Functional Analyses of Embryonic Cerebrospinal Fluid Proteins.

The embryonic cerebrospinal fluid (eCSF) influences neuroepithelial cell behavior, affecting proliferation, differentiation, and survival. One major question to resolve in the field is to precisely describe the eCSF molecules responsible and to understand how these molecules interact in order to exert their functions. Here we describe an in vitro protocol to analyze the influence of eCSF components on neuroepithelium development.

The emergence of mesencephalic trigeminal neurons.

BACKGROUND: The cells of the mesencephalic trigeminal nucleus (MTN) are the proprioceptive sensory neurons that innervate the jaw closing muscles. These cells differentiate close to the two key signalling centres that influence the dorsal midbrain, the isthmus, which mediates its effects via FGF and WNT signalling and the roof plate, which is a major source of BMP signalling as well as WNT signalling. METHODS: In this study, we have set out to analyse the importance of FGF, WNT and BMP signalling for the development of the MTN. We have employed pharmacological inhibitors of these pathways in explant cultures as well as utilising the electroporation of inhibitory constructs in vivo in the chick embryo. RESULTS: We find that interfering with either FGF or WNT signalling has pronounced effects on MTN development whilst abrogation of BMP signalling has no effect. We show that treatment of explants with either FGF or WNT antagonists results in the generation of fewer MTN neurons and affects MTN axon extension and that inhibition of both these pathways has an additive effect. To complement these studies, we have used in vivo electroporation to inhibit BMP, FGF and WNT signalling within dorsal midbrain cells prior to, and during, their differentiation as MTN neurons. Again, we find that inhibition of BMP signalling has no effect on the development of MTN neurons. We additionally find that cells electroporated with inhibitory constructs for either FGF or WNT signalling can differentiate as MTN neurons suggesting that these pathways are not required cell intrinsically for the emergence of these neurons. Indeed, we also show that explants of dorsal mesencephalon lacking both the isthmus and roof plate can generate MTN neurons. However, we did find that inhibiting FGF or WNT signalling had consequences for MTN differentiation. CONCLUSIONS: Our results suggest that the emergence of MTN neurons is an intrinsic property of the dorsal mesencephalon of gnathostomes, and that this population undergoes expansion, and maturation, along with the rest of the dorsal midbrain under the influence of FGF and WNT signalling.

Prenatal nicotine is associated with reduced AMPA and NMDA receptor-mediated rises in calcium within the laterodorsal tegmentum: a pontine nucleus involved in addiction processes.

Despite huge efforts from public sectors to educate society as to the deleterious physiological consequences of smoking while pregnant, 12-25% of all babies worldwide are born to mothers who smoked during their pregnancies. Chief among the negative legacies bestowed to the exposed individual is an enhanced proclivity postnatally to addict to drugs of abuse, which suggests that the drug exposure during gestation changed the developing brain in such a way that biased it towards addiction. Glutamate signalling has been shown to be altered by prenatal nicotine exposure (PNE) and glutamate is the major excitatory neurotransmitter within the laterodorsal tegmental nucleus (LDT), which is a brainstem region importantly involved in responding to motivational stimuli and critical in development of drug addiction-associated behaviours, however, it is unknown whether PNE alters glutamate signalling within this nucleus. Accordingly, we used calcium imaging, to evaluate AMPA and NMDA receptor-mediated calcium responses in LDT brain slices from control and PNE mice. We also investigated whether the positive AMPA receptor modulator cyclothiazide (CYZ) had differential actions on calcium in the LDT following PNE. Our data indicated that PNE significantly decreased AMPA receptor-mediated calcium responses, and altered the neuronal calcium response to consecutive NMDA applications within the LDT. Furthermore, CYZ strongly potentiated AMPA-induced responses, however, this action was significantly reduced in the LDT of PNE mice when compared with enhancements in responses in control LDT cells. Immunohistochemical processing confirmed that calcium imaging recordings were obtained from the LDT nucleus as determined by presence of cholinergic neurons. Our results contribute to the body of evidence suggesting that neurobiological changes are induced if gestation is accompanied by nicotine exposure. We conclude that in light of the role played by the LDT in motivated behaviour, the cellular changes in the LDT induced by exposures to nicotine prenatally, when combined with alterations in other reward-related regions, could contribute to the increased susceptibility to smoking observed in the offspring.

