Spatial restriction of spontaneous activity towards the rostral primary initiating zone during development of the embryonic mouse hindbrain

In the developing embryonic mouse hindbrain, we have previously shown that synchronized spontaneous activity is driven by midline serotonergic neurons at E11.5. This is mediated, at least in part, by the 5-HT2A receptor, which is expressed laterally in the hindbrain. Activity at E11.5 is widespread within the hindbrain tissue, propagating from the midline to more lateral regions. Using rapid acquisition of [Ca2+]i events along the midline, we now show that the rostral midline, primarily in the region of former rhombomere r2, is the primary initiating zone for this activity. We propose that at E11.5, the combined events along the rostral-caudal axis in combination with events propagating along the medial-lateral axis could assign positional information to developing neurons within the hindbrain. With further development, to E13.5, both the lateral and caudal dimensions of spontaneous activity retract to the rostral midline, occupying an area only 14% of that exhibited at E11.5. We also show that increased levels of [K+]o (to 8 mM) at E13.5 are able to increase the spread of spontaneous activity laterally and rostro-caudally. This suggests that spontaneous activity in the hindbrain depends in a dynamic way on the dominant initiating zone of the rostral midline, and that this relationship changes over development.     

Properties and mechanisms of spontaneous activity in the embryonic chick hindbrain

Spontaneous activity regulates many aspects of central nervous system development. We demonstrate that in the embryonic chick hindbrain, spontaneous activity is expressed between embryonic days (E) 6–9. Over this period the frequency of activity decreases significantly, although the events maintain a consistent rhythm on the timescale of minutes. At E6, the activity is pharmacologically dependent on serotonin, nACh, GABA_A, and glycine input, but not on muscarinic, glutamatergic, or GABA_B receptor activation. It also depends on gap junctions, t‐type calcium channels and TTX‐sensitive ion channels. In intact spinal cord‐hindbrain preparations, E6 spontaneous events originate in the spinal cord and propagate into lateral hindbrain tissue; midline activity follows the appearance of lateral activity. However, the spinal cord is not required for hindbrain activity. There are two invariant points of origin of activity along the midline, both within the caudal group of serotonin‐expressing cell bodies; one point is caudal to the nV exit point while the other is caudal to the nVII exit point. Additional caudal midline points of origin are seen in a minority of cases. Using immunohistochemistry, we show robust differentiation of the serotonergic raphe near the midline at E6, and extensive fiber tracts expressing GAD65/67 and the nAChR in lateral areas; this suggests that the medial activity is dependent on serotonergic neuron activation, while lateral activity requires other transmitters. Although there are differences between species, this activity is highly conserved between mouse and chick, suggesting that developmental event(s) within the hindbrain are dependent on expression of this spontaneous activity. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

Hes1 regulates the number and anterior-posterior patterning of mesencephalic dopaminergic neurons at the mid/hindbrain boundary (isthmus)

The lack of the Hes1 gene leads to the failure of cranial neurulation due to the premature onset of neural differentiation. Hes1 homozygous null mutant mice displayed a neural tube closure defect, and exencephaly was induced at the mid/hindbrain boundary. In the mutant mesencephalon, the roof plate was not formed and therefore the ventricular zone showing cell proliferation was displaced to the brain surface. Furthermore, the telencephalon and ventral diencephalon were defective. Despite the severe defects of neurogenesis in null mutants, the mesencephalic dopaminergic (mesDA) neurons were specified at the midline of the ventral mesencephalon in close proximity to two important signal centers — floor plate and mid/hindbrain boundary (i.e., the isthmic organizer). Using mesDA neuronal markers, tyrosine hydroxylase (TH) and Pitx3, the development of mesDA neurons was studied in Hes1 null mice and compared with that in the wild type. At early stages, between embryonic day (E) 11.5 and E12.5, mesDA neurons were more numerous in null mutants than in the wild type. From E13.5 onward, however, the cell number and fiber density of mesDA neurons were decreased in the mutants. Their distribution pattern was also different from that of the wild type. In particular, mesDA neurons grew dorsally and invaded the rostral hindbrain. 5-HT neurons were also ectopically located in the mutant midbrain. Thus, the loss of Hes1 resulted in disturbances in the inductive and repulsive activities of the isthmic organizer. It is proposed that Hes1 plays a role in regulating the location and density of mesDA neurons.     

