionic neurons are in alignment with radial glial processes in the embryonic spinal cord, an arrangement that is consistent with a hypothesis that glial elements might guide autonomic motor neurons during these periods of development. In contrast, during the second phase, the dorsal translocation of preganglionic neurons occurs in an orientation perpendicular to radial glial fibers, indicating that glial elements are not involved in the secondary migration of these cells. The results of previous investigations have provided evidence that, in addition to glial processes, axonal pathways might provide a substrate for neuronal migration. Logically, therefore, it is possible that the secondary dorsolateral translocation of autonomic preganglionic neurons could be directed along early forming circumferential axons of spinal association interneurons, and this hypothesis is supported by the fact that such fibers are appropriately arrayed in both developmental time and space to guide this movement.

Development of rostrocaudal dendritic bundles in rat thoracic spinal cord: analysis of cholinergic sympathetic preganglionic neurons.

Using monoclonal antibodies to choline acetyltransferase (ChAT) and glial fibrillary acidic protein (GFAP), we have analyzed the development of the dendritic bundles formed by cholinergic sympathetic preganglionic neurons (SPNs) in relationship to changes in the organization of glial fibers. In adult rat thoracic spinal cord, SPNs in the intermediolateral (IML) and central autonomic (CA) regions extend dendrites in both the mediolateral and rostrocaudal directions, forming a ladder-like pattern in horizontal sections of thoracic spinal cord. We report that, while the mediolateral dendrites form prenatally, the rostrocaudal dendritic bundles are not detected until at least a week later, during early postnatal life. The rostrocaudal dendrites develop rapidly during the first postnatal week, and achieve an adult-like pattern by postnatal day 14. The observed ontogenetic arrangements of dendritic bundles were correlated with the developing organization of astroglial processes with which they are intimately associated. While the appearance of mediolateral dendrites is consistent with the radial organization of glial in the embryonic spinal cord, the developmental time course of the rostrocaudal dendritic bundles coincides with the transformation of glial cells from this predominantly radial or transverse orientation to the randomly-oriented, stellate pattern of mature astrocytes. This temporal association suggests that ontogenetic changes in the organization of glial cells may contribute to the differential development of mediolateral and rostrocaudal dendritic patterns in the spinal cord.

Ultrastructural analysis of choline acetyltransferase-immunoreactive sympathetic preganglionic neurons and their dendritic bundles in rat thoracic spinal cord.

We have used a monoclonal antibody against choline acetyltransferase (ChAT) to aid in the identification of sympathetic preganglionic neurons (SPNs) and to examine their ultrastructure in rat thoracic spinal cord. The clusters of ChAT-immunoreactive (ChAT-IR) preganglionic cell bodies and their distinctive bundles of dendrites give rise to a ladder-like appearance in horizontal light microscopic sections. This organization also produced a characteristic appearance of preganglionic neuropil when viewed electron microscopically. The intermediolateral (IML) nucleus contained numerous rostrocaudally oriented ChAT-IR dendrites. In addition, mediolaterally oriented ChAT-IR dendrites extended between the IML and the central autonomic region. Both the ChAT-IR dendrites and somata of preganglionic neurons were intimately associated with astroglial processes. The cell bodies were typically covered over a large proportion of their surface by a thin astrocytic sheath, and this was associated with a paucity of axon terminals forming axosomatic synapses. Instead, the vast majority of synapses upon SPNs were of the axodendritic type. The most frequently observed type of axon terminal contained numerous round, clear vesicles along with several dense-core vesicles (DCVs). In addition, some boutons contained a predominance of DCVs. Serial section analysis revealed that these apparently diverse morphological classes of synaptic boutons may simply represent variability of structure throughout a single terminal, with a greater proportion of DCVs being located distal to the synaptic specialization and a greater number of round, clear vesicles being present adjacent to the synapse. Analysis of the dendritic bundles revealed that individual dendrites, like the cell bodies, were often isolated from each other and the surrounding neuropil by astrocytic processes. This arrangement usually was interrupted only at regions of synaptic contact where astrocytic processes surrounded the synaptic complex as a whole. Thus, astroglial ensheathment of SPNs seems designed to minimize cross-talk between the bundled dendrites, as well as to isolate or segregate the diverse afferent inputs known to impinge on these cells.

Astrocyte proliferation precedes a decrease in basket cells in the dentate fascia following chronic ethanol treatment in mice.