A novel TaulacZ allele reveals a requirement for Pitx2 in formation of the mammillothalamic tract.

The hypothalamic mammillary region is critical for spatial memory and vestibular processing. Pitx2 encodes a paired-like transcription factor that is highly expressed in the developing mammillary region and is required for subthalamic nucleus formation. Here we analyzed a loss of function Pitx2-TaulacZ knock-in allele to study the effects of Pitx2 deficiency on neuronal projections in the embryonic mammillary region. Pitx2-expressing neurons contribute axons to principal mammillary, mammillotegmental and mammillotectal tracts. Embryos with Pitx2 deficiency exhibit axonal fibers in the principal mammillary tract that are improperly bundled and disorganized, yet project caudally toward the tectum and tegmentum. Embryos with Nestin-Cre mediated conditional Pitx2 deficiency exhibit truncated mammillothalamic tracts (mtt) that fail to elongate, and reduced Pax6-positive cells at the branching point of the principal mammillary and mtt. These data suggest that Pitx2 mediates cell-autonomous and nonautonomous guidance cues necessary for mammillary collaterals destined to project to the anterior thalamus.

[Perinatal development of mammillotegmental connections in rats].

Development of direct axonal connections of the hypothalamic mammillary bodies with ventral and dorsal tegmental nuclei of Gudden was studied on fixed rat brains from day 14 of embryonic development until day 10 of postnatal development using the method of diffusion of the lipophilic fluorescent carbocyanine tracer 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate along the neuronal membranes. The tracer was inserted into the mammillary bodies or into the tegmentum and after incubation in a fixative fluorescent nerve cells and nerve fibers were visualized in the brain tissue. The mammillotegmental tract was found to start developing earlier than other conducting systems of the mammillary bodies. On days 14-15 of embryonic development, it was visualized as a bundle of axons running from the mammillary bodies caudally to the midbrain. A group of neurons in the midbrain tegmentum and their axons going to the mammillary bodies via the mammillary peduncle were first visualized on day 19 of embryonic development. The mammillotegmental tract and mammillary peduncle developed progressively from the moment of birth. Ventral and dorsal tegmental nuclei were formed in the midbrain by day 10 of the postnatal development. Thus, the formation of reciprocal connections of the mammillary bodies with midbrain tegmental nuclei was first described during perinatal development in rats.

Sim1 and Sim2 are required for the correct targeting of mammillary body axons.

The mammillary body (MB), and its axonal projections to the thalamus (mammillothalamic tract, MTT) and the tegmentum (mammillotegmental tract, MTEG), are components of a circuit involved in spatial learning. The bHLH-PAS transcription factors SIM1 and SIM2 are co-expressed in the developing MB. We have found that MB neurons are generated and that they survive at least until E18.5 in embryos lacking both Sim1 and Sim2 (Sim1(-/-);Sim2(-/-)). However, the MTT and MTEG are histologically absent in Sim1(-/-);Sim2(-/-) embryos, and are reduced in embryos lacking Sim1 but bearing one or two copies of Sim2, indicating a contribution of the latter to the development of MB axons. We have generated, by homologous recombination, a null allele of Sim1 (Sim1(tlz)) in which the tau-lacZ fusion gene was introduced, allowing the staining of MB axons. Consistent with the histological studies, lacZ staining showed that the MTT/MTEG is barely detectable in Sim1(tlz/tlz);Sim2(+/-) and Sim1(tlz/tlz);Sim2(-/-) brains. Instead, MB axons are splayed and grow towards the midline. Slit1 and Slit2, which code for secreted molecules that induce the repulsion of ROBO1-producing axons, are expressed in the midline at the level of the MB, whereas Robo1 is expressed in the developing MB. The expression of Rig-1/Robo3, a negative regulator of Slit signalling, is upregulated in the prospective MB of Sim1/Sim2 double mutants, raising the possibility that the growth of mutant MB axons towards the midline is caused by a decreased sensitivity to SLIT. Finally, we found that Sim1 and Sim2 act along compensatory, but not hierarchical, pathways, suggesting that they play similar roles in vivo.