Expression of Trk receptors in the developing mouse trigeminal ganglion: in vivo evidence for NT-3 activation of TrkA and TrkB in addition to TrkC

Animals lacking neurotrophin-3 (NT-3) are born with deficits in almost all sensory ganglia. Among these, the trigeminal ganglion is missing 70% of the normal number of neurons, a deficit which develops during the major period of neurogenesis between embryonic stages (E) 10.5 and E13.5. In order to identify the mechanisms for this deficit, we used antisera specific for TrkA, TrkB, and TrkC to characterize and compare the expression patterns of each Trk receptor in trigeminal ganglia of wild type and NT-3 mutants between E10.5 and E15.5. Strikingly, TrkA, TrkB, and TrkC proteins appear to be exclusively associated with neurons, not precursors. While some neurons show limited co-expression of Trk receptors at E11.5, by E13. 5 each neuron expresses only one Trk receptor. Neuronal birth dating and cell counts show that in wild-type animals all TrkB- and TrkC-expressing neurons are generated before E11.5, while the majority of TrkA-expressing neurons are generated between E11.5 and E13.5. In mice lacking NT-3, the initial formation of the ganglion, as assessed at E10.5, is similar to that in wild-type animals. At E11.5, however, the number of TrkC-expressing neurons is dramatically reduced and the number of TrkC-immunopositive apoptotic profiles is markedly elevated. By E13.5, TrkC-expressing neurons are virtually eliminated. At E11.5, compared to wild type, the number of TrkB-expressing neurons is also reduced and the number of TrkB immunoreactive apoptotic profiles is increased. TrkA neurons are also reduced in the NT-3 mutants, but the major deficit develops between E12.5 and E13.5 when elevated numbers of TrkA-immunoreactive apoptotic profiles are detected. Normal numbers of TrkA- and TrkB-expressing neurons are seen in a TrkC-deficient mutant. Therefore, our data provide evidence that NT-3 supports the survival of TrkA-, TrkB- and TrkC-expressing neurons in the trigeminal ganglion by activating directly each of these receptors in vivo.     

Spontaneous activity in the developing mouse midbrain driven by an external pacemaker

Central nervous system (CNS) development depends upon spontaneous activity (SA) to establish networks. We have discovered that the mouse midbrain has SA expressed most robustly at embryonic day (E) 12.5. SA propagation in the midbrain originates in midline serotonergic cell bodies contained within the adjacent hindbrain and then passes through the isthmus along ventral midline serotonergic axons. Once within the midbrain, the wave bifurcates laterally along the isthmic border and then propagates rostrally. Along this trajectory, it is carried by a combination of GABAergic and cholinergic neurons. Removing the hindbrain eliminates SA in the midbrain. Thus, SA in the embryonic midbrain arises from a single identified pacemaker in a separate brain structure, which drives SA waves across both regions of the developing CNS. The midbrain can self‐initiate activity upon removal of the hindbrain, but only with pharmacological manipulations that increase excitability. Under these conditions, new initiation foci within the midbrain become active. Anatomical analysis of the development of the serotonergic axons that carry SA from the hindbrain to the midbrain indicates that their increasing elongation during development may control the onset of SA in the midbrain. © 2009 Wiley Periodicals, Inc. Develop Neurobiol 2009     

Multiple influences on the migration of precerebellar neurons in the caudal medulla.

Neurons destined to form several precerebellar nuclei are generated in the dorsal neuroepithelium (rhombic lip) of caudal hindbrain. They form two ventrally directed migratory streams, which behave differently. While neurons in the superficial migration migrate in a subpial position and cross the midline to settle into the contralateral hindbrain, neurons in the olivary migration travel deeper in the parenchyma and stop ipsilaterally against the floor plate. In the present study, we compared the behavior of the two neuronal populations in an organotypic culture system that preserves several aspects of their in vivo environment. Both migrations occurred in mouse hindbrain explants dissected at E11.5 even when the floor plate was ablated at the onset of the culture period, indicating that they could rely on dorsoventral cues already distributed in the neural tube. Nevertheless, the local constraints necessary for the superficial migration were more specific than for the olivary migration. Distinct chemoattractive and chemorespulsive signal were found to operate on the migrations. The floor plate exhibited a strong chemoattractive influence on both migrations, which deviated from their normal path in the direction of ectopic floor plate fragments. It was also found to produce a short-range stop signal and to induce inferior olive aggregation. The ventral neural tube was also found to inhibit or slow down the migration of olivary neurons. Interestingly, while ectopic sources of netrin were found to influence both migrations, this effect was locally modulated and affected differentially the successive phases of migration. Consistent with this observation, while neurons in the superficial migration expressed the Dcc-netrin receptor, the migrating olivary neurons did not express Dcc before they reached the midline. Our observations provide a clearer picture of the hierarchy of environmental cues that influence the morphogenesis of these precerebellar nuclei.     

