Distinction in the immunoreactivities of two calcium-binding proteins and neuronal birthdates in the first and higher-order somatosensory thalamic nuclei of mice: Evolutionary implications.

Comparative embryonic studies are the most effective way to discern phylogenetic changes. To gain insight into the constitution and evolution of mammalian somatosensory thalamic nuclei, we first studied how calbindin (CB) and parvalbumin (PV) immunoreactivities appear during embryonic development in the first-order relaying somatosensory nuclei, i.e., the ventral posteromedial (VPM) and posterolateral (VPL) nuclei, and their neighboring higher-order modulatory regions, including the ventromedial or ventrolateral nucleus, posterior, and the reticular nucleus. The results indicated that cell bodies that were immunoreactive for CB were found earlier (embryonic day 12 [E12]) in the dorsal thalamus than were cells positive for PV (E14), and the adult somatosensory thalamus was characterized by complementary CB and PV distributions with PV dominance in the first-order relaying nuclei and CB dominance in the higher-order regions. We then labeled proliferating cells with [(3) H]-thymidine from E11 to 19 and found that the onset of neurogenesis began later (E12) in the first-order relaying nuclei than in the higher-order regions (E11). Using double-labeling with [(3) H]-thymidine autoradiography and CB or PV immunohistochemistry, we found that CB neurons were born earlier (E11-12) than PV neurons (E12-13) in the studied areas. Thus, similar to auditory nuclei, the first and the higher-order somatosensory nuclei exhibited significant distinctions in CB/PV immunohistochemistry and birthdates during embryonic development. These data, combined with the results of a cladistic analysis of the thalamic somatosensory nuclei, are discussed from an evolutionary perspective of sensory nuclei.

Living high training low induces physiological cardiac hypertrophy accompanied by down-regulation and redistribution of the renin-angiotensin system.

AIM: Living high training low" (LHTL) is an exercise-training protocol that refers living in hypoxia stress and training at normal level of O2. In this study, we investigated whether LHTL caused physiological heart hypertrophy accompanied by changes of biomarkers in renin-angiotensin system in rats. METHODS: Adult male SD rats were randomly assigned into 4 groups, and trained on living low-sedentary (LLS, control), living low-training low (LLTL), living high-sedentary (LHS) and living high-training low (LHTL) protocols, respectively, for 4 weeks. Hematological parameters, hemodynamic measurement, heart hypertrophy and plasma angiotensin II (Ang II) level of the rats were measured. The gene and protein expression of angiotensin-converting enzyme (ACE), angiotensinogen (AGT) and angiotensin II receptor I (AT1) in heart tissue was assessed using RT-PCR and immunohistochemistry, respectively. RESULTS: LLTL, LHS and LHTL significantly improved cardiac function, increased hemoglobin concentration and RBC. At the molecular level, LLTL, LHS and LHTL significantly decreased the expression of ACE, AGT and AT1 genes, but increased the expression of ACE and AT1 proteins in heart tissue. Moreover, ACE and AT1 protein expression was significantly increased in the endocardium, but unchanged in the epicardium. CONCLUSION: LHTL training protocol suppresses ACE, AGT and AT1 gene expression in heart tissue, but increases ACE and AT1 protein expression specifically in the endocardium, suggesting that the physiological heart hypertrophy induced by LHTL is regulated by region-specific expression of renin-angiotensin system components.

Sites of origin and developmental dynamics of the neurons in the core and shell regions of torus semicircularis in the Chinese softshell turtle (Pelodiscus sinensis).

To know the embryogenesis of the core and shell regions of the midbrain auditory nucleus, a single dose of [(3)H]-thymidine was injected into the turtle embryos at peak stages of neurogenesis in the shell and core of the torus semicircularis. Following sequential survival times, labeled neurons and the dynamics of cell proliferation were examined. The expression of vimentin (VM), reelin, calbindin, parvalbumin, and substance P were also studied. The results showed that: 1) progenitor cells for the core and shell regions were generated in different sites of the ventricular zone; 2) the length of the cell cycle or S-phase for the shell region were both longer than those for the core region (4.7 and 3.2 hours longer, respectively), suggesting that mitotic activity in the core region is higher than it is in the shell region; 3) the elongated cell bodies of the labeled core and shell cells had close apposition to VM fibers, suggesting that the migration of these cells is guided by VM fibers; 4) the germinal sites of the core and shell constructed by projecting the orientation of radial VM fibers back to the ventricular zone was consistent with those obtained by short and sequential survival [(3)H]-thymidine radiography; and 5) the beginning of positive staining for parvalbumin in the core region was interposed between those for calbindin and substance P in the shell regions. This study contributes to the understanding of how auditory nuclei are organized and how their components developed and evolved.