Identification of dopaminergic neurons of nigral and ventral tegmental area subtypes in grafts of fetal ventral mesencephalon based on cell morphology, protein expression, and efferent projections.

Transplants of fetal ventral mesencephalic tissue are known to contain a mixture of two major dopamine (DA) neuron types: the A9 neurons of the substantia nigra pars compacta (SNpc) and the A10 neurons of the ventral tegmental area (VTA). Previous studies have suggested that these two DA neuron types may differ in their growth characteristics, but, because of technical limitations, it has so far been difficult to identify the two subtypes in fetal ventral mesencephalon (VM) grafts and trace their axonal projections. Here, we have made use of a transgenic mouse expressing green fluorescent protein (GFP) under the tyrosine hydroxylase promoter. The expression of the GFP reporter allowed for visualization of the grafted DA neurons and their axonal projections within the host brain. We show that the SNpc and VTA neuron subtypes in VM grafts can be identified on the basis of their morphology and location within the graft, and their expression of a G-protein-gated inwardly rectifying K+ channel subunit (Girk2) and calbindin, respectively, and also that the axonal projections of the two DA neuron types are markedly different. By retrograde axonal tracing, we show that dopaminergic innervation of the striatum is derived almost exclusively from the Girk2-positive SNpc cells, whereas the calbindin-positive VTA neurons project to the frontal cortex and probably also other forebrain areas. The results suggest the presence of axon guidance and target recognition mechanisms in the DA-denervated forebrain that can guide the growing axons to their appropriate targets and indicate that cell preparations used for cell replacement in Parkinson's disease will be therapeutically useful only if they contain cells capable of generating the correct nigral DA neuron phenotype.

Developmental changes in pedunculopontine nucleus (PPN) neurons.

The developmental decrease in rapid-eye-movement (REM) sleep in man occurs between birth and after puberty. We hypothesize that if this decrease in REM sleep does not occur, lifelong increases in REM sleep drive may ensue. Such disorders are characterized by hypervigilance and sensory-gating deficits, such as are present in postpubertal onset disorders like schizophrenia, panic attacks (a form of anxiety disorder), and depression. The decrease in REM sleep in the rat occurs between 10 and 30 days of age. We studied changes in size and physiological properties of pedunculopontine nucleus (PPN) cells involved in the control of arousal, i.e., waking and REM sleep. During the largest decrease in REM sleep (12-21 days), cholinergic PPN neurons doubled in cell area, the hypertrophy peaking at 15-16 days, then decreasing in area by 20-21 days. Noncholinergic PPN cells did not change in area during this period. We confirmed the presence of two populations of PPN neurons based on action potential (AP) duration, with the proportion of short-AP-duration cells increasing and long AP duration decreasing between 12 and 21 days. Most cholinergic and noncholinergic cells had short AP durations. Afterhyperpolarization (AHP) duration became segregated into long and short AHP duration after 15 days. Cells with short AP duration also had short AHP duration. The proportion of PPN cells with Ih current increased gradually, peaking at 15 days, then decreased by 21 days. These changes in morphological and physiological properties are discussed in relation to the developmental decrease in REM sleep.

Arcuate plan of chick midbrain development.

In spinal cord and hindbrain development, neurons are generated as longitudinal cell columns aligned with the ventral and dorsal midlines. For rostral brain, however, the fundamental structure of early neuronal patterning remains poorly understood. We report here that, in the chick embryo, the ventral midbrain is remarkably regular in its cellular and molecular organization; it is arranged as a reiterative series of arcuate territories arrayed bilateral to the ventral midline. In the mantle layer of the ventral midbrain, an arcuate series of neuronal cell columns (midbrain arcs) is demonstrated by acetylcholinesterase histochemistry and gene expression for class III beta-tubulin, homeodomain transcription factors, and neurotransmitter synthetic enzymes. In the ventricular layer of midbrain progenitor cells, WNT and NOTCH ligand gene expression displays arcuate periodicities that form a tight three-dimensional registration with the arcs of the underlying mantle layer. Ventral midbrain arcuate patterning is even macroscopically visible, forming ridges along the ventricular surface. These observations establish that a single plan of arcuate organization governs the morphogenesis and cell-type specification of the ventral midbrain. Arcs are not restricted to the midbrain tegmentum but extend through the subthalamic tegmentum of the forebrain. Thus, the chick rostral brain, which is classically divided into midbrain and forebrain, can also be partitioned into the following: (1) a neuraxial region of arcs and (2) an anterodorsal cap that includes midbrain tectum and nonsubthalamic forebrain. We show that this partition of brain tissue is supported by the expression patterns of homologs of Drosophila gap genes.

