Hereditary congenital facial paresis is a rare autosomal dominantly inherited disorder, in which pathological changes in the brainstem affect the paired facial nuclei and nerves. Previously, the neuropilin-1 protein has been shown to control axon guidance and cell body position of facial motor neurons, and mice with a targeted disruption of neuropilin-1 present with developmental defects of the facial nerve nuclei. Plexin-A1 can function as a signal transducing subunit for the neuronal neuropilin receptor, and its gene is located in the linkage interval for hereditary congenital facial paresis at chromosome 3q21-q22 (MIM601471), making it an excellent candidate gene for this disorder. During mouse embryogenesis, the murine ortholog of plexin-A1 gene showed restricted spatial and temporal expression in the hindbrain, consistent with a role in cell body movement, or axonal guidance during facial nerve development. Sequence analysis of the plexin-A1 gene in patients from the 3q21-q22-linked hereditary congenital facial paresis family revealed several nucleotide changes. However, none of the nucleotide changes led to an amino acid substitution, and reverse transcriptase polymerase chain reaction analysis did not detect aberrant RNA processing. We therefore conclude that it is highly unlikely that Plexin-A1 is involved in the pathogenicity of hereditary congenital facial paresis.
Neurons in the hypothalamus sense changes in glucose concentration. Glucokinase (GK), a key enzyme for pancreatic (beta)-cell glucose sensing, was found in both the embryonic and adult hypothalamus. GK activity accounted for approximately 20% of total hexokinase (HK) activity in both embryonic and adult hypothalamus with no activity measured in cortical samples, indicating that glucose sensing in the hypothalamus initiates early in development and precedes the maturation of glucose signaling in liver.
We investigated several aspects of the dopaminergic and serotonergic functions throughout brain development in rats prenatally exposed to MDMA ("ecstasy"). Pregnant rats were treated with MDMA (10 mg/kg s.c.) or saline from the 13th to the 20th day of gestation and studies were conducted on the progeny from both groups: (i) quantification of whole brain contents of DA, 5-HT and metabolites from the 14th day of embryonic life (E-14) to weaning (21st day of postnatal life, P-21); (ii) quantification of DA and 5-HT membrane transporters by autoradiography from E-18 to adult age (P-70); (iii) measurement of pharmacologically induced release of DA and 5-HT using microdialysis on adult (P-70) freely moving rats; (iv) measurement of sucrose preference in adults (P-70).
The SoxE genes, Sox8, Sox9 and Sox10, have been shown to modulate glial specification and differentiation in the peripheral nervous system and spinal cord. However, little is known of their expression during brain development. We have therefore analyzed their expression in developing brain, focused on the cerebellum. In situ hybridization showed a striking degree of temporo-spatial variation in their expression in different glial lineages in both chick and mouse. Sox8 appeared both in oligodendrocytes and astrocytes, Sox9 was more abundant in astrocytes and Sox10 was restricted to later stages of oligodendrocyte development.
The effects of a single course of antenatal betamethasone on S100B protein concentration were investigated in Fisher 344 rats. On day 20 of gestation, pregnant rats were injected twice 8 h apart with either (1) 170 microg kg(-1) body weight betamethasone ("clinically-equivalent dose", equivalent to 12 mg twice, 24 h apart in humans), (2) half of this dose (equivalent to 6 mg) or (3) vehicle. We report reference values for S100B protein in the serum and different brain regions in both genders at 1, 2, and 21 days after birth. Interestingly, S100B concentration showed a time-dependent and brain region-specific pattern of expression. At P1, S100B was higher in the serum of males compared to females. In addition, we show that both doses of betamethasone decreased S100B concentration in the serum of males at P1, whereas in the hippocampus, it was reduced by the clinically-equivalent dose only. This suggests that lowering the dose of antenatal betamethasone may be less detrimental for brain maturation and therefore we reiterate the need for clinical trials with a low dose regimen.
