Appearance and differentiation of NADPH-D-neurons in ontogeny of the cerebral cortex in rats

The appearance of presumptive NO-ergic nerve cells and their differentiation in the rat neocortex were studied. For this purpose, a comparative analysis of the development and differentiation of NADPH-D-positive neurons in the neocortex transplants taken from the embryos of different ages and transplanted in the occipital cortex of adult rats and in the normally developing cerebral cortex. The nervous tissue was stained histochemically for NADPH-D. The results we obtained suggest that no NADPH-D-containing neurons were found in the transplants from 15-day embryos, while they developed in those from 18-day embryos. Hence, precursors of NO-ergic neurons were still absent in the presumptive neocortex of 15-day embryos and appeared only on day 16-18 of embryogenesis. Expression of NADPH-D begins in them only within four to five days, but the neurons are differentiated during a relatively short period of time. Most NADPH-D-positive neurons reach their structural-functional maturity already by the end of the first week of postnatal development, while their complete maturation takes place by the end of the second week of postnatal development.     

Phenotypic differentiation of neurons in intraocular transplants

Neurochemical differentiation of neurons in transplants developing in rat anterior eye chamber was studied. Pieces of the somatosensory neocortex area, isolated from 17-day fetuses of Wistar rats, were used for the transplantation. The general cytological analysis and immunochemical identification of GABAergic neurons in neocortical transplants and in the appropriate brain area of the recipient rats (control) were carried out after 6 months. Cytoarchitectonics typical for neocortex was not revealed in the transplants. Furthermore, a 1.4-fold decrease in numerical density of the entire neuron population was found compared to the control. The proportion of GABAergic nerve cells in the transplanted tissue was reduced even more dramatically— by 13.1 times. The dimensions of all types of neurons, especially GABAergic cells, were greater in the transplants in oculo compared to neocortex in situ. The increase in size occurred mostly due to the cytoplasm. Thus, the nuclei of GABA-positive neurons in the transplants were larger by 1.2 times compared to the control and their perikarya were larger by 1.5 times. The obtained results showed that the conditions in the anterior eye chamber the most dramatically affect the differentiation of GABAergic neurons, and cell hypertrophy, probably, is the functional compensation of the decrease in their number. Considering the literature data on the increased excitability and synchronized neuronal activity in the intraocular transplants, it can be assumed that these transplants can be used as a model for studying the cellular mechanisms of nervous tissue epileptization under disinhibition conditions.     

The development of thalamocortical connections studied by using carbocyanine dyes in the early ontogeny of rats]

We studied the differentiation of neurons and development of their connections in the occipital cortex and thalamic areas of the brain in early ontogenesis of rats: from day 11 of embryogenesis until day 19 of postnatal development. We used the method of staining of brain tissues by carbocyanine dyes after its preliminary fixation in aldehydes. Three carbocyanine dyes were used: DiI, DiO and DiA. We showed the dynamics of structural differentiation of the cortical neurons and lateral geniculate body of the thalamus and the specificity of formation of the axonal pathways between the neocortex and thalamic areas. The results obtained confirmed the hypothesis on ordered spatial-temporal growth of the cortical and thalamic fibers in early embryogenesis and revealed synchronous development of both classes of neurons of the lateral geniculate body. Retrograde and anterograde staining of the nerve cells processes by DiI and DiO showed fine morphological details of their structure. DiI provided for a good staining of the cells until day 19 of postnatal ontogenesis and DiO, until the end of embryogenesis, while DiA was not capable of diffusion in the fixed tissue.     

Neurons with different neurotransmitters in embryonic neocortical allografts in the rat sciatic nerve

Different subsets of interneurons in the Wistar rat neocortex and in neocortical transplants developing in a damaged nerve were identified by the following immunohistochemical markers: glutamate decarboxylase (GAD 67) for GABAergic nerve cells, NO-synthase (NOS) for NO-ergic neurons, choline acetyltransferase (ChAT) for cholinergic cells, and tyrosine hydroxylase for catecholaminergic structures. Twentyeight days after surgery, individual GAD 67-ir, NO-ir, ChAT-ir, and very rarely TH-ir cells were detected in the graft. It was shown that the number of GAD 67-ir neurons per unit area in the grafts was less than in the rat neocortex P20.     