Suspensional reaggregates of human foetal neocortex and tegmentum as objects of neurotransplantation.

Suspensional reaggregates were obtained from human neocortical and tegmental anlagen (7 weeks of gestation), using 0.1% tripsin solution, and cultivated in Medium 199. Suspensional reaggregates, formed after 2 days in vitro, were grafted into the Wistar rat striatum. Incipient stages of histogenesis in the reaggregates and their interaction with host brain were investigated using light and electron microscopy, with antibodies against vimentin, glial fibrillary acidic protein (GFAP), proliferating cell nuclear antigen (PCNA), ferritin, as well as lectin ricinus communis agglutinin (RCA). The reaggregates showed a low level of tissue organisation. An intermediate condition between suspension and the true tissue could be observed in them. These reaggregates had two evident features: a rather irregular cell arrangement (without parallel bundles of radial glia), and the presence of special intercellular junctions. Some cells made up fragments of neuroepithelial sheet in the form of true rosettes. The one-week-old grafts were integrated with the host brain as well as dissociated and contained host astrocytes. Degenerated cells and detritus appeared rarely. The data of this work let us conclude that the suspensional aggregate grafting combines some advantages of suspensional and solid grafting methods.

Control of chick tectum territory along dorsoventral axis by Sonic hedgehog.

Chick midbrain comprises two major components along the dorsoventral axis, the tectum and the tegmentum. The alar plate differentiates into the optic tectum, while the basal plate gives rise to the tegmentum. It is largely unknown how the differences between these two structures are molecularly controlled during the midbrain development. The secreted protein Sonic hedgehog (Shh) produced in the notochord and floor plate induces differentiation of ventral cell types of the central nervous system. To evaluate the role of Shh in the establishment of dorsoventral polarity in the developing midbrain, we have ectopically expressed Shh unilaterally in the brain vesicles including whole midbrain of E1.5 chick embryos in ovo. Ectopic Shh repressed normal growth of the tectum, producing dorsally enlarged tegmentum region. In addition, the expression of several genes crucial for tectum formation was strongly suppressed in the midbrain and isthmus. Markers for midbrain roof plate were inhibited, indicating that the roof plate was not fully generated. After E5, the tectum territory of Shh-transfected side was significantly reduced and was fused with that of untransfected side. Moreover, ectopic Shh induced a considerable number of SC1-positive motor neurons, overlapping markers such as HNF-3(beta) (floor plate), Isl-1 (postmitotic motor neuron) and Lim1/2. Dopaminergic and serotonergic neurons were also generated in the dorsally extended region. These changes indicate that ectopic Shh changed the fate of the mesencephalic alar plate to that of the basal plate, suppressing the massive cell proliferation that normally occurs in the developing tectum. Taken together our results suggest that Shh signaling restricts the tectum territory by controlling the molecular cascade for tectum formation along dorsoventral axis and by regulating neuronal cell diversity in the ventral midbrain.

MR imaging of the posterior fossa structures of human embryos and fetuses.

