Cathinone increases body temperature, enhances locomotor activity, and induces striatal c-fos expression in the Siberian hamster.

Cathinone is a ß-keto alkaloid that is the major active constituent of khat, the leaf of the Catha edulis plant that is chewed recreationally in East Africa and the Middle East. Related compounds, such as methcathinone and mephedrone have been increasing in popularity as recreational drugs, resulting in the recent proposal to classify khat as a Class C drug in the UK. There is still limited knowledge of the pharmacological effects of cathinone. This study examined the acute effects of cathinone on core body temperature, locomotor and other behaviors, and neuronal activity in Siberian hamsters. Adult male hamsters, previously implanted with radio telemetry devices, were treated with cathinone (2 or 5mg/kg i.p.), the behavioral profile scored and core body temperature and locomotor activity recorded by radio telemetry. At the end of the study, hamsters received vehicle or cathinone (5mg/kg) and neuronal activation in the brain was determined using immunohistochemical evaluation of c-fos expression. Cathinone dose-dependently induced significant (p<0.0001) increases in both temperature and locomotor activity lasting 60-90min. Cathinone (2mg/kg) increased rearing (p<0.02), and 5mg/kg increased both rearing (p<0.001) and lateral head twitches (p<0.02). Both cathinone doses decreased the time spent at rest (p<0.001). The number of c-fos immunopositive cells were significantly increased in the striatum (p<0.0001) and suprachiasmatic nucleus (p<0.05) following cathinone, indicating increased neuronal activity. There was no effect of cathinone on food intake or body weight. It is concluded that systemic administration of cathinone induces significant behavioral changes and CNS activation in the hamster.

Valproic acid reduces spatial working memory and cell proliferation in the hippocampus.

Valproic acid (VPA) is widely used clinically, as an anticonvulsant and mood stabilizer but is, however, also known to block cell proliferation through its ability to inhibit histone deacetylase enzymes. There have been a number of reports of cognitive impairments in patients taking VPA. In this investigation we examined the relationship between cognition and changes in cell proliferation within the hippocampus, a brain region where continued formation of new neurons is associated with learning and memory. Treatment of rats by i.p. injection of VPA, reduced cell proliferation in the sub granular zone of the dentate gyrus within the hippocampus. This was linked to a significant impairment in their ability to perform a hippocampus-dependent spatial memory test (novel object location). In addition, drug treatment caused a significant reduction in brain-derived neurotrophic factor (BDNF) and Notch 1 but not doublecortin levels within the hippocampus. These results support the idea that VPA may cause cognitive impairment and provide a possible mechanism for this by reducing neurogenesis within the hippocampus.

The transcription factor cSox2 and Neuropeptide Y define a novel subgroup of amacrine cells in the retina.

The retina has been extensively used as a model to study the mechanisms responsible for the production of different neural cell phenotypes. The importance of both extrinsic and intrinsic cues in these processes is now appreciated and numerous transcription factors have been identified which are required for both neuronal determination and cell differentiation. In this study we have analysed the expression of the transcription factor Sox2 during development of the chick retina. Expression was found in the proliferating cells of the retina during development and was down regulated by nearly all cell types as they started to differentiate and migrate to the different layers of the retina. In one cell type, however, Sox2 expression was retained after the cells have ceased division and migrated to their adult location. These cells formed two rows located on either side of the inner plexiform layer and were also positive for Neuropeptide Y, characteristics which indicate that they were a subpopulation of amacrine cells. The expression of Sox2 by only this population of post-mitotic neurones makes it possible to follow these cells as they migrate to their adult location and shows that they initially form a single row of cells which subsequently divides to form the double row seen in the adult tissue. We suggest that retained expression of Sox2 is involved in directing the differentiation of these cells and is an early marker of this cell type.

Rat embryonic myoblasts are restricted to forming primary fibres while later myogenic populations are pluripotent.

