Glutamatergic reticulospinal neurons in the mouse: developmental origins, axon projections, and functional connectivity.

Subcortical descending glutamatergic neurons, such as reticulospinal (RS) neurons, play decisive roles in the initiation and control of many motor behaviors in mammals. However, little is known about the mechanisms used by RS neurons to control spinal motor networks because most of the neuronal elements involved have not been identified and characterized. In this review, we compare, in the embryonic mouse, the timing of developmental events that lead to the formation of synaptic connections between RS and spinal cord neurons. We then summarize our recent research in the postnatal mouse on the organization of synaptic connections between RS neurons and lumbar axial motoneurons (MNs), hindlimb MNs, and commissural interneurons. Finally, we give a brief account of some of the most recent studies on the intrinsic capabilities for plasticity of the mammalian RS system. The present review should give an updated insight into how functional specificity in RS motor networks emerges.

Mesencephalic neuronal populations: new insights on the ventral differentiation programs.

The midbrain is a complex structure where different functions are located. This formation is mainly involved in the visual and auditory information process (tectum) and visual movements and motor coordination (tegmentum). Here we display a complete description of midbrain anatomy based on the prosomeric model and of the developmental events that take place to generate this structure. We also summarize the new data about the differentiation and specification of the basal populations of the midbrain. The neural tube suffers the influence of several secondary organizers. These signaling centers confer exact positional information to the neuroblasts. In the midbrain these centers are the Isthmic organizer for the antero-posterior axis and the floor and roof plates for the dorso-ventral axis. This segment of the brain contains, in the dorsal part, structures such as the collicula (superior and inferior), tectal grey and the preisthmic segment, and in the basal plate, neuronal populations such as the oculomotor complex, the dopaminergic substantia nigra and the ventral tegmental area, the reticular formation and the periacueductal grey. Knowledge of the genetic cascades involved in the differentiation programs of the diverse populations will be extremely important to understand not only how the midbrain develops, but how degenerative pathologies, such as Parkinson's disease, occurs. These cascades are triggered by signaling molecules such as Shh, Fgf8 or Wnt1 and are integrated by receptor complexes and transcription factors. These are directly responsible for the induction or repression of the differentiation programs that will produce a specific neuronal phenotype.

Inhibitory descending rhombencephalic projections in larval sea lamprey.

Lampreys are jawless vertebrates, the most basal group of extant vertebrates. This phylogenetic position makes them invaluable models in comparative studies of the vertebrate central nervous system. Lampreys have been used as vertebrate models to study the neuronal circuits underlying locomotion control and axonal regeneration after spinal cord injury. Inhibitory inputs are key elements in the networks controlling locomotor behaviour, but very little is known about the descending inhibitory projections in lampreys. The aim of this study was to investigate the presence of brain-spinal descending inhibitory pathways in larval stages of the sea lamprey Petromyzon marinus by means of tract-tracing with neurobiotin, combined with immunofluorescence triple-labeling methods. Neurobiotin was applied in the rostral spinal cord at the level of the third gill, and inhibitory populations were identified by the use of cocktails of antibodies raised against glycine and GABA. Glycine-immunoreactive (-ir) neurons that project to the spinal cord were observed in three rhombencephalic reticular nuclei: anterior, middle and posterior. Spinal-projecting GABA-ir neurons were observed in the anterior and posterior reticular nuclei. Double glycine-ir/GABA-ir spinal cord-projecting neurons were only observed in the posterior reticular nucleus, and most glycine-ir neurons did not display GABA immunoreactivity. The present results reveal the existence of inhibitory descending projections from brainstem reticular neurons to the spinal cord, which were analyzed in comparative and functional contexts. Further studies should investigate which spinal cord circuits are affected by these descending inhibitory projections.

Genetic identification of an embryonic parafacial oscillator coupling to the preBötzinger complex.