There have been few reports on MR imaging of the developing human fetal brain. The aim of this article is to establish a standard atlas of developing fetal brain, focusing in particular on posterior fossa structures. Eighty-eight formalin-fixed embryos and fetuses were examined using 1.5 Tesla MR units. Specimens ranged from Carnegie stage 17 to 28 gestational weeks. The morphologic changes in developing cerebellum, cerebellar fissures, pontine flexure, fourth ventricle, and cerebral aqueduct were observed in each developmental period. The height of the fourth ventricle and cerebral aqueduct and the thickness of the tectum and the tegmentum of the midbrain were measured. We obtained detailed MR images of the developmental changes in posterior fossa structures and produced an atlas of these images. Our study showed that the period of visualization of cerebellar structures and fissures was later on MR imaging than described in past anatomical and embryological studies. In addition, the sudden decrease in height of the fourth ventricle and the cerebral aqueduct found in our study might reflect the presence of communication between the fourth ventricle and subarachnoid space.

Calcium-binding proteins in the substantia nigra and ventral tegmental area during development: correlation with dopaminergic compartmentalization.

The importance of calcium in neuronal function has been amply demonstrated in recent years. The discovery of a class of proteins within neurons which bind calcium, therefore, has proven to be a catalyst for the generation of theories and hypotheses regarding mechanisms of neurotoxicity in the CNS. In addition, the distribution of certain calcium-binding proteins changes during neural development, suggesting that they may play a role in organization or pattern generation. We have examined the ontogeny of three related calcium-binding proteins, calbindin-D28, parvalbumin and calretinin, with respect to the ventral and dorsal compartments or tiers of the dopaminergic population in the ventral midbrain. Single and dual-label immunocytochemistry was employed to map the distributions of calcium-binding proteins and tyrosine hydroxylase from E18 through adulthood. The results show that each of the three proteins exhibits a unique developmental sequence and compartment preference, with calbindin D28 clearly related to the later-developing dorsal tier, and parvalbumin and calretinin to the ventral tier of the dopaminergic ventral mesencephalon.

Ontogenesis of embryonic porcine ventral mesencephalon in the perspective of its potential use as a xenograft in Parkinson's disease.

Human fetal neural dopaminergic tissue can be transplanted and can ameliorate neurological deficiencies in patients with Parkinson's disease (PD). Donor tissue from other species has been used experimentally for several years in animal experiments and is now being considered an attractive alternative, particularly from a donor species that breeds in large litters, e.g., the pig. We have studied the early ontogenetic development of the mesencephalic dopaminergic system in the pig, utilising an anti-tyrosine hydroxylase (TH) immunocytochemical technique, and demonstrated the earliest appearance of its cell bodies at embryonic day 20 (E20). We compared the porcine data with those of human fetal development, as revealed by the same technique. Embryonic dopaminergic cell groups resembling the A8, A9, and A10 of the rat are present in the pig and differentiate into the homologous cell groups of human, although interesting quantitative differences are apparent. In the pig, prolonged presence of immature characteristics of TH-immunoreactive (TH-i.r.) cell bodies was observed, notwithstanding the early outgrowth of TH-i.r. axons into the ganglionic eminence. In the human, on the other hand, cell divisions and maturation of dendrites have progressed to a further degree than in the pig, before such distinct outgrowth of axons takes place. In pig embryos of 28 days, cells in the ventral mesencephalon had differentiated into TH containing neurons, which indicates their potential to synthesize dopamine. In spite of their differentiation, these cells still showed immature morphological features (rounded cell bodies with undifferentiated, short processes). Dopamine synthesis by these cells was demonstrated in previous studies by the high performance liquid chromatographic technique (HogenEsch et al. [1993] Can. J. Neurol. Sci. 20(suppl. 4):P.S. 235). In a separate paper, we have described that these porcine 28-day dopaminergic cells retain their potential for development and outgrowth in culture (van Roon et al. [1995] Res. Neurol. Neurosci. 7:199-205). We conclude that the ventral mesencephalon in pig embryos of 28 days is a potential source of dopaminergic neurons to be used as a xenograft in PD.

Differential binding profile and internalization process of neurotensin via neuronal and glial receptors.