Three populations of myoblasts, embryonic, foetal and adult, appear sequentially during myogenesis. The present study uses retroviruses to mark myoblasts clones in vivo from these populations. Myoblasts labelled at E15 (embryonic) contributed to primary fibres only. The majority of marked primary fibres were slow but a small number of clones contained marked primaries which were no longer slow at E19. Myoblasts labelled at E17 (foetal) fused with both primary and secondary fibres and most clones contained both fast and slow fibres. Similarly, adult myoblasts marked at P0 fused with all fibre types. These results indicate that embryonic myoblasts are restricted to producing only primary fibres which are initially slow but which can convert to being fast. Clones of foetal and adult myoblasts fuse with both primary and secondary fibres which may be either fast or slow.

Differential response of fetal and neonatal myoblasts to topographical guidance cues in vitro.

Fusion of mononucleated myoblasts into parallel arrays of mutinucleated myotubes is an essential step in skeletal myogenesis. The formation of such a highly ordered structure requires myoblasts to come together, orient and align in the correct location prior to fusion. We report here that fetal and neonatal myoblasts can use topographical features as strong guidance cues in vitro. Myoblasts were cultured on multiple grooved substrata of varying dimensions, and the axial orientations of individual cells were recorded. Both fetal and neonatal myoblasts aligned parallel with the direction of deep grooves (2.3-6.0 micron), which is correlated well with the location of myoblasts in similar sized grooves during secondary myogenesis. Fetal myoblasts also responded to shallower grooves (0. 04-0.14 micron) by aligning parallel or perpendicular to the direction of the grooves, indicating the ability of these cells to respond to fine elements normally encountered within the developing muscle architecture. In contrast, neonatal myoblasts failed to respond to shallow grooves, adding to the suggestion that fetal and neonatal myoblasts may represent separate populations of myoblasts. Overall, the results demonstrate that myoblasts respond to large and small features of the physical topography in vitro and indicate that structural elements in the microenvironment of the muscle may play a critical role in myoblast spatial organization during myogenesis.

The generation of fiber diversity during myogenesis.

Adult muscle is composed of different fiber types distinguished by their speed of contraction and metabolism. The generation of these differences is related both to the sequence in which muscle fibers form and to differences between the myogenic cells involved. Fibers form in two successive waves (primary and secondary) whose time of appearance can be correlated with the existence of successive populations of myogenic cells (embryonic and fetal). The differences between fibers arise through an interplay between heritable cellular commitment, where cells are preprogrammed to produce particular types of fiber and influences from the limb environment. The techniques of genetically marking cells and clonal analysis in vivo and in vitro are starting to reveal the relationship between these different influences. Although the process of myogenesis is similar in birds and mammals it is likely that cell autonomous behaviour plays a more important role during avian development as compared to mammals. The identification of muscle specific transcription factors has provided some clues to the mechanisms by which development is controlled but the expression of relatively few of these has been correlated with the sequence of events seen in myogenesis.

Dynamic expression of chicken Sox2 and Sox3 genes in ectoderm induced to form neural tissue.

The chick genes, cSox2 and cSox3, are members of a large family of genes that encode transcription factors. Previous studies have shown that these genes are predominantly expressed in the central nervous system during embryonic development. We show that cSox3 is expressed throughout the ectoderm that is competent to form nervous tissue before neural induction. The expression of cSox3 is lost from cells as they undergo gastrulation to form nonectodermal tissues; the transcription factor, Brachyury, appears in cells about to undergo gastrulation a short time before cSox3 transcripts are lost. Therefore, Brachyury expression may act functionally upstream of cSox3 downregulation. cSox3 expression is also lost from non-neuronal ectoderm shortly after the neural plate becomes morphologically apparent. cSox2 expression increases dramatically in the central nervous system as neural ectoderm is established. The appearance of cSox2 in neural ectoderm represents one of the earliest molecular responses to neural induction documented thus far.

After embryonic day 17, distribution of cells on surface of primary muscle fibres in mouse is non-random.