An in vitro model of human neocortical development using pluripotent stem cells: cocaine-induced cytoarchitectural alterations

Neocortical development involves ordered specification of forebrain cortical progenitors to various neuronal subtypes, ultimately forming the layered cortical structure. Modeling of this process using human pluripotent stem cells (hPSCs) would enable mechanistic studies of human neocortical development, while providing new avenues for exploration of developmental neocortical abnormalities. Here, we show that preserving hPSCs aggregates – allowing embryoid body formation – while adding basic fibroblast growth factor (bFGF) during neuroepithelial development generates neural rosettes showing dorsal forebrain identity, including Mash1⁺ dorsal telencephalic GABAergic progenitors. Structures that mirrored the organization of the cerebral cortex formed after rosettes were seeded and cultured for 3 weeks in the presence of FGF18, BDNF and NT3. Neurons migrated along radial glia scaffolding, with deep-layer CTIP2⁺ cortical neurons appearing after 1 week and upper-layer SATB2⁺ cortical neurons forming during the second and third weeks. At the end of differentiation, these structures contained both glutamatergic and GABAergic neurons, with glutamatergic neurons being most abundant. Thus, this differentiation protocol generated an hPSC-based model that exhibits temporal patterning and a neuronal subtype ratio similar to that of the developing human neocortex. This model was used to examine the effects of cocaine during neocorticogenesis. Cocaine caused premature neuronal differentiation and enhanced neurogenesis of various cortical neuronal subtypes. These cocaine-induced changes were inhibited by the cytochrome P450 inhibitor cimetidine. This in vitro model enables mechanistic studies of neocorticogenesis, and can be used to examine the mechanisms through which cocaine alters the development of the human neocortex.KEY WORDS: Neocortical development, Dorsal forebrain model, hPSCs, Cocaine, Premature neuronal differentiation     

Electrophysiological research into afferent connections of embryonic neocortex allotransplants grafted into the association cortex of adult rats

Grafts of the rat fetal neocortex at the 17–18th day of gestation were placed in the cavity made by aspiration in the primary visual or somatosensory cortex of adult rats. Findings from electrophysiological research performed 3–3.5 months after this transplant showed that neurons of this transplant responded to sensory stimulation specific to the cortical regions replaced by the transplant in 50% of animals. This response was evoked by stimulating local receptive fields displaying a topical organization pattern in a proportion of the animals. Neuronal response in the transplant indicated that the usual field of vision previously existing on the replaced portions of visual cortex had been restored. Electrical stimulation applied locally to a number of brain structures showed that the transplants received afferent inputs from the thalamic nucleus normally projecting to the cortical region replaced by the graft, as well as from homotopic sites on the contralateral cortex. Latencies and time course of neuronal response to stimulating these regions of the host brain resemble those observed in the normal. Afferent inputs from the host brain to cortical transplants thus emulate normal cortical input. Possible mechanisms underlying reinnervation of the grafts are discussed.N. I. Vavilov Institute of General Genetics, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 20, No. 4, July–August, 1988, pp. 448–456.     

The development of peptide-containing neurons within neocortical transplants in adult mice

Transplantation of embryonic neocortex into adult host neocortex leads to the survival of many donor cells, with the subsequent differentiation of the cortical neurons within a loosely laminated cellular pattern. We wanted to know whether peptide-containing neurons that are known to exist in normal neocortex would survive in the transplants, and if so, whether they would differentiate into morphological cell types that normally contain these peptides in cortex. By 30 days after transplantation, the implants were well vascularized and the donor neurons appeared healthy in Nissl-stained preparations. AChE-positive axons grew across the interface and innervated the transplant in moderate densities. Immunocytochemical localization of peptides in the transplant revealed that processes containing the four peptides normally present in cortex also develop in the transplants. These were vasoactive intestinal polypeptide, cholecystokinin, pancreatic polypeptide and somatostatin. Other peptides not yet demonstrated in and presumably not present in neocortex, did not develop in the transplants. These included alpha-melanocyte stimulating hormone, arginine-vasopressin, corticotropin releasing factor, beta-endorphin and substance P. The results demonstrate that peptide-immunoreactive neurons survive in neural transplants, where they develop complicated patterns of axonal arborization. The conditions used in these experiments produced no evidence that peptidergic neurons within the transplant grow out of the transplant and into the host brain within six weeks. Similarly, host peptidergic axons were never seen crossing the interface zone and entering the transplant in any significant numbers.     

Quantitative morphologic studies on the myocardium in early stages of ontogenesis and after application of various cardioactive agents