LIFR beta plays a major role in neuronal identity determination and glial differentiation in the mouse facial nucleus

In the hindbrain, generation of the facial nucleus involves complex developmental processes that will lead to the formation of a structure composed of motor neurons, astrocytes and oligodendrocytes. The implication of LIF-related cytokines in the development of this nucleus came to light with the analysis of mice mutant for the receptor of these cytokines, LIFR beta, which exhibit a massive loss of facial branchiomotor (fbm) neurons at birth and a severe decrease in GFAP expression, a marker of astrocytes. To uncover the cellular mechanisms regulated by LIFR beta during facial nucleus development, we first analyzed its expression pattern in the hindbrain. lifr beta is first expressed at E11.5 in the hindbrain neuroepithelium. The receptor is absent during the migration of fbm post-mitotic neurons but is strongly expressed when fbm neurons have reached rhombomere 6 at E12.5, and its expression is maintained until E18.5. From the analysis of lifr beta mutant embryos, we established that LIFR beta is necessary for fbm neurons' identity determination. We also show that LIFR beta is implicated in astrocyte and oligodendrocyte differentiation, specifically within the facial nucleus.     

Serotonin patterns locomotor network activity in the developing zebrafish by modulating quiescent periods

Developing neural networks follow common trends such as expression of spontaneous, recurring activity patterns, and appearance of neuromodulation. How these processes integrate to yield mature, behaviorally relevant activity patterns is largely unknown. We examined the integration of serotonergic neuromodulation and its role in the functional organization of the accessible locomotor network in developing zebrafish at behavioral and cellular levels. Locally restricted populations of serotonergic neurons and their projections appeared in the hindbrain and spinal cord of larvae after hatching (approximately day 2). However, 5-HT affected the swimming pattern only from day 4 on, when sustained spontaneous swimming appeared. 5-HT and its agonist quipazine increased motor output by reducing intervals of inactivity, observed behaviorally (by high-speed video) and in recordings from spinal neurons during fictive swimming (by whole-cell current clamp). 5-HT and quipazine had little effect on the properties of the activity periods, such as the duration of swim episodes and swim frequency. Further, neuronal input resistance, rheobasic current, and resting potential were not affected significantly. The 5-HT antagonists methysergide and ketanserin decreased motor output by prolonging the periods of inactivity with little effect on the active swim episode or neuronal properties. Our results suggest that 5-HT neuromodulation is integrated early in development of the locomotor network to increase its output by reducing periods of inactivity with little effect on the activity periods, which in contrast are the main targets of 5-HT neuromodulation in neonatal and adult preparations.     

The Wheels mutation in the mouse causes vascular, hindbrain, and inner ear defects

In a screen for mouse mutations with dominant behavioral anomalies, we identified Wheels, a mutation associated with circling and hyperactivity in heterozygotes and embryonic lethality in homozygotes. Mutant Wheels embryos die at E10.5-E11.5 and exhibit a host of morphological anomalies which include growth retardation and anomalies in vascular and hindbrain development. The latter includes perturbation of rhombomeric boundaries as detected by Krox20 and Hoxb1. PECAM-1 staining of embryos revealed normal formation of the primary vascular plexus. However, subsequent stages of branching and remodeling do not proceed normally in the yolk sac and in the embryo proper. To obtain insights into the circling behavior, we examined development of the inner ear by paint-filling of membranous labyrinths of Whl/+ embryos. This analysis revealed smaller posterior and lateral semicircular canal primordia and a delay in the canal fusion process at E12.5. By E13.5, the lateral canal was truncated and the posterior canal was small or absent altogether. Marker analysis revealed an early molecular phenotype in heterozygous embryos characterized by perturbed expression of Bmp4 and Msx1 in prospective lateral and posterior cristae at E11.5. We have constructed a genetic and radiation hybrid map of the centromeric portion of mouse Chromosome 4 across the Wheels region and refined the position of the Wheels locus to the approximately 1.1-cM region between D4Mit104 and D4Mit181. We have placed the locus encoding Epha7, in the Wheels candidate region; however, further analysis showed no mutations in the Epha7-coding region and no detectable changes in mRNA expression pattern. In summary, our findings indicate that Wheels, a gene which is essential for the survival of the embryo, may link diverse processes involved in vascular, hindbrain, and inner ear development.     

