Patterns of cell division and interkinetic nuclear migration in the chick embryo hindbrain

Early in its development, the chick embryo hindbrain manifests an axial series of bulges, termed rhombomeres. Rhombomeres are units of cell lineage restriction, and both they and their intervening boundaries form a series that reiterates various features of neuronal differentiation, cytoarchitecture, and molecular character. The segmented nature of hindbrain morphology and cellular development may be related to early patterns of cell division. These were explored by labeling with BrdU to reveal S-phase nuclei, and staining with basic fuchsin to visualise mitotic cells. Whereas within rhombomeres, S-phase nuclei were located predominantly toward the pial surface of the neuroepithelium, at rhombomere boundaries S-phase nuclei were significantly closer to the ventricular surface. The density of mitotic figures was greater toward the centres of rhombomeres than in boundary regions. Mitotic cells did not show any consistent bias in the orientation of division, either in the centres of rhombomeres, or near boundaries. Our results are consistent with the idea that rhombomeres are centres of cell proliferation, while boundaries contain populations of relatively static cells with reduced rates of cell division.     

Bilaterally propagating waves of spontaneous activity arising from discrete pacemakers in the neonatal mouse cerebral cortex

Spontaneous electrical activity that moves in synchronized waves across large populations of neurons plays widespread and important roles in nervous system development. The propagation patterns of such waves can encode the spatial location of neurons to their downstream targets and strengthen synaptic connections in coherent spatial patterns. Such waves can arise as an emergent property of mutually excitatory neural networks, or can be driven by a discrete pacemaker. In the mouse cerebral cortex, spontaneous synchronized activity occurs for approximately 72 h of development centered on the day of birth. It is not known whether this activity is driven by a discrete pacemaker or occurs as an emergent network property. Here we show that this activity propagates as a wave that is initiated at either of two homologous pacemakers in the temporal region, and then propagates rapidly across both sides of the brain. When these regions of origin are surgically isolated, waves do not occur. Therefore, this cortical spontaneous activity is a bilateral wave that originates from a discrete subset of pacemaker neurons. © 2009 Wiley Periodicals, Inc. Develop Neurobiol, 2009     

Bimodal septal and cortical triggering and complex propagation patterns of spontaneous waves of activity in the developing mouse cerebral cortex

Spontaneous waves of activity that propagate across large structures during specific developmental stages play central roles in CNS development. To understand the genesis and functions of these waves, it is critical to understand the spatial and temporal patterns of their propagation. We recently reported that spontaneous waves in the neonatal cerebral cortex originate from a ventrolateral pacemaker region. We have now analyzed a large number of spontaneous waves using calcium imaging over the entire area of coronal slices from E18‐P1 mouse brains. In all waves, the first cortical region active is this ventrolateral pacemaker. In half of the waves, however, the cortical pacemaker activity is itself triggered by preceding activity in the septal nuclei. Most waves are restricted to the septum and/or ventral cortex, with only some invading the dorsal cortex or the contralateral hemisphere. Waves fail to propagate at very stereotyped locations at the boundary between ventral and dorsal cortex and at the dorsal midline. Waves that cross these boundaries pause at these same locations. Waves at these stages are blocked by both picrotoxin and CNQX, indicating that both GABA_A and AMPA receptors are involved in spontaneous activity. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 679–692, 2010     

Direct effects of serotonin and ketanserin on the functional morphology of embryonic chick skin in vitro

Summary Different organotypical culture methods are used to test the direct effects of serotonin and ketanserin, a S₂, α₁, and H₁ receptor antagonist in vascular tissue, on fibroblasts and epidermal cells of embryonic chick skin in vitro. From light microscopic and electron microscopic analyses, we learn that serotonin enhances keratinization and differentiation, whereas ketanserin reduces differentiation in comparison to the control cultures. Incorporation data of fragments cultured with [³H]thymidine show that ketanserin, within a dose range from 0.05 to 5 μg/ml, stimulates proliferation. Serotonin at a concentration of 10 μg/ml slightly slows down proliferation, whereas lower doses of 0.1 and 1 μg/ml result in tritium activities that do not differ from control cultures. This investigation was financially supported by the National Fund of Scientific Research, Belgium, 3.0022.87.     