The hindbrain transcription factors Phox2b and Egr2 (also known as Krox20) are linked to the development of the autonomic nervous system and rhombomere-related regulation of breathing, respectively. Mutations in these proteins can lead to abnormal breathing behavior as a result of an alteration in an unidentified neuronal system. We characterized a bilateral embryonic parafacial (e-pF) population of rhythmically bursting neurons at embryonic day (E) 14.5 in mice. These cells expressed Phox2b, were derived from Egr2-expressing precursors and their development was dependent on the integrity of the Egr2 gene. Silencing or eliminating the e-pF oscillator, but not the putative inspiratory oscillator (preBötzinger complex, preBötC), led to an abnormally slow rhythm, demonstrating that the e-pF controls the respiratory rhythm. The e-pF oscillator, the only one active at E14.5, entrained and then coupled with the preBötC, which emerged independently at E15.5. These data establish the dual organization of the respiratory rhythm generator at the time of its inception, when it begins to drive fetal breathing.

The precerebellar linear nucleus in the mouse defined by connections, immunohistochemistry, and gene expression.

The linear nucleus (Li) is a prominent cell group in the caudal hindbrain, which was first described in a study of cerebellar afferents in the rat by [Watson, C.R.R., Switzer, R.C. III, 1978. Trigeminal projections to cerebellar tactile areas in the rat origin mainly from N. interpolaris and N. principalis. Neurosci. Lett. 10, 77-82.]. It was named for its elongated appearance in transverse sections. Since this original description in the rat, reference to the nucleus seems to have been largely absent from experimental studies of mammalian precerebellar nuclei. We therefore set out to define the cytoarchitecture, cerebellar connections, and molecular characteristics of Li in the mouse. In coronal Nissl sections at the level of the rostral inferior olive, it consists of two parallel bands of cells joined at their dorsal apex by a further band of cells, making the shape of the Greek capital letter pi. Our three-dimensional reconstruction demonstrated that the nucleus is continuous with the lateral reticular nucleus (LRt) and that the ambiguus nucleus sits inside the arch of Li. Cerebellar horseradish peroxidase injections confirmed that the cells of Li project to cerebellum. We have shown that Li cells express Atoh1 and Wnt1 lineage markers that are known to label the rhombic lip derived precerebellar nuclei. We have examined the relationship of Li cells to a number of molecular markers, and have found that many of the cells express a nonphosphorylated epitope in neurofilament H (SMI 32), a feature they share with the LRt. The mouse Li therefore appears to be a rostrodorsal extension of the LRt.

Origin of the earliest correlated neuronal activity in the chick embryo revealed by optical imaging with voltage-sensitive dyes.

Spontaneous correlated neuronal activity during early development spreads like a wave by recruiting a large number of neurons, and is considered to play a fundamental role in neural development. One important and as yet unresolved question is where the activity originates, especially at the earliest stage of wave expression. In other words, which part of the brain differentiates first as a source of the correlated activity, and how does it change as development proceeds? We assessed this issue by examining the spatiotemporal patterns of the depolarization wave, the optically identified primordial correlated activity, using the optical imaging technique with voltage-sensitive dyes. We surveyed the region responsible for the induction of the evoked and spontaneous depolarization waves in chick embryos, and traced its developmental changes. The results showed that the wave initially originated in a restricted area near the obex and was generated by multiple regions at later stages. We suggest that the upper cervical cord/lower medulla near the obex is the kernel that differentiates first as the source of the correlated activity, and that regional and temporal differences in neuronal excitability might underlie the developmental profile of wave generation in early chick embryos.

Enhancer detection in zebrafish permits the identification of neuronal subtypes that express Hox4 paralogs.