Two G-protein-coupled receptors for the tridecapeptide neurotensin (NT) have been identified and cloned in mammalian brain: a high-affinity (Kd = 0.3 nM) receptor, sensitive to the antagonist SR 48692 but insensitive to levocabastine, and a lower-affinity (Kd = 2-4 nM) receptor, sensitive to levocabastine but with poor affinity for SR 48692. Although there is good evidence that the high-affinity site is predominantly expressed in neurons, little is known of the cellular localization of the low-affinity receptor. In the present study, we identify by confocal microscopy selective levocabastine-sensitive, SR 48692-resistant binding of a fluorescent derivative of NT (fluo-NT) to a subpopulation of glial fibrillary acidic protein-immunoreactive glial cells grown in culture from the midbrain and cerebral cortex of embryonic and neonatal rats, respectively. We also demonstrate, by combining fluo-NT detection with tyrosine hydroxylase immunofluorescence, that these glial binding sites are differentially regulated from the SR 48692-sensitive NT receptor expressed in the same cultures by mesencephalic dopamine neurons. Whereas the latter undergoes rapid ligand-induced internalization followed by centripetal mobilization of ligand-receptor complexes from processes to perikarya and from perikaryal periphery to cell center, the former induces the formation of cell-surface clusters that fail to internalize. It is concluded that NT may exert its effects on both neurons and astrocytes in the CNS. Whereas NT neural signaling is exerted through high-affinity receptors and may be partly effected through internalization of receptor-ligand complexes, glial signaling is exerted through low-affinity NT receptors and appears to be transduced exclusively at the level of the plasma membrane.

A morphometric evaluation of morphological types of microglia and astroglia in human fetal mesencephalon.

Quantitative changes associated with the differentiation of microglial and astroglial cells in 48 fetal mesencephalons between 8 and 22 week of gestation (GW) were studies. Using lectin (RCA-1) labelling, two morphological types of RCA-1 positive cells, ameboid microglia (AM) and ramified microglia (RM) were identified on the basis of the cell body shape and the configuration of cytoplasmic processes. Astrocytes (AS) were identified by immunocytochemical labelling of GFAP. Measurements of microglial and astroglial cells were carried out in mesencephalon tectum and tegmentum under the microscope in sequential segments corresponding to equal sized non-overlapping areas. The quantitative data about concerning the number and percentage distribution of AM, RM and AS from each of the grid-quartiles were analyzed. The study revealed a time-sequence of appearance, characteristic pattern of distribution along perpendicular and longitudinal axes originating from aqueduct and changes in percent distribution of both types of microglial cells and astrocytes. Ameboid type of microglia was already present in 8 GW fetus in tectum (22.5 cells/mm2) and in tegmentum (10.2 cells/mm2). During the fetal development the number of AM peaked about 8-9 GW in tectum, accounting for 29 cells/mm2 close to aqueduct, and about 11-12 weeks in tegmentum, reaching 10.2-11 cells/mm2 close to aqueduct. As the fetus development advanced, the number of AM cells both in tectum and tegmentum was slowly dropped down reaching about 1.0 cells/mm2 in 20-22 GW. The ramified microglial cells as well as astroglial cells emerged in the fetal mesencephalon after 11 GW. A quantitative study also revealed a rapid increase in the density of RM in 13-16 GW (6.3 cells/mm2-close to aqueduct) and AS cells in 13-16 GW (36 cells/mm2-close to aqueduct) and later a decrease in cell number was observed. Similarly to AM, changes in the number of RM and AS cells were stabilized to about 20-22 GW. The percentage distribution of each type of microglia and astrocyte cells both in tectum and tegmentum differed markedly during the fetus development depending on the localization along the axes.

Early developmental changes in [3H]nicotine binding in the human brainstem.