Tonic nicotinic transmission enhances spinal GABAergic presynaptic release and the frequency of spontaneous network activity

Synaptically driven spontaneous network activity (SNA) is observed in virtually all developing networks. Recurrently connected spinal circuits express SNA, which drives fetal movements during a period of development when GABA is depolarizing and excitatory. Blockade of nicotinic acetylcholine receptor (nAChR) activation impairs the expression of SNA and the development of the motor system. It is mechanistically unclear how nicotinic transmission influences SNA, and in this study we tested several mechanisms that could underlie the regulation of SNA by nAChRs. We find evidence that is consistent with our previous work suggesting that cholinergically driven Renshaw cells can initiate episodes of SNA. While Renshaw cells receive strong nicotinic synaptic input, we see very little evidence suggesting other spinal interneurons or motoneurons receive nicotinic input. Rather, we found that nAChR activation tonically enhanced evoked and spontaneous presynaptic release of GABA in the embryonic spinal cord. Enhanced spontaneous and/or evoked release could contribute to increased SNA frequency. Finally, our study suggests that blockade of nAChRs can reduce the frequency of SNA by reducing probability of GABAergic release. This result suggests that the baseline frequency of SNA is maintained through elevated GABA release driven by tonically active nAChRs. Nicotinic receptors regulate GABAergic transmission and SNA, which are critically important for the proper development of the embryonic network. Therefore, our results provide a better mechanistic framework for understanding the motor consequences of fetal nicotine exposure. © 2015 Wiley Periodicals, Inc. Develop Neurobiol 76: 298–312, 2016     

Control of spontaneous activity during development

Electrical activity participates in the development of the nervous system and comes in two general forms. Use-dependent or experience-driven activity occurs relatively late in development, and is important in events of terminal nervous system differentiation, such as stabilization of synaptic connections. Earlier in development, activity is spontaneous, occurring independently of normal sensory input and motor output. Spontaneous activity participates in many of the initial events of axon outgrowth, pruning of synaptic connections, and maturation of neuronal signaling properties. Despite its importance, the genesis of spontaneous activity is poorly understood. What is clear is that spontaneous activity must be regulated by the patterns with which voltage- and ligand-gated ion channels develop in individual neurons. This review explores how that regulation most likely occurs. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 97–109, 1998     

Segmentation and compartition in the early avian hindbrain

For the comparative embryologists of the early 20th century, the segment-like bulges that appear transiently during the early stages of vertebrate hindbrain development were both the object of fascination and the subject of vigorous dispute. Conflicting views were held as to the significance of these 'rhombomeres' to brain development and their more general relevance to head evolution. Whether rhombomeres are inconsequential bumps in the embryonic brain or true segments-iterative or metameric units-has only recently been resolved. A number of studies using more modern techniques (such as immunohistochemistry, in situ hybridisation, axonal tracing, single cell labelling, heterotopic and orthotopic grafting, and the manipulation of gene expression by electroporation) have shown that the hindbrain has a truly metameric cellular organisation. The avian embryo has played a particularly prominent role in such studies by virtue of its large size and accessibility, its amenability to microsurgery, and its well-described anatomy. Furthermore, electrophysiological studies, also on avian embryos, have shown that segmentation of the parent neuroepithelium into rhombomeres plays a crucial part in establishing the functional organization of the hindbrain. Segmentation suggests the early allocation of defined sets of precursor cells and is therefore presumed to allow a specific identity for each successive segment to emerge from a common ground plan. This short review will focus on the contribution the avian embryo has made to our understanding of this fly-like region of the vertebrate brain, in respect of its morphology and neuronal architecture, the cellular and molecular mechanisms involved in establishing and maintaining the segments, and the molecular controls of segmental identity.     