Activity of zebrafish hoxb4a in the developing brain was analyzed in comparison to hoxa4a and hoxd4a using unique enhancer detection transgenes. Cytoplasmic YFP revealed shape and axonal projections of neurons in animals with insertions near the Hox4 genes and provided a means for the identification of neuronal subtypes. Despite an early activity of the genes in neuroepithelial cells and later in immature postmitotic neurons, we found reporter expression in distinct neuronal subtypes in the r7-r8-derived hindbrain. Most strikingly, hoxb4a neuronal subtypes projected through the vagus and into the pectoral fin while others formed symmetrically located fiber tracts innervating the cerebellum and the tectum, features that are partially shared by the other two paralogs. Collectively, our expression analysis indicates that hoxb4a in combination with its paralogs may play a significant role in the development of precerebellar, vagal, and pectoral fin neuronal subtypes.

Molecular mechanisms controlling midline crossing by precerebellar neurons.

Precerebellar neurons of the inferior olive (IO) and lateral reticular nucleus (LRN) migrate tangentially from the rhombic lip toward the floor plate following parallel pathways. This process is thought to involve netrin-1 attraction. However, whereas the cell bodies of LRN neurons cross the midline, IO neurons are unable to do so. In many systems and species, axon guidance and cell migration at the midline are controlled by Slits and their receptor Robos. We showed previously that precerebellar axons and neurons do not cross the midline in the absence of the Robo3 receptor. To determine whether this signaling by Slits and the two other Robo receptors, Robo1 and Robo2, also regulates precerebellar neuron behavior at the floor plate, we studied the phenotype of Slit1/2 and Robo1/2/3 compound mutants. Our results showed that many IO neurons can cross the midline in absence of Slit1/2 or Robo1/2, supporting a role for midline repellents in guiding precerebellar neurons. We also show that these molecules control the development of the lamellation of the inferior olivary complex. Last, the analysis of Robo1/2/3 triple mutants suggests that Robo3 inhibits Robo1/2 repulsion in precrossing LRN axons but not in IO axons in which it has a dominant and distinct function.

The development of descending projections from the brainstem to the spinal cord in the fetal sheep.

BACKGROUND: Although the fetal sheep is a favoured model for studying the ontogeny of physiological control systems, there are no descriptions of the timing of arrival of the projections of supraspinal origin that regulate somatic and visceral function. In the early development of birds and mammals, spontaneous motor activity is generated within spinal circuits, but as development proceeds, a distinct change occurs in spontaneous motor patterns that is dependent on the presence of intact, descending inputs to the spinal cord. In the fetal sheep, this change occurs at approximately 65 days gestation (G65), so we therefore hypothesised that spinally-projecting axons from the neurons responsible for transforming fetal behaviour must arrive at the spinal cord level shortly before G65. Accordingly we aimed to identify the brainstem neurons that send projections to the spinal cord in the mature sheep fetus at G140 (term = G147) with retrograde tracing, and thus to establish whether any projections from the brainstem were absent from the spinal cord at G55, an age prior to the marked change in fetal motor activity has occurred. RESULTS: At G140, CTB labelled cells were found within and around nuclei in the reticular formation of the medulla and pons, within the vestibular nucleus, raphe complex, red nucleus, and the nucleus of the solitary tract. This pattern of labelling is similar to that previously reported in other species. The distribution of CTB labelled neurons in the G55 fetus was similar to that of the G140 fetus. CONCLUSION: The brainstem nuclei that contain neurons which project axons to the spinal cord in the fetal sheep are the same as in other mammalian species. All projections present in the mature fetus at G140 have already arrived at the spinal cord by approximately one third of the way through gestation. The demonstration that the neurons responsible for transforming fetal behaviour in early ontogeny have already reached the spinal cord by G55, an age well before the change in motor behaviour occurs, suggests that the projections do not become fully functional until well after their arrival at the spinal cord.

Changes within maturing neurons limit axonal regeneration in the developing spinal cord.