Little is known about the developmental profile of nicotinic cholinergic receptors in the developing human brain, despite the potential importance of such information in understanding the pathogenesis of neurological abnormalities or increased risk for the sudden infant death syndrome in offspring exposed to nicotine in utero. In this study, we determined the distribution of [3H]nicotine binding in the developing human brainstem by quantitative tissue autoradiography. In midgestational fetuses, [3H]nicotine binding sites were heavily concentrated in tegmental nuclei related to cardiopulmonary integration, arousal, attention, rapid eye movement sleep, and somatic motor control. Over the last half of gestation, [3H]nicotine binding decreased 60-70% in the tegmental nuclei, with a significant difference in binding between midgestation and early infancy. In contrast, there was essentially no change in [3H]nicotine binding in the major cerebellar-relay nuclei (principal inferior olive and griseum pontis) between the same time-points. Tritium quenching by increasing lipid (myelin) content in tissue sections did not account for the decreases in [3H]nicotine binding in tegmental nuclei. Based upon the high levels of [3H]nicotine binding at midgestation, combined with experimental data demonstrating trophic properties for acetylcholine, we postulate that nAChRs a role in the development of the brainstem tegmentum during this period, and that once this role is fulfilled, nicotinic cholinergic binding decreases and remains low thereafter. Alternatively, nicotinic cholinergic receptors may be critical for other developmentally related functions and/or neurotransmission in the brainstem tegmentum at midgestation. The high levels of [3H]nicotine binding in the brainstem tegmentum at midgestation and its rapidly changing profile over late gestation further suggest that mid-to-late gestation is a developmental period during which this region is likely to be most vulnerable to the harmful effects of nicotine in maternal cigarette smoke. The baseline information provided in this study is potentially relevant towards understanding attention deficits and risk for the sudden infant death syndrome in offspring exposed to cigarette smoke in utero.

Emergence of connectivity in the embryonic rat parietal cortex.

In order to understand how cortical circuitry is put together, we examined the emergence of corticofugal projection cells and the arrival of subcortical afferents in the presumptive parietal cortex of the embryonic rat cerebrum. Afferent and efferent projections were selectively labeled by applications of the lipophilic tracers DiI and DiA in aldehyde-fixed brains of 12-18-d-old rat embryos (E12-E18; gestation: 21 d). On E12 and E13, the neocortical anlage consists of a ventricular zone and a preplate, with no extracortical connections. By E14, just prior to the appearance of the cortical plate, polymorphic cells located in the ventrolateral preplate of the telencephalic vesicle send out the first group of corticofugal axons toward the ganglionic eminence. Shortly thereafter, the cortical plate emerges as a dense band of radially oriented cells that also contribute to the corticofugal projection. By E15, axons of the early cortical projection cells cascade through the striatal primordium, the future site of the internal capsule. At the time of cortical plate formation and initial corticofugal axon outgrowth, ascending corticopetal axon systems have not yet arrived in the neocortex. Double-labeling experiments in which one dye is placed in the neocortex and the other in the ipsilateral dorsal thalamus reveal that cortical efferents encounter the first ascending wave of thalamofugal axons at the level of the striatum. Collectively, these two axonal systems bridge the necortex and the diencephalon. Upon their arrival in the neocortex on E16, thalamic axons follow a ventrolateral to dorsomedial course within the intermediate zone. Thalamic axons are the first subcortical afferent system to arrive in the neocortex. Other ascending afferent systems arising from the midbrain tegmentum enter the neocortex after E17. Comparison of thalamocortical and tegmentocortical projections in two halves of the same brain and across various embryonic ages clearly reveals that the two projection systems differ in their trajectories as well as in their time of arrival. Present observations challenge the view that the precocious arrival of subcortical axons provides the impetus for cortical maturation, and suggest that cortical plate differentiation and the initial organization of corticofugal projection patterns occur independent of ascending pathways.

Further data on the development of SRIF-like immunoreactive nerve cell populations in the chick embryo brain stem. II. Midbrain tegmentum.

Further immunocytochemical analysis of the neuroblasts with SRIF-like immunoreactivity (ir) was carried out on the chick embryo midbrain tegmentum. 5 or 100 microns mesencephalon sections were obtained from 60 White Leghorn chick embryos at stages (E = Embryonic days) ranging from E4 1/2 to E18 and incubated with rabbit polyclonal antibodies against synthetic cyclic Somatostatin-14, according to PAP-DAB technique. In the midbrain tegmentum the ir appeared as from E12. From E12 to E13 1/2-E14 the ir distribution gradually changed. From E14 to E18 numbers and spatial arrangement of the positive neuroblast groups did not show substantial changes; in these respects the ir distributional pattern proved to be similar to the one observed by the Authors in adult animals. From E17 to E18 a decrease in the positive neuroblast density appeared to occur, particularly in a ventrally placed group. These results are consistent with a possible local regulative role of the SRIF.