Development of glutamate-induced intracellular Ca2+ rise in the embryonic chick retina

Neurotransmitters affect neuronal development by regulating intracellular Ca²⁺ concentrations. We studied spatiotemporal pattern of the development of glutamate-induced intracellular Ca²⁺ rise in the embryonic chick retina, where developmental changes in mitotic activity, cell death, and synapse formation have been well established. Glutamate was bath-applied to the central part of the retina dissected at embryonic day 3 (E3) to E13, and changes in intracellular Ca²⁺ concentration were measured with Fura-2 fluorescence. The Ca²⁺ rise to glutamate first appeared at E6, reached a maximum at E9–10, and then declined before the appearance of synaptic structures (E12). Ca²⁺ rises to kainate (KA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) appeared earlier and were larger in amplitude than those to N-methyl-D -aspartic acid. The KA/AMPA receptor of the E9 chick retina was permeable for Ca²⁺, suggesting the functional expression of Ca²⁺-permeable KA/AMPA receptors at the stage of retinal cell death. The Ca²⁺rise to glutamate and KA occurred intensely at the inner plexiform layer, the inner part of inner nuclear layer, and the ganglion cell layer, where the cell death occurs. The Ca²⁺ rise to high K²⁺, in contrast, occurred intensely at the nerve fiber layer and the ganglion cell layer, developing continuously from E3 until E11. Our study shows that the Ca²⁺ rise to glutamate develops with the decline of the mitotic activity of the retinal cells and is transiently enhanced during the period of cell death in the embryonic chick retina. © 1998 John Wiley & Sons, Inc. J Neurobiol 34: 113–125, 1998     

Subepithelial type II collagen deposition during embryonic chick limb development

Summary Type II collagen is a major component of hyaline cartilage but recent studies have demonstrated the presence of this protein in a variety of interfaces that separate epithelia from mesenchyme, particularly in early stages of embryonic chick development. In the present study an immunohistochemical analysis of the distribution of type II collagen was performed on closely staged wing buds of early chick embryo. This report describes how using two different monoclonal antibodies against type II collagen and the peroxidase or fluorescence staining technique reveals that deposition of type II collagen at the ectoderm-mesenchyme interface occurs in the proximal part of the limb coincidentally with the appearance of this protein in the proximal core region, where chondrogenesis begins (stage 25). Then the staining in the subepithelial region spreads distallly with time, following the progression of the formation of cartilage rudiments. At about 7 days of development type II collagen is present under the apical ectoderm ridge and surrounds completely the wing bud underneath the epithelium. At the same time, another antibody directed against the cartilage-specific proteoglycan core protein only stains the chondrogenic central core of the limb and not the subepithelium. Although type II collagen and cartilage-specific proteoglycan are closely associated in the cartilage, the observations presented here suggest that the deposition of these proteins can be regulated independently during limb formation. The role of type II collagen at the epithelium-mesenchyme interface during limb formation is still to be determined.     

Roles of glutamate and GABA receptors in setting the developmental timing of spontaneous synchronized activity in the developing mouse cortex

Spontaneous, synchronized electrical activity (SSA) plays important roles in nervous system development, but it is not clear what causes it to start and stop at the appropriate times. In previous work, we showed that when SSA in neonatal mouse cortex is blocked by TTX in cultured slices during its normal time of occurrence (E17-P3), it fails to stop at P3 as it does in control cultured slices, but instead persists through at least P10. This indicates that SSA is self-extinguishing. Here we use whole-cell recordings and [Ca2+]i imaging to compare control and TTX-treated slices to isolate the factors that normally extinguish SSA on schedule. In TTX-treated slices, SSA bursts average 4 s in duration, and have two components. The first, lasting about 1 s, is mediated by AMPA receptors; the second, which extends the burst to 4 s and is responsible for most of the action potential generation during the burst, is mediated by NMDA receptors. In later stage (P5-P9) control slices, after SSA has declined to about 4% of its peak frequency, bursts lack this long NMDA component. Blocking this NMDA component in P5-P9 TTX-treated slices reduces SSA frequency, but not to the low values found in control slices, implying that additional factors help extinguish SSA. GABA(A) inhibitors restore SSA in control slices, indicating that the emergence of GABA(A)-mediated inhibition is another major factor that helps terminate SSA.     