Embryonic birds and mammals display a remarkable ability to regenerate axons after spinal injury, but then lose this ability during a discrete developmental transition. To explain this transition, previous research has emphasized the emergence of myelin and other inhibitory factors in the environment of the spinal cord. However, research in other CNS tracts suggests an important role for neuron-intrinsic limitations to axon regeneration. Here we re-examine this issue quantitatively in the hindbrain-spinal projection of the embryonic chick. Using heterochronic cocultures we show that maturation of the spinal cord environment causes a 55% reduction in axon regeneration, while maturation of hindbrain neurons causes a 90% reduction. We further show that young neurons transplanted in vivo into older spinal cord can regenerate axons into myelinated white matter, while older axons regenerate poorly and have reduced growth cone motility on a variety of growth-permissive ligands in vitro, including laminin, L1, and N-cadherin. Finally, we use video analysis of living growth cones to directly document an age-dependent decline in the motility of brainstem axons. These data show that developmental changes in both the spinal cord environment and in brainstem neurons can reduce regeneration, but that the effect of the environment is only partial, while changes in neurons by themselves cause a nearly complete reduction in regeneration. We conclude that maturational events within neurons are a primary cause for the failure of axon regeneration in the spinal cord.

Homeodomain transcription factors in the development of subsets of hindbrain reticulospinal neurons.

Hindbrain reticulospinal neurons are involved in complex neural functions that are mediated by spinal elements, including posture control and modulation of respiration and cardiovascular function. Recent descriptive studies with chick, mouse, and rat embryos have provided anatomical insight into the development of the different reticulospinal nuclei and the establishment of their axonal projection pathways into the spinal cord. In this study, we have addressed the molecular control of this process. Retrograde labeling of reticulospinal neurons in chick and mouse embryos combined with immunostaining for the homeodomain factors Lhx1/Lhx5, Lhx3/Lhx4, and Chx10 have defined transcriptional codes that label subsets of neurons with different axon projection patterns. Gain of function and loss of function experiments using in ovo electroporation implicate these transcription factors in the determination of reticulospinal neuron identity. Furthermore, our studies reveal novel gene interactions between the transcription factors analyzed that may determine the final patterns of reticulospinal axon projection.

Relationship between bioelectric activity of neurons in the gigantocellular nucleus of the medulla oblongata and spontaneous movements of chick embryo.

Electrophysiological study revealed a correlation between changes in bioelectric activity of the reticular gigantocellular nucleus and movements of chick embryos during ontogeny (16-20 days). This relationship increased by the end of embryogenesis. The reticular gigantocellular nucleus is the major source of supraspinal influences on motor activity during ontogeny. Blockade of proprioceptive impulses with myorelaxin inhibited bioelectric activity of the regulatory gigantocellular nucleus, which attests to the activating effect of proprioception.

Morphological study of the perireticular nucleus in human fetal brains.

Abstract The perireticular nucleus consists of scattered neurons that are located in the internal capsule. The presence of perireticular neurons in the rat, ferret, cat and human has been described previously. Evidence suggests that the perireticular neurons in various species decrease in number with increasing gestation, but in humans this finding has not been supported by quantitative data. This study aimed to investigate (1) the morphology of the human fetal perireticular neurons, (2) the average number of perireticular neurons within the anterior and posterior crus of the internal capsule per unit area, and (3) the magnitude and the stage of neuronal loss in the human perireticular nucleus subsequent to maturation. Nissl-stained sections of the internal capsule of human fetal brains of 24, 26.5, 32, 35, 37 and 39 weeks of gestation showed a number of clearly distinguishable large perireticular and small microglia cells. A regular increase of both perireticular and microglial cells was observed up to 32 weeks of gestation, after which a dramatic reduction in the number of both perireticular and microglia cells was observed. The average number of perireticular and the microglia cells per unit area, located within the posterior crus, was more than in the anterior crus of the internal capsule. In the adult, no perireticular neurons were detected within the internal capsule. The results show that perireticular neurons are not restricted to the region lateral to the thalamus and medial to the globus pallidus (posterior crus) but are also present at the region lateral to the caudate nucleus and medial to the globus pallidus (anterior crus).

Expression of neuropeptide FF, prolactin-releasing peptide, and the receptor UHR1/GPR10 genes during embryogenesis in the rat.