A rat short-tail mutation with neurological defects (named queue courte, qc) was discovered. Histopathology in adult qc/qc rats revealed hypoplasia of the cerebellum and hippocampus, maldevelopment of the choroid plexus and corpus callosum. These abnormalities are strongly reminiscent of the phenotypic abnormalities found in the shaker short-tail or dreher (dr) mouse mutation at the LIM homeobox transcription factor 1 alpha locus (Lmx1a). The qc mutation is an autosomal recessive and has been mapped to the dr homologous region on rat chromosome 13, and Northern blot analysis demonstrated no expression of Lmx1a in qc/qc rats. Narrowing and distortion of the ventricles were observed from embryonic day 17 (E17) in qc/qc rats. From E17, fusion of the opposing neuroepithelium and formation of neuroepithelial rosettes were also found. Arrangements of neuroepithelial cells were disturbed and processes of radial glia were disoriented in the fused lesions. Neuronal migration analysis using BrdU immunohistochemistry revealed defective migration from the neuroepithelium toward the neocortex and mesencephalon in qc/qc rats. These findings suggest that the qc mutation is involved in development of the ventricular system and dorsal migration of neurons.
In the chick retina, dopaminergic cells are generated between embryonic days 3 and 7 (E3/E7). However, the expression of tyrosine hydroxylase (TH), the first enzyme in the catecholamine synthetic pathway, is only detected after E11/E12. During the interval comprising E7 to E12, signals conveyed by cAMP are important to determine the TH phenotype. The present study shows that pituitary adenylyl cyclase-activating polypeptide (PACAP), via cAMP, is a major endogenous component in defining the TH phenotype of retina dopaminergic cells during development. PACAP type 1 receptor and its mRNA were detected in retinas since E6. PACAP was also immunodetected in cells localized in the inner nuclear layer of retinas since E8. This peptide promoted greater than 10-fold increase in cAMP accumulation of retinas obtained from embryos since E8, an effect that was blocked by PACAP6-38 (PAC1 receptor antagonist). In cultured retina cells from E8 and E9, maintained for 6 days in vitro with 10 nM PACAP (for 5 days), the number of dopaminergic cells expressing tyrosine hydroxylase increased 2.4-fold. The cAMP analog, 8-Br-cAMP and 3-isobutyl-1-methylxanthine (IBMX, a phosphodiesterase inhibitor) also increased the number of tyrosine hydroxylase-positive cells by 4- to 6-fold. IBMX plus PACAP treatment resulted in 17-fold increase in the number of cells positive for tyrosine hydroxylase. Under this condition the amount of tyrosine hydroxylase expression, as detected by western blot analysis, was also increased. The protein kinase-A inhibitor, rp-cAMPS, significantly reduced the effect of PACAP. Our data show that this peptide is an important factor influencing the definition of the tyrosine hydroxylase phenotype of retina dopaminergic cells within a narrow window of development.
Previously, this laboratory demonstrated that developing serotonin (5-HT) neurons and other fetal rhombencephalic neurons are reduced by in vivo and in vitro exposure to ethanol, effects that are related to ethanol's augmentation of apoptosis. We also found that 5-HT1A agonists diminished the ethanol-associated reduction of 5-HT neurons and other fetal rhombencephalic neurons by attenuating the pro-apoptotic effects of ethanol. Presently, we investigated the hypothesis that the protective/anti-apoptotic effects of a 5-HT1A agonist on fetal rhombencephalic neurons are mediated by activation of the phosphatidylinositol 3' kinase (PI-3K) and/or the mitogen-activated protein kinase kinase (MAPKK) pathway. Apoptotic and non-apoptotic fetal rhombencephalic neurons were quantitated in primary cultures that were treated with 50 mM ethanol and with 100 nM of a 5-HT1A agonist such as 8-OH-DPAT [8-hydroxy 2-(di-n-propylamino)tetralin], ipsapirone, or buspirone. Analysis of neurons stained with Hoechst 33342 demonstrated the anti-apoptotic effects of 5-HT1A agonists and implicated the involvement of the PI-3K pathway and possibly the MAPKK pathway with the protective effects of these drugs. The protective effects were blocked by a 5-HT1A antagonist (WAY 100635), an inhibitor of PI-3K (LY294002), and an inhibitor of MAPKK (PD98059). Western blot analyses showed that ethanol treatment reduces basal pAkt levels. These analyses also provide support for the involvement of the PI-3K pathway; ipsapirone stimulated the phosphorylation of Akt in control and ethanol-treated neurons, and these effects were antagonized by LY294002.