Interaction between hindbrain and spinal networks during the development of locomotion in zebrafish

Little is known about the role of the hindbrain during development of spinal network activity. We set out to identify the activity patterns of reticulospinal (RS) neurons of the hindbrain in fictively swimming (paralyzed) zebrafish larvae. Simultaneous recordings of RS neurons and spinal motoneurons revealed that these were coactive during spontaneous fictive swim episodes. We characterized four types of RS activity patterns during fictive swimming: (i) a spontaneous pattern of discharges resembling evoked high-frequency spiking during startle responses to touch stimuli, (ii) a rhythmic pattern of excitatory postsynaptic potentials (EPSPs) whose frequency was similar to the motoneuron EPSP frequency during swim episodes, (iii) an arrhythmic pattern consisting of tonic firing throughout swim episodes, and (iv) RS cell activity uncorrelated with motoneuron activity. Despite lesions to the rostral spinal cord that prevented ascending spinal axons from entering the hindbrain (normally starting at approximately 20 h), RS neurons continued to display the aforementioned activity patterns at day 3. However, removal of the caudal portion of the hindbrain prior to the descent of RS axons left the spinal cord network unable to generate the rhythmic oscillations normally elicited by application of N-methyl-d-aspartate (NMDA), but in approximately 40% of cases chronic incubation in NMDA maintained rhythmic activity. We conclude that there is an autonomous embryonic hindbrain network that is necessary for proper development of the spinal central pattern generator, and that the hindbrain network can partially develop independently of ascending input.     

Regulation of SC1/DM-GRASP during the migration of motor neurons in the chick embryo brain stem

The hindbrain of the chick embryo contains three classes of motor neurons: somatic, visceral, and branchial motor. During development, somata of neurons in the last two classes undergo a laterally directed migration within the neuroepithelium; somata translocate towards the nerve exit points, through which motor axons are beginning to extend into the periphery. All classes of motor neuron are immunopositive for the SC1/DM-GRASP cell surface glycoprotein. We have examined the relationship between patterns of motor neuron migration, axon outgrowth, and expression of the SC1/DM-GRASP mRNA and protein, using anterograde or retrograde axonal tracing, immunohistochemistry, and in situ hybridization. We find that as motor neurons migrate laterally, SC1/DM-GRASP is down-regulated, both on neuronal somata and axonal surfaces. Within individual motor nuclei, these lateral, more mature neurons are found to possess longer axons than the young, medial cells of the population. Labelling of sensory or motor axons growing into the second branchial arch also shows that motor axons reach the muscle plate first, and that SC1/DM-GRASP is expressed on the muscle at the time growth cones arrive. 1994 John Wiley & Sons, Inc.     

Early events in the induction of glutamine synthetase activity by hydrocortisone in embryonic chick neural retina

Summary Primary cultures of 10-day embryonic chick neural retinas were used to investigate early aspects of the mechanism of hydrocortisone action on glutamine synthetase activity. As little as 2 hr of hydrocortisone exposure served to initiate significant increases in the glutamine synthetase activity levels assayed after 24 hr culture. Time course studies indicated that the increase in glutamine synthetase activity observed after 24 hr in culture resulted from a two-phase rise in activity and that cycloheximide was effective in suppressing the second-phase rise. Additional inhibition studies demonstrated that the second-phase increase in enzyme activity required continuous protein synthesis during the initial 6 hr. The evidence suggests a mechanism of hydrocortisone action involving the production of a protein which is important for the induction of glutamine synthetase activity by hydrocortisone. This work was supported by a National Science Foundation (U.S.A.) Training Grant.     

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     

Calcium channels and GABA receptors in the early embryonic chick retina

The properties of calcium channels were studied at the period of neurogenesis in the early embryonic chick retina. The whole neural retina was isolated from embryonic day 3 (E3) chick and loaded with a Ca²⁺-sensitive fluorescent dye (Fura-2). The retinal cells were depolarized by puff application of high-K⁺ solutions. Increases in intracellular Ca²⁺ concentrations were evoked by the depolarization through calcium channels. The type of calcium channel was identified as l-type by the sensitivity to dihydropyridines. The Ca²⁺ response was completely blocked by 10 μM nifedipine, whereas it was remarkably enhanced by 5 μM Bay K 8644. Then we sought a factor to activate the calcium channel and found that GABA could activate it by membrane depolarization at the E3 chick retina. Puff application of 100 μM GABA raised intracellular Ca²⁺ concentrations, and this Ca²⁺ response to GABA was also sensitive to the two dihydropyridines. Intracellular potential recordings verified clear depolarization by bath-applied 100 μM GABA. The Ca²⁺ response to GABA was mediated by GABA_A receptors, since the GABA response was blocked by 10 μgM bicuculline or 50 μM picrotoxin, and mimicked by muscimol but not by baclofen. Neither glutamate, kainate, nor glycine evoked any Ca²⁺ response. We conclude that l-type calcium channels and GABA_A receptors are already are already expressed before differentiation of retinal cells and synapse formation in the chick retina. A possibility is proposed that GABA might act as a trophic factor by activating l-type calcium channels via GABA_A receptors during the early period of retinal neurogenesis. © 1993 John Wiley & Sons, Inc.     