Distinct expression of midkine and pleiotrophin in the spinal cord and placental tissues during early mouse development

Midkine and pleiotrophin comprise a family of heparin-binding growth factors, and are expressed in overlapping tissues during the mid- to late-gestation periods of mouse development. Their distinct expression during early mouse development, as revealed by in situ hybridization, was reported. Midkine was expressed in the embryonic ectoderm from as early as embryonic day (E5.5). In the neural tube midkine was expressed specifically in the neuroepithelium, that is, in the whole area of the neural tube at E9.5, and in the ventricular zone from E10.5-13.5. At E15.5, when the neuroepithelium disappeared, midkine concomitantly became undetectable. In contrast, pleiotrophin expression started exclusively in the neural plate at E8.5, and in the lateral plate of the neural tube at E9.5. It then became restricted to a dorsal ventricular zone from E11.5-13.5, and finally to the central gray neurons at E15.5. Moreover, pleiotrophin was expressed in the ventral horns. Among placental tissues, midkine was detected in the chorion, the fetal component of the placenta, whereas pleiotrophin was found in the decidua basalis, the maternal component of the placenta. The distinct expression of midkine and pleiotrophin suggests their differential role in early development.     

Region- and cell type-selective expression of the evolutionarily conserved Nolz-1/zfp503 gene in the developing mouse hindbrain

Nolz-1/Zfp503, a zinc finger-containing gene, is a mammalian member of the SP1-related nocA/elb/tlp-1 gene family. Previous studies have shown that Nolz-1 homologs are important for patterning the rhombomeres in zebrafish hindbrain. We therefore studied the expression pattern of Nolz-1 in the developing mouse hindbrain. Nolz-1 mRNA expression was detected in the prospective rhombomere 3, 5 and caudal regions as early as E8.75. After E11.5, Nolz-1-positive cells were organized as distinct cell clusters, and they were largely non-overlapped with either Pax2-positive or Phox2b-positive domains. Most interestingly, we found that Nolz-1 was specifically expressed by Phox2b-negative/Isl1/2-positive somatic motor neurons, but not by Phox2b-positive/Isl1/2-positive branchial and visceral motor neurons, suggesting that Nolz-1 may regulate development of somatic motor neurons in the hindbrain. In addition to be expressed in differentiating post-mitotic neurons, Nolz-1 was also expressed by progenitor cells in the ventricular zone located in the dorsal part of aqueduct and the alar plates of hindbrain, which suggests a regulatory role of Nolz-1 in the germinal zone. Taken together, based on its domain- and cell type-selective pattern, Nolz-1 may involve in regulation of various developmental processes, including regional patterning and cell-type specification and differentiation in the developing mouse hindbrain.     

Distribution of serotonin immunoreactive neurons in the brainstem of the hamster, guinea pig, rabbit, and rat

Immunohistochemical techniques were employed to study the distribution of serotonin (5-HT) immunoreactive neurons in the brainstem of the hamster, guinea pig, rabbit and rat. 5-HT neurons were principally found to be concentrated in the midline raphe nuclei, particularly, the raphe pallidus, raphe obscurus, raphe magnus, raphe median, raphe pontis and raphe dorsalis nuclei. Characteristically, these cell bodies are displayed in bands or wing-like patterns which extend laterally from the raphe into reticular formations. The formations often appear to blend with the catecholamine system. They are particularly evident in the brainstems of the rabbit and hamster which contain wider and more lateral extensions of the serotonergic (5-HT) neurons than those observed in the brainstems of the rat and guinea pig. The widespread distribution of 5-HT immunoreacted cell bodies in the brainstem shows that there are significant prospects of 5-HT in neuronal activities.     