The anesthetic, propofol, effectively suppresses excitatory synaptic transmission and facilitates long-term depression (LTD) in the CA1 region of the hippocampus. Here, we have examined whether these effects are different in the developing hippocampus. We found that propofol in suppressing whole-cell excitatory postsynaptic currents (EPSC) was more effective in 21 day old rats than either in 7 day old rats or under the condition of high intracellular chloride concentration in 21 day old rats. Furthermore, the propofol concentration to facilitate the NMDA receptor-dependent LTD was lower at postnatal day 21 than at postnatal day 7. Interestingly, the decay time of EPSC was decreased during the development from postnatal day 7 to 21, but it was increased by the recording condition of high intracellular chloride concentration or by propofol administration. All these effects of propofol were dependent on the chloride channel opening. These observations suggest that propofol may induce differential anesthetic effects in the developing hippocampus, at least partially, depending on the intracellular chloride concentration.
During early postnatal development, afferent neurons of the cochlear (spiral) ganglion progressively refine their projections to auditory hair cells so that, by hearing onset, most cochlear nerve fibers innervate a single hearing receptor. One mechanism that might contribute to these changes in cochlear innervation is the programmed cell death (apoptosis) of developing neurons within the spiral ganglion. In the present study, we used the TUNEL method and morphological criteria to identify apoptotic cells within the spiral ganglion of the Mongolian gerbil during the first week of postnatal life when afferent projections to the cochlea are actively refined in this species. The locations of individual apoptotic spiral ganglion cells were mapped onto three-dimensional reconstructions of the entire ganglion for an age-graded series of gerbils to produce the first high-resolution, spatiotemporal maps of apoptotic ganglion cell death for the postnatal cochlea. We observed a significant increase in apoptosis in the spiral ganglion from postnatal day (P) 4 through P6. During this time, the most intense apoptotic activity occurred in regions of the spiral ganglion providing innervation to the lower middle and apical turns of the cochlea. The time course and regional variation of programmed cell death within the developing gerbil spiral ganglion are discussed in terms of the postnatal refinement of cochlear innervation and its possible functional significance for hearing in gerbils.
The comparative study of anti- and pro-saccade task performance contributes to our functional understanding of the frontal lobes, their alterations in psychiatric or neurological populations, and their changes during the life span. In the present study, we apply regression analysis to model life span developmental effects on various pro- and anti-saccade task parameters, using data of a non-representative sample of 327 participants aged 9 to 88 years. Development up to the age of about 27 years was dominated by curvilinear rather than linear effects of age. Furthermore, the largest developmental differences were found for intra-subject variability measures and the anti-saccade task parameters. Ageing, by contrast, had the shape of a global linear decline of the investigated saccade functions, lacking the differential effects of age observed during development. While these results do support the assumption that frontal lobe functions can be distinguished from other functions by their strong and protracted development, they do not confirm the assumption of disproportionate deterioration of frontal lobe functions with ageing. We finally show that the regression models applied here to quantify life span developmental effects can also be used for individual predictions in applied research contexts or clinical practice.
Early postnatal developmental changes in N-methyl-d-aspartate (NMDA) receptor (NR) subunits regulate cerebellar granule cell maturation and potentially Purkinje cell development. We therefore investigated Purkinje cell morphology in slice cultures from mice with genetic subunit exchange from NR2C to NR2B (NR2C-2B). NR2C-2B Purkinje cells after 12 days in vitro showed a significantly impaired dendritic arbour complexity with reduced branching density as compared to wild-type cells, a phenotype that was reversed by NMDA treatment. These data support the concept that in cerebellar slice cultures, Purkinje cell dendritic outgrowth is regulated by granule cell inputs.