Recently, several RF-amide peptides have been identified in mammals. These peptides have a similar C-terminal RF-motif and share some G-protein coupled receptors. Neuropeptide FF (NPFF) and prolactin-releasing peptide (PrRP) are expressed in the same brain areas in the adult rat and act both in prolactin release and cardiovascular regulation. Here, we characterized the embryonal expression from embryonal day 14 to postnatal day 0 of both peptide mRNAs and the mRNA distribution of UHR1/GPR10-like receptor by using in situ hybridization (ISH) and quantitative reverse transcriptase-polymerase chain reaction. NPFF mRNA was found in the spinal cord, caudal solitary tract nucleus, and surprisingly, in the medullary reticular formation. The only peripheral organs displaying NPFF mRNA expression were the lungs and the spleen. PrRP gene expression was seen in the caudal solitary tract nucleus, medullary reticular formation, pontine isthmus and liver, kidney, and testis. The receptor UHR1/GPR10 gene was expressed consistently in the medullary reticular formation and the adrenal gland but also transiently in several locations. All three genes showed weak but even ISH signal in the pituitary. These findings suggest different roles for the peptides during development and indicate that UHR1/GPR10-like receptor could also bind other ligands in addition to PrRP.

Diffusible signals and fasciculated growth in reticulospinal axon pathfinding in the hindbrain.

We have addressed the control of longitudinal axon pathfinding in the developing hindbrain, including the caudal projections of reticular and raphe neurons. To test potential sources of guidance signals, we assessed axon outgrowth from embryonic rat hindbrain explants cultured in collagen gels at a distance from explants of midbrain-hindbrain boundary (isthmus), caudal hindbrain, or cervical spinal cord. Our results showed that the isthmus inhibited caudally directed axon outgrowth by 80% relative to controls, whereas rostrally directed axon outgrowth was unaffected. Moreover, caudal hindbrain or cervical spinal cord explants did not inhibit caudal axons. Immunohistochemistry for reticular and raphe neuronal markers indicated that the caudal, but not the rostral projections of these neuronal subpopulations were inhibited by isthmic explants. Companion studies in chick embryos showed that, when the hindbrain was surgically separated from the isthmus, caudal reticulospinal axon projections failed to form and that descending pioneer axons of the medial longitudinal fasciculus (MLF) play an important role in the caudal reticulospinal projection. Taken together, these results suggest that diffusible chemorepellent or nonpermissive signals from the isthmus and substrate-anchored signals on the pioneer MLF axons are involved in the caudal direction of reticulospinal projections and might influence other longitudinal axon projections in the brainstem.

Compartments in the lamprey embryonic brain as revealed by regulatory gene expression and the distribution of reticulospinal neurons.

The vertebrate neural tube consists of a series of neuromeres along its anteroposterior axis. Between amphioxus that possesses no neuromeres and gnathostomes, the lamprey occupies a critical position in the phylogeny for the origin of the segmented brain. To clarify the rhombomeric configuration of the Japanese lamprey, Lampetra japonica, we injected rhodamine- and fluorescein-labeled dextrans into the larval spinal cord, and retrogradely labeled the reticulospinal neurons. We also isolated prosomere marker genes from the embryonic cDNA library of L. japonica, and performed in situ hybridization on the embryonic brain. Of the genes examined, LjOtxA, LjPax6, LjPax2/5/8, LjDlx1/6, and LjTTF-1 were expressed in clearly demarcated polygonal domains. In the telencephalon, LjDlx1/6, LjPax6, and a putative paralogue of LjEmx were expressed in different domains; the LjEmx paralogue was expressed in the dorsal region, and LjDlx1/6 and LjPax6 in a complimentary fashion of the middle part. These expression patterns implied existence of a tripartite configuration of the lamprey telencephalon similar to that in gnathostomes. All these evidences strongly suggest that the segmental and compartmental architecture of the vertebrate brain was already established before the divergence of agnathans and gnathostomes.