Primary role of the serotonergic midline system in synchronized spontaneous activity during development of the embryonic mouse hindbrain

In the developing embryonic mouse hindbrain, we have shown that previously widespread synchronized spontaneous activity at E11.5 retracts to the initiating zone of the rostral hindbrain by E13.5, and ceases completely by E14.5. We now confirm that at E11.5 and E13.5, the primary driver of spontaneous activity is serotonergic input, while other transmitters (GABA, glutamate, NE, and ATP) have only modulatory roles. Using immunocytochemistry, we also show that at E13.5, 5-HT-positive neurons in the midline extend over a larger rostro-caudal distance than at E11.5, and that in the presumptive initiating zone, cell bodies occupy a band that extends 200 microm laterally on each side of the midline, with extensive axonal processes. The 5-HT2A receptor retains expression in lateral tissue over this developmental time. We find that in addition to being sensitive to 5-HT receptor antagonists, spontaneous activity is also abolished by blockers of gap junctions, and is increased in frequency and lateral spread by application of ammonium, presumably via increased intracellular pH augmenting gap junction conductance. Thus, 5-HT neurons of the midline remain the primary drivers of spontaneous activity at several stages of development in the hindbrain, relying in part on gap junctional communication during initiation of activity.     

Circadian control of spontaneous flight activity in the blowfly, Lucilia cuprina

ABSTRACT. Under laboratory light: dark cycles, the flight activity of adult Lucilia cuprina (Wied.) was low during darkness and uniformity high during light. This pattern persisted as a rhythm both in constant darkness and in constant light of intensity up to 1lx, with a period of approximately 22 h in each. Light pulses of 15 min at l00lx applied to the free-running rhythm in constant darkness generated phase shifts of up to 60°, 12-h light pulses of the same intensity generated maximal (180°) phase shifts. The phase response curves had shapes similar to those of a number of other insect rhythms. When exposed to light periods (70 lx) of greater than 12 h followed by constant darkness, the rhythm reinitiated at the light-dark transition from a constant phase equivalent to that at the time of the light-dark transition in the LD 12:12 cycle.     

Pre‐/post‐otic rhombomeric interactions control the emergence of a fetal‐like respiratory rhythm in the mouse embryo

How regional patterning of the neural tube in vertebrate embryos may influence the emergence and the function of neural networks remains elusive. We have begun to address this issue in the embryonic mouse hindbrain by studying rhythmogenic properties of different neural tube segments. We have isolated pre‐ and post‐otic hindbrain segments and spinal segments of the mouse neural tube, when they form at embryonic day (E) 9, and grafted them into the same positions in stage‐matched chick hosts. Three days after grafting, in vitro recordings of the activity in the cranial nerves exiting the grafts indicate that a high frequency (HF) rhythm (order: 10 bursts/min) is generated in post‐otic segments while more anterior pre‐otic and more posterior spinal territories generate a low frequency (LF) rhythm (order: 1 burst/min). Comparison with homo‐specific grafting of corresponding chick segments points to conservation in mouse and chick of the link between the patterning of activities and the axial origin of the hindbrain segment. This HF rhythm is reminiscent of the respiratory rhythm known to appear at E15 in mice. We also report on pre‐/post‐otic interactions. The pre‐otic rhombomere 5 prevents the emergence of the HF rhythm at E12. Although the nature of the interaction with r5 remains obscure, we propose that ontogeny of fetal‐like respiratory circuits relies on: (i) a selective developmental program enforcing HF rhythm generation, already set at E9 in post‐otic segments, and (ii) trans‐segmental interactions with pre‐otic territories that may control the time when this rhythm appears. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006