Synaptic development and neuronal myelination are altered with growth restriction in fetal guinea pigs.

This study examines aberrant synaptogenesis and myelination of neuronal connections as possible links to neurological sequelae in growth-restricted fetuses. Pregnant guinea pig sows were subjected to uterine blood flow restriction or sham surgeries at midgestation. The animals underwent necropsy at term with fetuses grouped according to body weight and brain-to-liver weight ratios as follows: appropriate for gestational age (n = 12); asymmetrically fetal growth restricted (aFGR; n = 8); symmetrically fetal growth restricted (sFGR; n = 8), and large for gestational age (n = 8). Fetal brains were perfusion fixed and paraffin embedded to determine immunoreactivity for synaptophysin and synaptopodin as markers of synaptic development and maturation, respectively, and for myelin basic protein as a marker for myelination, which was further assessed using Luxol fast blue staining. The most pertinent findings were that growth-restricted guinea pig fetuses exhibited reduced synaptogenesis and synaptic maturation as well as reduced myelination, which were primarily seen in subareas of the hippocampus and associated efferent tracts. These neurodevelopmental changes were more pronounced in the sFGR compared to the aFGR animals. Accordingly, altered hippocampal development involving synaptogenesis and myelination may represent a mechanism by which cognitive deficits manifest in human growth-restricted offspring in later life.

Placodal sensory ganglia coordinate the formation of the cranial visceral motor pathway.

The parasympathetic reflex circuit is controlled by three basic neurons. In the vertebrate head, the sensory, and pre- and postganglionic neurons that comprise each circuit have stereotypic positions along the anteroposterior (AP) axis, suggesting that the circuit arises from a common developmental plan. Here, we show that precursors of the VIIth circuit are initially aligned along the AP axis, where the placode-derived sensory neurons provide a critical "guidepost" through which preganglionic axons and their neural crest-derived postganglionic targets navigate before reaching their distant target sites. In the absence of the placodal sensory ganglion, preganglionic axons terminate and the neural crest fated for postganglionic neurons undergo apoptosis at the site normally occupied by the placodal sensory ganglion. The stereotypic organization of the parasympathetic cranial sensory-motor circuit thus emerges from the initial alignment of its precursors along the AP axis, with the placodal sensory ganglion coordinating the formation of the motor pathway.

Cooperation between GDNF/Ret and ephrinA/EphA4 signals for motor-axon pathway selection in the limb.

Establishment of limb innervation by motor neurons involves a series of hierarchical axon guidance decisions by which motor-neuron subtypes evaluate peripheral guidance cues and choose their axonal trajectory. Earlier work indicated that the pathway into the dorsal limb by lateral motor column (LMC[l]) axons requires the EphA4 receptor, which mediates repulsion elicited by ephrinAs expressed in ventral limb mesoderm. Here, we implicate glial-cell-line-derived neurotrophic factor (GDNF) and its receptor, Ret, in the same guidance decision. In Gdnf or Ret mutant mice, LMC(l) axons follow an aberrant ventral trajectory away from dorsal territory enriched in GDNF, showing that the GDNF/Ret system functions as an instructive guidance signal for motor axons. This phenotype is enhanced in mutant mice lacking Ret and EphA4. Thus, Ret and EphA4 signals cooperate to enforce the precision of the same binary choice in motor-axon guidance.

The determination of projection neuron identity in the developing cerebral cortex.

Here we review the mechanisms that determine projection neuron identity during cortical development. Pyramidal neurons in the mammalian cerebral cortex can be classified into two major classes: corticocortical projection neurons, which are concentrated in the upper layers of the cortex, and subcortical projection neurons, which are found in the deep layers. Early progenitor cells in the ventricular zone produce deep layer neurons that express transcription factors including Sox5, Fezf2, and Ctip2, which play important roles in the specification of subcortically projecting axons. Upper layer neurons are produced from progenitors in the subventricular zone, and the expression of Satb2 in these differentiating neurons is required for the formation of axonal projections that connect the two cerebral hemispheres. The Fezf2/Ctip2 and Satb2 pathways appear to be mutually repressive, thus ensuring that individual neurons adopt either a subcortical or callosal projection neuron identity at early times during development. The molecular mechanisms by which Satb2 regulates gene expression involves long-term epigenetic changes in chromatin configuration, which may enable cell fate decisions to be maintained during development.

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.

Laminar specificity of extrinsic cortical connections studied in coculture preparations.