During the formation of skeletal muscle, secondary fibres form, by cell fusion, on the surface of primary fibres. Three-dimensional reconstructions of primary fibres with the secondary fibres and cells on their surfaces were produced from spaced serial transmission electron micrographs. Reconstructions were made of fibres from embryonic day (E) 17, E19, and E21 of the Extensor Digitorum muscle of Balb/c mice. Cell distribution was analysed in two ways. Firstly, nearest neighbour analysis was used to see if cells were randomly arranged or clustered. Secondly, the association of cells and secondary fibres was tested by measuring the distances between cells and each secondary fibre. Cells were found to be randomly distributed on the surface of primaries at E17 but significantly clustered, and associated with smaller secondary fibres at E19 and E21. Cells were not associated with the ends of secondary fibres. Cells associated with secondary fibres lay in the groove formed by contact between adjacent primary and secondary fibres. This apparent response to the topography of the surface on which the cells are lying has previously been mimicked in vitro by growing cells on grooved surfaces. It is likely that cells associated with secondaries will fuse laterally with these fibres and their response to topography is part of the process of bringing them into correct alignment with the fibre before fusion.

The effect of neonatal monocular enucleation on the optic nerves of the rat.

It is known that removal of one eye in the rat in early postnatal life results in an aberrant ipsilateral projection from the remaining eye. We have estimated the number of optic nerve fibres in 36 days old Black and White Hooded rats that had been unilaterally enucleated at birth. There was no significant difference in the transverse cross-sectional area of the optic nerves of control and enucleated rats. Control animals had an average of about 109,000 myelinated fibres and about 11,000 unmyelinated fibres. The mean minimum diameter of the myelinated fibres was 0.73 microns. The corresponding values in the unilaterally enucleated animals were not significantly different from control animals. These results are discussed in the context of the previously reported increased ipsilateral projection in unilaterally enucleated rats.

Placental growth in the pig.

The growth of the porcine placenta from 38 days until term is described. Measurements were made of its area, circumference and in situ length. Placental area increased during the period of study due to increases in the uterine circumference at the sites of conceptuses. No change was found in uterine horn length or placental length with age. Fetal weight correlated well with placental area but poorly with the other parameters. Placental length was shorter in more crowded horns and showed a U shaped distribution with position within uterine horns. These results are discussed in terms of competition for space within the uterus as a cause of the within litter variation in fetal size.

Muscle development in large and small pig fetuses.

The largest and smallest littermates were chosen by weight from litters of 38 days' gestation to 1 day post partum. Complete frozen sections of the semitendinosus muscle were used to provide a qualitative and quantitative account of the development of the primary and secondary generations of muscle fibres. The results showed that the time of formation of primary and secondary fibres, and the numbers of primary fibres formed, were the same in both large and small littermates. The number of secondary fibres formed, however, was lower in the smaller fetuses and resulted in there being a 17% difference in total fibre number at birth. Primary fibres in small fetuses were smaller, due to the smaller central myofibril-free region. This small size may have restricted the available surface area for secondary fibre formation. Fibre hyperplasia was found to cease between 85 and 95 days' gestation, and so the fibre number difference is likely to be permanent.

DNA, RNA and protein in skeletal muscle of large and small pig fetuses.

The largest and smallest littermates were chosen by weight from litters of pigs from 38 days' gestation until 1 day post partum. DNA, RNA and two fractions of protein (sarcoplasmic and fibrillar) were measured in the semitendinosus muscle. Total DNA, RNA and protein were found to increase throughout gestation and to have significantly higher values in the larger littermates. DNA concentration reached a maximum at 80-100 days' gestation and then declined. RNA concentration declined throughout gestation while protein concentration fell to a minimum value at 54 days and then increased until term. No difference could be demonstrated between large and small littermates in the concentrations of the above constituents. The RNA/DNA ratio remained relatively constant throughout gestation but was significantly higher in the larger littermates. The protein/DNA ratio increased towards the end of gestation but showed no difference between large and small animals. These results are discussed in terms of the histology of developing muscle and the nutritional status of large and small littermates.