Germinal sites and migrating routes of cells in the mesencephalic and diencephalic auditory areas in the African clawed frog (Xenopus laevis).

There is a clear core-shell organization in the auditory nuclei of amniotes. However, such organization only exists in the mesencephalic, but not in the diencephalic auditory regions of amphibians. To gain insights into how this core-shell organization developed and evolved, we injected a small dose of [(3)H]-thymidine into tadpoles of Xenopus laevis at peak stages of neurogenesis in the mesencephalic and diencephalic auditory areas. Following different survival times, the germinal sites and migrating routes of cells were examined in the shell (laminar nucleus, Tl; magnocellular nucleus, Tmc) and core (principal nucleus, Tp) regions of the mesencephalic auditory nucleus, torus semicircularis (Ts), as well as in the diencephalic auditory areas (posterior thalamic nucleus, P; central thalamic nucleus, C). Double labeling for [(3)H]-thymidine autoradiography and immunohistochemistry for vimentin was also performed to help determine the routes of cell migration. We found three major results. First, the germinal sites of Tp were intercalated between Tl and Tmc, arising from those of the shell regions. Second, although the germinal sites of Tl, Tmc, and Tp were located in the same brain levels (at rostromedial or caudomedial levels of Ts), neurogenesis in Tl or Tmc started earlier than that in Tp. Finally, the P and C were also generated in different ventricle sites. However, unlike Ts their neurogenesis showed no obvious temporal differences. These data demonstrate that a highly differentiated auditory region, such as Tp in Ts, is lacking in the diencephalon of amphibian. Our data are discussed from the view of the constitution and evolutionary origins of auditory nuclei in vertebrates.

Evolutionary significance of delayed neurogenesis in the core versus shell auditory areas of Mus musculus.

Early comparative embryogenesis can reflect the organization and evolutionary origins of brain areas. Neurogenesis in the auditory areas of sauropsids displays a clear core-to-shell distinction, but it remains unclear in mammals. To address this issue, [3H]-thymidine was injected into pregnant mice on consecutive embryonic (E) days (E10-E19) to date neuronal birthdays. Immunohistochemistry for substance P, calbindin, and parvalbumin was conducted to distinguish the core and shell auditory regions. The results showed that: 1) cell generation began at E13 in the external or dorsal nucleus of the inferior colliculus (IC), but it did not start in the caudomedial portion of the central nucleus of IC, and significantly fewer cells were produced in the medial and rostromedial portions of the central nucleus of IC; 2) cells were generated at E11 in the dorsal and medial divisions of the medial geniculate complex (MGd and MGm, respectively), whereas cell generation was absent in the medial and rostromedial portions of the ventral medial geniculate complex (MGv), and fewer cells were produced in the caudomedial portion of MGv; 3) in the telencephalic auditory cortex, cells were produced at E11 or E12 in layer I and the subplate, which receive projections from the MGd and MGm. However, cell generation occurred at E13-E18 in layers II-VI, including the area receiving projections from the MGv. The core-to-shell distinction of neurogenesis is thus present in the mesencephalic to telencephalic auditory areas in the mouse. This distinction of neurogenesis is discussed from an evolutionary perspective.

Neurogenic development of the auditory areas of the midbrain and diencephalon in the Xenopus laevis and evolutionary implications.

To study whether the core-versus-shell pattern of neurogenesis occurred in the mesencephalic and diencephalic auditory areas of amniotes also appears in the amphibian, [(3)H]-thymidine was injected into tadpoles at serial developmental stages of Xenopus laevis. Towards the end of metamorphism, [(3)H]-thymidine labeling was examined and led to two main observations: 1) neuron generation in the principal nucleus (Tp) started at stage 50, and peaked at stage 53, whereas it began at stage 48.5, and peaked around stage 49 in the other two mesencephalic auditory areas, the laminar nucleus (Tl) and the magnocellular nucleus (Tmc). 2) Neuron generation appeared at stage 40, and peaked around stage 52 in the posterior thalamic nucleus (P) and the central thalamic nucleus (C). Our study revealed that, like the cores of mesencephalic auditory nuclei in amniotes, Tp showed differences from Tl and Tmc in the onset and the peak of neurogenesis. However, such differences did not occur in the P and C. Our neurogenetic data were consistent with anatomical and physiological reports indicating a clear distinction between the mesencephalic, but not the diencephalic auditory areas of the amphibian. Our data are helpful to get insights into the organization of auditory nuclei and its evolution in vertebrates.