The formation of specific neural connections in the cerebral cortex was studied using organotypic coculture preparations composed of subcortical and cortical regions. Morphological and electrophysiological analysis indicated that several cortical efferent and afferent connections, such as the corticothalamic, thalamocortical, corticocortical, and corticotectal connections, were established in the cocultures with essentially the same laminar specificity as that found in the adult cerebral cortex, but without specificity of sensory modality. This suggests the existence of a cell-cell recognition system between cortical or subcortical neurons and their final targets. This interaction produces lamina-specific connections, but is probably insufficient for the formation of the modality-specific connections.

Development of the deep cerebellar nuclei: transcription factors and cell migration from the rhombic lip.

The deep cerebellar nuclei (DCN) are the main output centers of the cerebellum, but little is known about their development. Using transcription factors as cell type-specific markers, we found that DCN neurons in mice are produced in the rhombic lip and migrate rostrally in a subpial stream to the nuclear transitory zone (NTZ). The rhombic lip-derived cells express transcription factors Pax6, Tbr2, and Tbr1 sequentially as they enter the NTZ. A subset of rhombic lip-derived cells also express reelin, a key regulator of Purkinje cell migrations. In organotypic slice cultures, the rhombic lip was necessary and sufficient to produce cells that migrate in the subpial stream, enter the NTZ, and express Pax6, Tbr2, Tbr1, and reelin. In later stages of development, the subpial stream is replaced by the external granular layer, and the NTZ organizes into distinct DCN nuclei. Tbr1 expression persists to adulthood in a subset of medial DCN projection neurons. In reeler mutant mice, which have a severe cerebellar malformation, rhombic lip-derived cells migrated to the NTZ, despite reelin deficiency. Studies in Tbr1 mutant mice suggested that Tbr1 plays a role in DCN morphogenesis but is not required for reelin expression, glutamatergic differentiation, or the initial formation of efferent axon pathways. Our findings reveal underlying similarities in the transcriptional programs for glutamatergic neuron production in the DCN and the cerebral cortex, and they support a model of cerebellar neurogenesis in which glutamatergic and GABAergic neurons are produced from separate progenitor compartments.

Growth hormone and its receptor in projection neurons of the chick visual system: retinofugal and tectobulbar tracts.

Recent studies have shown the presence of growth hormone (GH) in the retinal ganglion cells (RGCs) of the neural retina in chick embryos at the end of the first trimester [embryonic day (E) 7] of the 21 day incubation period. In this study the presence of GH in fascicles of the optic fiber layer (OFL), formed by axons derived from the underlying RGCs, is shown. Immunoreactivity for GH is also traced through the optic nerve head, at the back of the eye, into the optic nerve, through the optic chiasm, into the optic tract and into the stratum opticum and the retinorecipient layer of the optic tectum, where the RGC axons synapse. The presence of GH immunoreactivity in the tectum occurs prior to synaptogenesis with RGC axons and thus reflects the local expression of the GH gene, especially as GH mRNA is also distributed within this tissue. The distribution of GH-immunoreactivity in the visual system of the E7 embryo is consistent with the distribution of the GH receptor (GHR), which is also expressed in the neural retina and tectum. The presence of a GH-responsive gene (GHRG-1) in these tissues also suggests that the visual system is not just a site of GH production but a site of GH action. These results support the possibility that GH acts as a local growth factor during early embryonic development of the visual system.

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.

Ontogeny of the projections from the anteroventral periventricular nucleus of the hypothalamus in the female rat.

Neurons in the anteroventral periventricular nucleus of the hypothalamus (AVPV) mediate a variety of autonomic functions. In adults they primarily innervate neuroendocrine nuclei in the periventricular zone of the hypothalamus, including the paraventricular and arcuate nuclei (PVH, ARH). Ascending projections from the AVPV also provide inputs to the ventrolateral septum (LSv) and the principal division of the bed nuclei of the stria terminalis (BSTp). Consistent with a role in regulating preovulatory luteinizing hormone secretion, rostral projections from the AVPV contact gonadotropin-releasing hormone (GnRH) neurons surrounding the vascular organ of the lamina terminalis (OVLT). To study the development of these pathways, we placed implants of the lipophilic tracers DiI and CMDiI into the AVPV of female rats ranging in age from embryonic day 19 (E19) through adulthood. The earliest projections targeted a population of GnRH neurons, with apparent contacts from labeled fibers observed as early as E19. These connections appeared to be fully developed before birth, as similar numbers of appositions from AVPV projections onto the GnRH-immunoreactive cells were observed at all ages examined. Caudal projections were delayed relative to projections to the OVLT. Labeled AVPV fibers reached the PVH during the first postnatal week, and fibers targeting the BSTp and LSv were not observed until the second and third postnatal weeks, respectively. Labeled AVPV fibers were not seen in the ARH of animals at any age. Our results demonstrate that projections from the AVPV develop with both spatial and temporal specificity, innervating each target with a unique developmental profile.