We report here the histogenesis of the brainstem of the trout (Salmo trutta fario) and the medaka (Oryzias latipes) chosen as examples of teleosts with slow and fast growth, respectively. Our results reveal that the sequence of formation of brain structures is rather similar in the teleosts species examined so far, but some interspecific differences do exist in terms of brainstem maturation at particular developmental stages, such as the end of the gastrulation and hatching periods. This sequence includes the subdivision of the brainstem in different transverse segments and longitudinal zones, where morphologically discernible boundaries are observed along the caudorostral and ventrodorsal axis. The boundary formation and subsequent subdivision of the trout and medaka brainstems, together with the proliferation pattern observed by immunohistochemistry with an antibody against the proliferating cell nuclear antigen (PCNA), support a segmental model throughout the brainstem. The spatiotemporal pattern of PCNA immunoreactivity is similar in the mesencephalon and rhombencephalon of the two teleosts species studied, although proliferation centers are less clearly defined in the medaka. Moreover, the segmental appearance of the brainstem, as revealed by PCNA immunohistochemistry, is blurred earlier in the medaka than in the trout. Thus, the trout brain appears a suitable model for morphogenetic studies because it allows more gradual survey of the changes throughout development.
We investigated the immunohistochemical expression of the tyrosine hydroxylase (TH) enzyme and the morphometric parameters of the human locus coeruleus (LC) in the brainstems of 32 subjects aged from 17 gestational weeks to 12 postnatal month, died of unknown (sudden unexplained perinatal and infant deaths) and known causes. The goals of this study were: (1) to obtain basic information about the structure and physiology of the LC during the first phases of human nervous system development; (2) to evaluate whether there is altered expression of TH and/or structural alterations of the LC in cases of sudden perinatal and infant death; and (3) to verify if morphological and/or physiological abnormalities of the LC could be related to maternal cigarette smoking. Morphometric analysis showed homogeneous data in cases of sudden perinatal and infant death and in age-matched controls who had died of known aetiology. However, immunohistochemistry demonstrated in a wide subset of sudden and unexplained deaths a negativity or low positivity of TH. High distribution of TH protein were instead detectable in the LC neurons of foetuses aged 17–18 gestational weeks who had died of known causes. Therefore, we postulate the functional importance of the LC in the early phases of central nervous system development. Besides, the observation of a significant correlation between sudden unexplained death, negativity of TH staining and maternal smoking, prompted us to suppose a close relation between smoking in utero and a decrease of the noradrenergic activity of the LC, leading to sudden death in the last part of pregnancy and in the first year of life.
We have generated 362 bp and 547 bp partial sequences for Rana pipiens ephrin-A2 and ephrin-A5 mRNA, respectively. Translation homologies for the comparable segments of cDNA of chicken, mouse and human are 90.8, 86.9 and 84.4% for the ephrin-A2 sequence and 85.7, 85.0 and 85.0% for the ephrin-A5 sequence. Digoxigenin-labeled riboprobes were prepared and applied by means of in situ hybridization to whole-mounts of the brains of mature adults and expression patterns in tadpoles were also explored. The RNA probes revealed similar posterior (high) to anterior (low) expression gradients in the adult tectum, demonstrating that both ephrin-As are expressed in the adult Ranid frog tectum. Only the ephrin-A2 probe was tested on tadpole brain, yielding an appropriately graded expression pattern similar to the adult.
The present work was conducted to study the cellular composition and developmental capacity of retinal neurospheres. Furthermore, the ability of grafted neurospheres to integrate into adult retinal tissue was studied in an in vitro model. Retinal progenitor cells isolated from rat embryos were expanded into neurospheres in vitro in the presence of basic fibroblast growth factor (bFGF), epidermal growth factor (EGF) and leukemia inhibitory factor (LIF). Neurospheres labeled with a lipophilic dye were placed onto explants, and tissue interactions were analyzed after 2–6 days of culture. Immunocytochemical analysis of neurospheres revealed the presence of neuronal and glial cells. Proliferating neuronal and glial cells were observed after 2 weeks, whereas the neuronal cell proliferation declined considerably after 4 weeks. Few apoptotic cells were observed in the neurospheres. Neurospheres cultured on explanted adult retina engrafted with the surrounding tissue, but progenitor cell migration into the explants was low. However, the grafted neurospheres appeared to limit the experimentally induced photoreceptor apoptosis in the surrounding explant tissue.