The effect of chronic ethanol administration on the density of basket cells in the dentate gyrus of mice selectively bred for their sensitivity to acute ethanol exposure (long-sleep, LS and short-sleep, SS) was assessed in two experiments. In addition, the effect of chronic ethanol on the density of dentate granule cells and astrocytes was examined. In the first experiment, mice received 3 weeks of a liquid ethanol diet with 35% of their calories derived from ethanol (EDC). In this experiment, LS mice did not demonstrate a change in the density of granule cells or in the density of basket cells. There was, however, a significant increase in the density of astrocytes as a result of this treatment for the LS mice. The SS mice were unaffected on all measures. In the second experiment, portions of which have been reported previously, mice received a diet with 23% EDC for 3 months. As a result of this exposure, LS mice showed a significant decrease in the density of basket cells, but there was no change in the density of granule cells or astrocytes. There was no difference between controls and experimental mice from the SS group on any of these parameters. These results suggest that at least in the dentate gyrus, chronic ethanol treatment selectively reduces the density of basket cells but only in mice that are more sensitive to the hypnotic effects of acute ethanol exposure. Furthermore, this effect seems to be preceded by an apparent increase in the density of astrocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of chronic ethanol consumption on the fine structure of the CA1 stratum oriens in short-sleep and long-sleep mice: short-term and long-term exposure.

The effect of chronic ethanol administration on the fine structure of the hippocampal CA1 stratum oriens was examined in two lines of mice selectively bred for their differential sensitivity to acute ethanol exposure (long-sleep, LS and short-sleep, SS mice). Two experiments were performed. In the first experiment, mice received a liquid diet for 3 weeks with the final amount of ethanol being 35% ethanol-derived calories. In the second experiment, mice received 23.5% ethanol-derived calories for 3 months. Quantitative electron microscopy of the dendritic spines and synaptic appositions in the stratum oriens of CA1 revealed an interaction between diet and line of mice, but only in the 3-month exposure condition. This difference was due to a significant decrease in the density of spines and synaptic appositions in the LS mice receiving ethanol. Additionally, baseline differences between lines indicate that the lines are differing in the density of spine synapses in the absence of ethanol treatment. The possible interaction between acute sensitivity to ethanol and differences in fine structure are examined.

Effect of chronic ethanol consumption on the fine structure of the dentate gyrus in long-sleep and short-sleep mice.

The effect of short- and long-term chronic ethanol consumption on the fine structure of the dentate gyrus was examined in two lines of mice selected for their differential sensitivity to acute ethanol administration. Quantitative electron microscopic analysis of dendritic spines, axon terminals, and synaptic appositions revealed significant differences between the long-sleep and short-sleep mice. In control preparations, long-sleep mice were found to have larger spine areas and perimeters, larger axon terminals, and longer synaptic appositions than short-sleep mice. In addition, the shape of dendritic spines in the long-sleep mice was significantly more complex than those of short-sleep mice. Ethanol tended to increase this complexity in long-sleep mice only. Ethanol had only a limited effect on the other anatomical measures. The results provide evidence for ultrastructural differences between the nervous systems of these lines of mice which may have a role in their differential sensitivity to acute ethanol administration.

Changes in the frequency of basket cells in the dentate fascia following chronic ethanol administration in mice.

The frequency of basket cells in the granule cell layer of the dentate fascia of Short Sleep (SS) and Long Sleep (LS) mice was determined following 3 months of ethanol exposure. These mice were bred for their differential susceptibility to the narcotic effects of acute doses of ethanol. The ethanol-insensitive SS mice were unaffected by the treatment while the ethanol-sensitive LS mice that received ethanol showed a significant decrease in basket cell frequency over their control group counterparts. These basket cells are thought to control the tonic level of activity of the granule cells. Thus, a decrease in basket cell frequency might lead to higher granule cell activity following chronic ethanol exposure. This effect could counteract the assumed stronger depressant effect of ethanol in the relatively ethanol-sensitive LS mice.

Actin filament organization within dendrites and dendritic spines during development.

The myosin S-1 subfragment was used to label actin filaments in the developing rat brain. The results show actin filaments present throughout the dendritic region with highest concentrations within growth cones and regions of spine development. Between 6 and 25 days postnatal, spines became more complex and actin filaments within them increased in number and formed a complex network. The observed organization of actin supports the hypothesis that actin has a role in the protrusion of spines from the dendrite during development.

Semen collection and spermatozoa characteristics in budgerigars (Melopsittacus undulatus).

Semen samples were obtained from budgerigars by applying gentle pressure to both sides of the cloaca. The semen appeared to be stored in the seminal glomera, two structures on either side of the proctodeum. These structures have been described before in Passeriformes. The spermatozoon in budgerigars is similar to the spermatozoon of the domestic fowl, but differs from the spermatozoon of Passeriformes.

Calcium in the spine apparatus of dendritic spines in the dentate molecular layer.

With a pyroantimonate precipitation technique, we have demonstrated Ca2+ in the sacs of the dendritic spine apparatus in the SER of dendrites and axon terminals, in synaptic vesicles, multivesicular bodies, mitochondria, and glial processes of the dentate molecular layer. It is speculated that the spine apparatus may be a Ca2+ sequestering organelle which may regulate levels of intraspinal and intradendritic Ca2+ during synaptic activity.