Development of the monosynaptic stretch reflex circuit.

Significant advances have been made during the past few years in our understanding of how the spinal monosynaptic reflex develops. Transcription factors in the Neurogenin, Runt, ETS, and LIM families control sequential steps of the specification of various subtypes of dorsal root ganglia sensory neurons. The initiation of muscle spindle differentiation requires neuregulin 1, derived from Ia afferent sensory neurons, and signaling through ErbB receptors in intrafusal muscle fibers. Several retrograde signals from the periphery are important for the establishment of late connectivity in the reflex circuit. Finally, neurotrophin 3 released from muscle spindles regulates the strength of sensory-motor connections within the spinal cord postnatally.

Er81 is expressed in a subpopulation of layer 5 neurons in rodent and primate neocortices.

Laminar organization is a fundamental cytoarchitecture in mammalian CNS and a striking feature of the neocortex. ER81, a transcription factor, has recently been utilized as a marker of cells in the layer 5 of the neocortex. We further pursued the distribution of ER81 to investigate the identity of the ER81-expressing cells in the brain. Er81 transcript was expressed in a subset of pyramidal cells that were scattered throughout the entire width of layer 5. In the rat cortex, Er81 transcripts were first detected in the ventricular zone at E15, remained expressed in putative prospective layer 5 neurons during infant and juvenile stages. The ER81-expressing subpopulation in adult layer 5 neurons did not segregate with the phenotypes of the projection targets. By retrograde labeling combined with immunohistochemistry or reverse transcription-polymerase chain reaction analysis, we found ER81 expression in nearly all of the layer 5 neurons projecting to the spinal cord or to the superior colliculus, while in only one-third of the layer 5 neurons projecting to the contralateral cortex. Er81 was also detected in layer 5 neurons in a P2 Japanese macaque monkey but not in adult monkey cortices. These findings suggest that a neuron class defined by a molecular criterion does not necessarily segregate with that defined by an anatomical criterion, that ER81 is involved in cell differentiation of a subset of layer 5 projection neurons and that this mechanism is conserved among rodents and primates.

Adrenaline contributes to prenatal respiratory maturation in rat medulla-spinal cord preparation.

Adrenaline is a potent respiratory regulator. However, adrenergic contribution to the developing respiratory center has not been studied extensively. Adrenaline application on embryonic day 17 medulla-spinal cord block preparations abolished non-respiratory activity and enhanced respiratory frequency. Phentolamine application on neonatal blocks that produced stable neonatal respiration resulted in respiratory destabilization. These results suggest that central adrenergic modulation is involved in fetal respiratory development and maintenance of stable respiration.

Ontogeny of modulatory inputs to motor networks: early established projection and progressive neurotransmitter acquisition.

Modulatory information plays a key role in the expression and the ontogeny of motor networks. Many developmental studies suggest that the acquisition of adult properties by immature networks involves their progressive innervation by modulatory input neurons. Using the stomatogastric nervous system of the European lobster Homarus gammarus, we show that contrary to this assumption, the known population of projection neurons to motor networks, as revealed by retrograde dye migration, is established early in embryonic development. Moreover, these neurons display a large heterogeneity in the chronology of acquisition of their full adult neurotransmitter phenotype. We performed retrograde dye migration to compare the neuronal population projecting to motor networks located in the stomatogastric ganglion in the embryo and adult. We show that this neuronal population is quantitatively established at developmental stage 65%, and each identified projection neuron displays the same axon projection pattern in the adult and the embryo. We then combined retrograde dye migration with FLRFamide-like, histamine, and GABA immunocytochemistry to characterize the chronology of neurotransmitter expression in individual identified projection neurons. We show that this early established population of projection neurons gradually acquires its neurotransmitter phenotype complement. This study indicates that (1) the basic architecture of the known population of projection inputs to a target network is established early in development and (2) ontogenetic plasticity may depend on changes in neurotransmitter phenotype expression within preexisting neurons rather than in the addition of new projection neurons or fibers.