Differences in neurogenesis differentiate between core and shell regions of auditory nuclei in the turtle (Pelodiscus sinensis): evolutionary implications.

There is a clear core-versus-shell distinction in cytoarchitecture, electrophysiological properties and neural connections in the mesencephalic and diencephalic auditory nuclei of amniotes. Determining whether the embryogenesis of auditory nuclei shows a similar organization is helpful for further understanding the constituent organization and evolution of auditory nuclei. Therefore in the present study, we injected [(3)H]-thymidine into turtle embryos (Pelodiscus sinensis) at various stages of development. Upon hatching, [(3)H]-thymidine labeling was examined in both the core and shell auditory regions in the midbrain, diencephalon and dorsal ventricular ridge. Met-enkephalin and substance P immunohistochemistry was used to distinguish the core and shell regions. In the mesencephalic auditory nucleus, the occurrence of heavily labeled neurons in the nucleus centralis of the torus semicircularis reached its peak at embryonic day 9, one day later than the surrounding shell. In the diencephalic auditory nucleus, the production of heavily labeled neurons in the central region of the reuniens (Re) was highest at embryonic day (E) 8, one day later than that in the shell region of reuniens. In the region of the dorsal ventricular ridge that received inputs from the central region of Re, the appearance of heavily labeled neurons also reached a peak one day later than that in the area receiving inputs from the shell region of reuniens. Thus, there is a core-versus-shell organization of neuronal generation in reptilian auditory areas.

Sex difference in cellular proliferation within the telencephalic ventricle zone of Bengalese finch.

Cellular proliferation within the ventricular zone (VZ) may contribute to sex differences through the net addition of neurons in song control nuclei. To address this issue, we administered [(3)H]thymidine to Bengalese finches of both sexes, and estradiol benzoate (EB) to females 15 days post hatching. The birds were killed 2h later to examine thymidine labeled cells within the VZ at three brain levels, HVC, anterior commissure and Area X. Our results indicated that: (1) cell proliferation in the VZ was significantly higher in the three studied brain levels in males and EB implant females relative to intact or empty implant females, respectively; (2) proliferation in the dorsal half of the VZ, in proximity to HVC, was notably higher than that in the ventral half of the VZ; (3) proliferation in the ventral VZ (VVZ), which is relatively close to Area X was higher relative to other subregions of VZ (dorsal and intermediate). Our study suggests that sex differences in cell proliferation in the VZ may contribute to the net growth of HVC and Area X in males, and estradiol may play an important role in sexual difference in cellular proliferation within the VZ.

Comparative analyses of song complexity and song-control nuclei in fourteen oscine species.

Most studies on the relationship between measures of song behavior and the sizes of song control nuclei have focused on one or two oscine species, and often show inconsistent results. To address this issue, we first measured four variables for song complexity, i.e., song repertoire size, syllable repertoire size, the mean number of syllables per phrase (MNS) and the number of syllables in the longest phrase (NSLP), and the sizes of three song control nuclei, i.e., HVC, RA (the robust nucleus of the arcopallium), and Area X in 14 oscine species from eight families. To tackle the problem of statistical non-independence that probably existed among the closely related species, we reconstructed the phylogeny of the species studied using mitochondrial cytochrome b DNA sequences from GenBank. By using the methods adopted in most previous reports, we tested the relationship between song complexity and the sizes of the song control nuclei. We found that: 1) the absolute sizes of RA and Area X, but not of HVC, were positively correlated to the three measures of song complexity, but that only the residual size of RA in regard to telencephalon size was significantly correlated to the song measures; 2) independent contrasts analysis showed RA and Area X to be significantly associated with NSLP. Our results indicated that the relationship between song behavior and its neural structures varied among song nuclei, suggesting that each song control nucleus may play a different role in song behavior.

[The production and distribution of the newborn neural precursor in the telencephalon of female Melanocorypha mongolica and compared with female Lonchura striata].