The cytoarchitecture of dorsal cochlear nucleus (DCN), characterized by a distinct laminar structure similar to the cerebellar cortex of the normal mouse, is known to be disrupted in the Reelin-deficient mouse, reeler. Here, we have reexamined both the cytoarchitecture and myeloarchitecture of this nucleus and described expression pattern of Reelin protein during perinatal periods. Reelin-immunopositive granule cells were firstly recognized in the external granular layer of the DCN at embryological day 16 (E16). Next, we examined the cytoarchitecture of the DCN of the normal and reeler mice with Ca2+/calmodulin-dependent protein kinase IIα (CaMKIIα) immunostaining. CaMKIIα-immunoreactive cartwheel cells were laminarly distributed in the layer II of the normal DCN, but scattered throughout the reeler DCN. Injection of retrograde tracer, Fluoro-Gold (FG) into the inferior colliculus of the reeler mouse resulted in that retrogradely labeled neurons in the DCN were radially scattered instead of being confined to a single layer as seen in the normal mouse. To examine whether CaMKIIα-immunopositive cartwheel cells are neurons projecting to the inferior colliculus or not, double labeling with CaMKIIα immunohistochemistry and retrograde labeling with an injection of FG into the inferior colliculus were made, which revealed that CaMKIIα-immunoreactive cartwheel cells do not send axons to the inferior colliculus. The present findings imply that Reelin may have some roles in the formation of laminar structures of the DCN.
The development of the serotonergic (5HT) and dopaminergic (DA) systems may contribute to the onset of psychotic disorders during late adolescence and early adulthood. Previous studies in our laboratory have suggested that these systems may compete for functional territory on neurons during development, as lesions of the serotonergic system at postnatal day 5 (P5) result in an increase in the density of dopaminergic fibers in rat medial prefrontal cortex (mPFC). In the present study, the dopaminergic system of P5 rats was lesioned with intracisternal injections of 6-hydroxydopamine (6-OHDA). Quantification of serotonin-immunoreactivity (5HT-IR) in mPFC at adulthood (P70) revealed a significant decrease in fiber density within layers II and III of the Cg3 subdivision of mPFC in lesioned rats compared to sham controls. We propose that the decrease in serotonergic fibers in mPFC in response to a neonatal depletion of dopamine may be due to the loss of a trophic effect of this system on 5HT neurons and/or fibers during development. Taken together with previous findings, our data suggest that there may be an "inverse trophic" relationship between the cortical DA and 5HT systems whereby dopamine facilitates the ingrowth of 5HT fibers, while serotonin suppresses the ingrowth of DA fibers. We present a model based on neurotrophic interactions at the cortical and brainstem levels that could potentially explain these unexpected results.
This study was conducted to characterize the post-pubertal developmental aspects on seizure susceptibility and severity as well as calcium/calmodulin protein kinase type II (CaM kinase II) activity in status epilepticus (SE). Thirty- to ninety-day-old rats, in 10-day increments, were studied. This corresponds to a developmental age group that has not received thorough attention. The pilocarpine model of SE was characterized both behaviorally and electrographically. Seven criteria were analyzed for electrographical characterization: seizure severity, SE susceptibility, the average number of discrete seizures, average time until first seizure, average time to SE, average time from first discrete seizure to SE, and death. After 1 h of SE, specific brain regions were isolated for biochemical study. Phosphate incorporation into a CaM kinase II-specific substrate, autocamtide III, was used to determine kinase activity. There was no developmental effect on the average number of discrete seizures, average time until first seizure, average time to SE, average time from first discrete seizure to SE, and death; however, there was a significant effect on SE probability and seizure severity. Once SE was expressed, all animals showed a decrease in both cortical and hippocampal CaM kinase II activities. Conversely, seizure activity in the absence of SE did not result in a decrease in CaM kinase II activity. The data suggest that there is a gradual age-dependent modulation of SE susceptibility and seizure severity within the developmental stages studied. Additionally, once status epilepticus is observed at any age, there is a corresponding SE-induced inhibition of CaM kinase II.