Development of connections in the human visual system during fetal mid-gestation: a DiI-tracing study.

Animal studies have shown that connections between the retina, lateral geniculate nucleus (LGN), and visual cortex begin to develop prenatally. To study the development of these connections in humans, regions of fixed brain from fetuses of 20-22 gestational weeks (GW) were injected with the fluorescent tracer DiI. Placement of DiI in the optic nerve or tract labeled retinogeniculate projections. In the LGN, these projections were already segregated into eye-specific layers by 20 GW. Retinogeniculate segregation thus preceded cellular lamination of the LGN, which did not commence until 22 GW. Thalamocortical axons, labeled from DiI injections into the optic radiations, densely innervated the subplate, but did not significantly innervate the cortical plate. This pattern was consistent with observations of a "waiting period" in animals, when thalamocortical axons synapse in the subplate for days or weeks before entering the cortical plate. Cortical efferent neurons (labeled retrogradely from the optic radiations) were located in the subplate and deep layers of the cortical plate. In summary, human visual connections are partially formed by mid-gestation, and undergo further refinement during and after this period. The program for prenatal development of visual pathways appears remarkably similar between humans and other primates.

Pattern of development of ascending and descending fibers in embryonic spinal cord of chick: I. Role of position information.

Previous studies on the rigid time and position pattern of embryonic spinal cord development are extended to include the pattern of growth of axonal processes. This study was designed to analyze the position of axons in the marginal zone as they course (fasciculate) in the ascending and descending direction from a single spinal segment. Injections of 3H-proline were made into L1 or T6 of chick embryos of stages 21--29 (3.5--6 days of incubation). The location of radioactive label in the marginal zone was analyzed autoradiographically to show the location of labeled axons originating from the injected segment. In the early stages of development (stage 21), the labeled descending axons were located in the most ventromedial aspect of the marginal zone. The labeled ascending axons were located more laterally and to the perimeter of this zone. In stages 27--29, the labeled axons near the site of injection were located in the inner two-thirds of the marginal zone, and in the progressively more rostral levels the label shifted laterally and to the perimeter in a laminar pattern. It was concluded that the labeled ascending axons from an individual spinal segment layer upon the axons which originate from the more rostral segments. How this rigid time and position pattern of axonal growth can impart a topographic order into fiber tracts of the spinal cord was discussed.

The early development of thalamocortical and corticothalamic projections.

The early development of thalamocortical and corticothalamic projections in hamsters was studied to compare the specificity and maturation of these pathways, and to identify potential sources of information for specification of cortical areas. The cells that constitute these projections are both generated prenatally in hamsters and they make reciprocal connections. Fluorescent dyes (DiI and DiA) were injected into the visual cortex or lateral geniculate nucleus in fixed brains of fetal and postnatal pups. Several issues in axonal development were examined, including timing of axon outgrowth and target invasion, projection specificity, the spatial relationship between the two pathways, and the connections of subplate cells. Thalamic projections arrive in the visual cortex 2 days before birth and begin to invade the developing cortical plate by the next day. Few processes invade inappropriate cortical regions. By postnatal day 7 their laminar position is similar to mature animals. By contrast, visual cortical axons from subplate and layer 6 cells reach posterior thalamus at 1 day after birth in small numbers. By 3 days after birth many layer 5 cell projections reach the posterior thalamus. On postnatal day 7, there is a sudden increase in the number of layer 6 projections to the thalamus. Surprisingly, these layer 6 cells are precisely topographically mapped with colabeled thalamic afferents on their first appearance. Subplate cells constitute a very small component of the corticothalamic projection at all ages. Double injections of DiI and DiA show that the corticofugal and thalamocortical pathways are physically separate during development. Corticofugal axons travel deep in the intermediate zone to the thalamic axons and are separate through much of the internal capsule. Their tangential distribution is also distinct. The early appearance of the thalamocortical pathway is consistent with an organizational role in the specification of some features of cortical cytoarchitecture. The specific initial projection of thalamocortical axons strongly suggests the recognition of particular cortical regions. The physical separation of these two pathways limits the possibility for exchange of information between these systems except at their respective targets.

Pre- and postnatal development of efferent connections of the cochlear nucleus in the rat.