The production of labeled precursors in adult female Melanocorypha mongolica and Lonchura striata were investigated by using bromodeoxyuridine (BrdU) and ABC immunohistochemical method to mark new synthesized DNA. Then, the distribution of newborn precursor was compared between the two kinds of songbirds. The results reveal that: 1) During 1-4 days after BrdU intramuscular injection, there were a great number of labeled cells in lateral ventricular zone (LVZ),and forming proliferation 'hot spots' in the ventricular zone (VZ) of the ventral striatum and neostriatum. The newborn labeled precursor in the two adult songbirds originated from ventricular zone in telencephalon. In the two adult songbirds, there were a great number of labeled precursors in the ventricular zone (VZ) of the medial striatum and lateral striatum and forming proliferation 'the first hot spots'; in the top of LVZ and forming proliferation 'the second hot spots'. In Melanocorypha mongolica,there were a lot of labeled precursors in the tail of VZ in lateral striatum and forming proliferation 'the third hot spots'. But in Lonchura striata, there were a small number BrdU labeled precursors in the tail of VZ. 2) From 5 days after BrdU intramuscular injection in Melanocorypha mongolica, a great number of labeled precursors in the LVZ began to migrate into the other region of the telencephalon. During 5-30 day after BrdU intramuscular injection, some of labeled cells emerged into the nucleus of high vocal center (HVc), and robust nucleus of the archistriatum (RA) in Melanocorypha mongolica. But there were no BrdU labeled cells in the HVc and RA in female Lonchura striata during the 30 days after BrdU intramuscular injection. New neurons, which are added to HVc and RA continuously in adult female Melanocorypha mongolica, may be necessary for learning the new songs of adult male birds. HVc and RA of adult Lonchura striata have few new neurons to replace older neurons.

The distribution of substance P and met-enkephalin in vocal control nuclei among oscine species and its relation to song complexity.

Substance P (SP) and methionine-enkephalin (ENK) have been reported to appear in song control nuclei of oscine species. However, it remains unknown whether or not SP and ENK location in song control nuclei is correlated with song behavior. To address this issue, the present study first measured two variables for song complexity, i.e., song repertoire sizes, and syllable repertoire sizes in 11 oscine species. Then, we examined the distribution of SP and ENK in four control nuclei, two in the motor pathway, i.e., HVC and the robust nucleus of arcopallium (RA), and the other two in the forebrain pathway, i.e., Area X and the lateral magnocellular nucleus of the anterior nidopallium (LMAN). Finally, we measured the relative amounts of immunoreactivity for SP and ENK in song control nuclei, and tested whether they were correlated with song complexity. Our results showed that: (1) SP and ENK were broadly distributed in the song control nuclei of studied species. However, SP immunohistochemistry was more robust in comparison with ENK, and SP is generally more abundant in the two song learning nuclei than those in the two song producing ones; (2) SP and ENK staining patterns in song control nuclei did not show any obvious phylogenetic relationship among studied oscine species; (3) there was a significant correlation between the relative amounts of immunoreactivity for SP and the song and syllable repertoire sizes. Our results suggest that SP or ENK might be involved in song behavior, such as birdsong learning or memory.

[The localization and the sexual differences of substance P in vocal control and part auditory nuclei of Carduelis spinus].

The expression of substance P in the vocal control and auditory nuclei was investigated by wsing immunohistochemical methods, and the gray density of numerical value was measured with the image processing system in females and males of Carduelis spinus. Then, the distribution and the gray density of substance P were compared between males and females. The results indicate that: 1) Substance P labeled terminal and part cells were distributed in the Area X; 2) Substance P labeled cells were distributed in the nucleus high vocal center (HVc), magnocellular nucleus of the anterior neostriatum (MAN), robust nucleus of the archistriatum (RA) and dorsolateral nucleus of the anterior thalamus (DLM); 3) Substance P labeled terminal and fibers were distributed in the vocal control nuclei such as nucleus dorsalis medialis (DM) and the nucleus hypoglossi, pars tracheosyringealis (nXI-Its), and in the auditory nuclei such as the nucleus ovidalisashell (Ov shell), the shell regions of mesencephalicus lateralis, pars dorsalis (MLd shell) and the nucleus intercollicularis (ICo). The values of gray degree of substance P labeled cells or fibers were significantly higher in males than that in females. The present study indicates that the distribution of substance P exhibits significantly sexual difference in the songbird. The presence of substance P in most auditory and vocal control nuclei suggests that substance P may play an important physiological role in the auditory perception and vocal production.