[Synapse architectonics of the cerebral cortex in the evolutionary aspect]. / Sinapsoarkhitektonika kory bol'shogo mozga v évoliutsionnom aspekte.

The formation of synapses in ontogenesis is an important problem of neuromorphology. The paper shows that the bulk of synapses of the developing brain is formed on the basis of previous specialization of membranes. Early in ontogenesis, most formed synapses shows asymmetric contacts. In postnatal ontogenesis, the formation of synapses proceeds due to the simultaneous occurrence of specialization of membranes and synaptic vesicles, by forming a "dot" active zone. Systemogenesis, such as the general law of brain development, plays an important role in understanding the functional significance of the shown mechanisms of maturation of synapses.

Generation of a novel functional neuronal circuit in Hoxa1 mutant mice.

Early organization of the vertebrate brainstem is characterized by cellular segmentation into compartments, the rhombomeres, which follow a metameric pattern of neuronal development. Expression of the homeobox genes of the Hox family precedes rhombomere formation, and analysis of mouse Hox mutations revealed that they play an important role in the establishment of rhombomere-specific neuronal patterns. However, segmentation is a transient feature, and a dramatic reconfiguration of neurons and synapses takes place during fetal and postnatal stages. Thus, it is not clear whether the early rhombomeric pattern of Hox expression has any influence on the establishment of the neuronal circuitry of the mature brainstem. The Hoxa1 gene is the earliest Hox gene expressed in the developing hindbrain. Moreover, it is rapidly downregulated. Previous analysis of mouse Hoxa1(-/-) mutants has focused on early alterations of hindbrain segmentation and patterning. Here, we show that ectopic neuronal groups in the hindbrain of Hoxa1(-/-) mice establish a supernumerary neuronal circuit that escapes apoptosis and becomes functional postnatally. This system develops from mutant rhombomere 3 (r3)-r4 levels, includes an ectopic group of progenitors with r2 identity, and integrates the rhythm-generating network controlling respiration at birth. This is the first demonstration that changes in Hox expression patterns allow the selection of novel neuronal circuits regulating vital adaptive behaviors. The implications for the evolution of brainstem neural networks are discussed.

Anatomic relationships of the human arcuate nucleus of the medulla: a DiI-labeling study.

The arcuate nucleus (ARC) at the ventral surface of the human medulla has been historically considered a precerebellar nucleus. More recently, it has been implicated in central chemoreception, cardiopulmonary coupling and blood pressure responses. A deficiency of the ARC has been reported in a subset of putative human developmental disorders of ventilatory function. To investigate anatomic relationships of the ARC with brainstem regions involved in cardiorespiratory control, we applied crystals of DiI, a lipophilic dye which labels cells and cell processes by lateral diffusion along cell membranes, to 23 paraformaldehyde-fixed human fetal brainstems at 19 to 22 weeks postconceptional age. After 7 to 15.5 months diffusion, serial frozen sections were examined by florescence microscopy. DiI diffusion from the ARC labeled fibers and cell bodies in the medullary raphé, and the external arcuate fibers. Diffusion from the medullary raphé [corrected] labeled the reticular formation, medullary raphé, and the ARC. Diffusion from the pyramid and the basis pontis (negative control) labeled the corticospinal tract, with no labeling of the medullary raphé or ARC. The results suggest the existence of cellular connections between the ARC and the caudal raphé, a region implicated in cardiorespiratory control.

The use of tracers in explants of the developing reticular formation and spinal cord of Xenopus laevis.

The development of reticulospinal projections to the lumbar spinal cord is studied by using a collagen co-culture system. Outgrowing reticulospinal fibers seem to grow out in a straightforward direction, without a specific preference for the lumbar or tail spinal cord. Carbocyanine tracers such as DiI, DiO and DiA are used to label the outgrowing fibers or their parent cell bodies. In double labeling studies contacts of outgrowing reticulospinal fibers with lumbar motoneurons are analyzed. The advances of a confocal laser scanning microscope for such studies are illustrated.