Although the connections of the auditory brainstem nuclei are well described in adult mammals, almost nothing is known concerning how and when these connections develop. The purpose of the present study was to describe the development of the efferent projections of the cochlear nucleus (CN), the first central relay station in the ascending auditory pathway of mammals. We used two tracers in rats aged between embryonic day 15 (E15) and postnatal day 14 (P14; birth in the rat is at E22 = P0). The carbocyanine dye DiI was applied into the CN in aldehyde-fixed tissue. The second tracer, biocytin, was applied into the ventral acoustic stria in an in vitro slice preparation. The ontogeny of the efferent projections from the CN could be divided into three periods. The first period (E15-E17) is characterized by axonal outgrowth. Axons traverse nuclei in the superior olivary complex and the lateral lemniscus and finally grow up into the inferior colliculus, but axon collaterals do not form during this period. The second period (E18-P5) is marked by pronounced collateral branching of CN fibers in auditory brainstem nuclei. Collateralisation in the contralateral inferior colliculus starts shortly before that in the ipsilateral superior olivary complex. The remaining auditory nuclei become successively innervated, as indicated by collaterals found in them. During the third period (P5-P14) terminal structures mature further, as shown by the morphological changes of the calyces of Held in the medial nucleus of the trapezoid body. In conclusion, our results show that the efferent connections from the cochlear nucleus form over a period of almost two weeks and are laid down without forming aberrant internuclear connections. On a nuclear level, an adult-like projection pattern is already achieved one week prior to the onset of physiological hearing.

Early development of efferent projections from the chick tectum.

The early development of the uncrossed tectobulbar and the crossed tectospinal tracts was studied. These two projections arise from the same structure, the mesencephalon, and develop during the same time period, but follow divergent courses. We have traced the pathways followed by these projections and identified the positions at which axon guidance decisions are made. The first neurons differentiate either side of the entire rostrocaudal extent of the dorsal midline and initiate axons that extend dorsoventrally across the surface of the tectum. At the ventral edge of the tectum these axons turn abruptly and fasciculate to form a caudal descending projection to the hindbrain. These axons extend to the caudal hindbrain and do not project to the periphery along cranial nerve roots. We therefore consider this tract to be the tectobular, rather than the mesencephalic division of the trigeminal. While the tectobulbar projection is still developing, a second wave of axons is initiated, which arises from only the rostral part of the tectum. These axons grow beyond the tectobulbar turn point and continue toward the ventral midline, where they cross the floor plate, before turning caudally at the lateral edge of the main descending hindbrain tract, the ventrolateral tract. We discuss the development of these tracts with reference to possible guidance cues mediating their course.

The genesis of efferent connections from the visual cortex of the fetal rhesus monkey.

The prenatal development of the cortical projections to the dorsal lateral geniculate nucleus (LGN), superior colliculus (SC) and pulvinar was studied by autoradiography of orthogradely transported 3H-proline injected into the occipital cortex of fetal rhesus monkeys aged from embryonic day 63 (E63) to E95. Differentiation of pyramidal neurons situated in the infragranular strata of the cortical plate (prospective layers 5 and 6, which give rise to these efferent projections) was also examined in Golgi preparations prepared from specimens of corresponding embryonic ages. In autoradiographs of the E63 fetus, no radioactive label was seen in subcortical structures. In two specimens injected around E70, label was present in the prospective magnocellular layers of the LGN and within the immediately surrounding cell-poor zones. At these young fetal ages, the presence of topographic order in the corticogeniculate projection could not be determined due to the large size of the injection sites relative to the small cerebral vesicles. By E84 the portion of the prospective parvocellular layers adjacent to the white matter also contained label which was characteristically wedge-shaped and appropriately located with respect to the site of the cortical injection, suggesting that topographic order is established. In the oldest fetus (E95) label in the LGN assumed a configuration similar to that seen in the adult. The cortical projection also invades the SC and pulvinar around E70. In the SC, label was initially confined to the stratum opticum, but by E84 it extended into the superficial gray. Thus, all known classes of efferent pathways from the visual cortex to subcortical structures are present by the middle of the 165-day gestational period in rhesus monkey. The one-month period, E63-E97, during which these efferent visual connections are established is characterized by the considerable growth and increased complexity of the dendritic arborization of pyramidal cells in the infragranular cortical layers of area 17. Thus the development of visual cortical efferents occurs in rough synchrony with the genesis of the afferent pathway from the LGN (Rakic, '76a; '79) and with the onset of morphological differentiation of pyramidal neurons in the infragranular cortical layers.