GABAergic Neurons in the Embryonic Olfactory Pit/Vomeronasal Organ: Maintenance of Functional GABAergic Synapses in Olfactory Explants

In previous work, we showed a robust γ-aminobutyric acid (GABAergic) synaptic input onto embryonic luteinizing hormone-releasing hormone (LHRH) neurons maintained in olfactory explants. In this study, we identify GABAergic neurons in olfactory pit (OP) of embryonic micein vivoand study, using patch-pipet whole-cell current and voltage clamp techniques, synaptic interactions of these neurons in explant cultures.In vivo,glutamate decarboxylase (GAD, the enzyme which synthesizes GABA) mRNA was first detected in nasal regions on Embryonic Day (E) 11.5. From E12.5 to E13.5, robust GAD expression was localized to cells primarily in the ventral aspect of the OP. GAD mRNA was not detected over dorsally located cells in olfactory sensory or respiratory epithelium. In addition, GAD mRNA was not observed in cells along olfactory axons. GAD mRNA was dramatically reduced in the OP/vomeronasal organ by E16.5. Using antibodies against both GABA and GAD, immunopositive axonal-like tracts were detected in the nasal septum on E12.5. GABAergic staining decreased by E13.5. To examine synaptic interactions of these GABAergic cells, embryonic olfactory explants were generated and maintained in serum-free media. As explants spread, neuron-like cells migrated into the periphery, sometimes forming ganglion-like clusters. Cells were recorded, marked intracellularly with Lucifer Yellow and post-fixation, immunocytochemically examined. Forty-six cells, typically multipolar, were GABAergic, had resting potentials around −50 mV, and exhibited spontaneous action potentials which were generated by spontaneous depolarizing GABAergic (GABA_A) synaptic activity. OP neurons depolarized in response to GABA by increasing Cl⁻conductance. The biophysical properties of OP-derived GABAergic neurons were distinct from those reported for olfactory receptor neurons but similar to embryonic LHRH neurons. However, unlike LHRH neurons, GABAergic neurons did not migrate large distances in olfactory explants or appear to leave the olfactory pitin vivo.     

Effects of 5-hydroxytryptamine on the neuronal firing rate of bulbar reticular neurons

The effects of 5-hydroxytryptamine (5-HT) on neuronal firing rate were studied in the reticular gigantocellular nucleus (GRN) and, for a comparison, in the interstitial (IRN), the parvicellular (PRN) and the lateral (LRN) nuclei, sharing some of GRN functional characteristics. Unitary extracellular recordings performed in anesthetized rats demonstrated that microiontophoretic application of 5-HT modulated the background firing rate in 92% of GRN, in 100% of IRN and LRN, and in 77% of PRN tested neurons. In GRN, 5-HT application induced excitatory responses in 49% of the neurons tested and inhibitions in 43% of them. Both types of effects were dose dependent and appeared scattered throughout the nucleus. Enhancements and decreases of firing rate in response to 5-HT application were also recorded in IRN (58% and 42% respectively), LRN (43% and 57%) and PRN (36% and 41%). The 5-HT(1A) receptor agonist 8-hydroxy-2(di-n-propylamino)tetralin (8-OH-DPAT) mimicked 5-HT evoked inhibitions in all the nuclei tested and induced weak inhibitory responses also in neurons excited by 5-HT. The 5-HT2A receptor agonist alphamethyl-5-hydroxytryptamine (alpha-me-5-HT) mimicked excitatory as well as inhibitory responses to 5-HT, the former prevailing in GRN and the latter in the remaining reticular nuclei. Both excitatory and inhibitory responses to 5-HT were partially or totally blocked by the application of 5-HT2 receptor antagonist ketanserin. It is concluded that an extended, strong and differentiated control is exerted by 5-HT on the electrical activity of bulbar reticular neurons. Both 5-HT(1A) and 5-HT(2A) receptors mediate these effects, but the involvement of other receptors appears probable.     

The slit receptor Rig-1/Robo3 controls midline crossing by hindbrain precerebellar neurons and axons

During development, precerebellar neurons migrate dorsoventrally from the rhombic lip to the floor plate. Some of these neurons cross the midline while others stop. We have identified a role for the slit receptor Rig-1/Robo3 in directing this process. During their tangential migration, neurons of all major hindbrain precerebellar nuclei express high levels of Rig-1 mRNA. Rig-1 expression is rapidly downregulated as their leading process crosses the floor plate. Interestingly, most precerebellar nuclei do not develop normally in Rig-1-deficient mice, as they fail to cross the midline. In addition, inferior olivary neurons, which normally send axons into the contralateral cerebellum, project ipsilaterally in Rig-1 mutant mice. Similarly, neurons of the lateral reticular nucleus and basilar pons are unable to migrate across the floor plate and instead remain ipsilateral. These results demonstrate that Rig-1 controls the ability of both precerebellar neuron cell bodies and their axons to cross the midline.