The authors have morphometrically studied the differentiation of the myocardium in dynamic phases of the embryonic and postnatal development in chickens and Syrian Hamsters. Moreover, they investigated the action of the beta-adrenalytic substances Practolol and Trimepranol on ultrastructure of the cardiac muscle in adult animals. The volume of mitochondria in myocardial cells in 6-day old chicken embryos amounts to 5.65% of the total cell volume, in 12-day old embryos 14.35%, in 18-day old embryos 19.60%, in 1-day old chickens 23.24% which is nearly as much as in adult animals. The volume of myofibrils in 6-day old embryos is about 3.2%, in 12-day old embryos about 7.4%, in 18-day old embryos about 16.4% and in 1-day old chickens about 21.2%. The differences between individual groups are statistically significant. The dynamics of differentiation of the myocardium in Syrian Hamsters was studied in 5 phases, namely in 14-day old embryos and in postnatal phases on the 2nd, 5th, 14th and 21st days after birth. Most cells in 14-day old embryos are rather immature. Participation of the volume of mitochondria, myofibrils, equipment of mitochondria with cristae etc. considerably increase in postnatal phases. These findings suggest that the heart of mammals is rather immature at birth and will differentiate mainly in the postnatal developmental phases. Many morphometric findings, as regards the action of beta-adrenalytic drugs on the ultrastructure of the myocardium in adult rabbits, point to the fact that application of these substances will give rise to degenerative alterations in approximately 10% of myocardial cells. Theoretical explantation of these mechanisms is being discussed.     

Synapses of the human brain in early prenatal ontogeny

Synaptogenesis in presumptive cerebral cortex was studied in 7-8 week human embryos by electron microscopy. The first synapses in the regions studied appeared in 7-week embryos. These synapses were detected only in the marginal zone of the developing human cerebral cortex, where they were localized on the somas and processes of the young Cajal-Retzius' neurons. A functional role of early embryonic synaptogenesis in the developing human cerebral cortex is suggested.     

Expression of alpha2A adrenoceptors during rat neocortical development

Norepinephrine has been suggested to play a neurotrophic role during development and is present in the brain as early as embryonic day (E) 12. We have recently demonstrated that the alpha2A adrenoceptor subtype is widely expressed during times of neuronal migration and differentiation throughout the developing brain. Here, we report the temporal and spatial expression pattern of alpha2A adrenoceptors in neocortex during late embryonic and early postnatal development using in situ hybridization and receptor autoradiography. Functional alpha2 receptors in embryonic rat cortex were also detected using agonist stimulated [35S]GTPgammaS autoradiography. Both alpha2A mRNA and protein expression were strongly increased by E19 and E20, respectively. The increased expression was in the cortical plate and intermediate and subventricular zones, corresponding to tiers of migrating and differentiating neurons. This transient up-regulation of alpha2A adrenoceptors was restricted to the lateral neocortex. At E20, functional alpha2 adrenoceptors were also detected in deep layers of lateral neocortex. During the first week of postnatal development, the expression of alpha2A mRNA and protein changed markedly, giving rise to a more mature pattern of anatomical distribution. The temporal and spatial distribution of alpha2A adrenoceptors in developing neocortex is consistent with expression of functional proteins on migrating and differentiating layer IV to II neurons. These findings suggest that alpha2A receptors may mediate a neurotrophic effect of norepinephrine during fetal cortical development. The early delineation of the lateral neocortex, which will develop into somatosensory and auditory cortices, suggests an intrinsic regulation of alpha2A mRNA expression.     

Distribution of neuronal nitric oxide synthase-immunoreactive neurons in the cerebral cortex and hippocampus during postnatal development

Although many reports have argued a role for nitric oxide (NO) during postnatal development, there has been no combined demonstration in the cerebral cortex and hippocampus. We have investigated the distribution and morphology of neurons and fibers expressing neuronal NO synthase (nNOS) in the cerebral cortex and hippocampal formation of rats during the postnatal development, and correlated these findings with developmental events taking place in these regions. In the cerebral cortex, the nNOS-immunoreactive cells could be divided into two classes : heavily stained neurons and lightly stained neurons. For the lightly stained nNOS-positive neurons, only the cell bodies were observed, whereas for the heavily stained neurons, the cell bodies and their dendrites were visible. During the postnatal days, heavily stained neurons reached their typical morphology in the second week and appeared in all layers except for layer I. In the hippocampus, there was a transient expression of nNOS in the pyramidal cell layer at P3â€P7, and this expression disappeared during following days. The adult pattern of staining developed gradually during the postnatal period. This study suggested that these alterations might reflect a region-specific role of NO and a potential developmental role in the postnatal cerebral cortex and hippocampus     

Behavior and Differentiation of the Neural Stem Cells in vivo

We studied the behavior and differentiation of human and rat neural stem cells after transplantation in the adult rat brain without immunosuppression. The rat stem cells were isolated from the presumptive neocortex of 15-day-old embryos. The human cells were isolated from the ventricular brain zone of 9-week-old embryos and cultivated for two weeks before transplantation. The results of histomorphological studies suggest that the microenvironment factors did not suppress the growth or development of transplanted stem cells. Both rat and human embryonic multipotent neural cells showed similar behavior and differentiation into neurons and glial cells. After transplantation, they continued to mitotically divide and migrated from the graft area to the surrounding tissue of a recipient brain. The presumptive glial cells migrated preferentially along the capillaries and fibrous structures of the recipient brain. Similar behavior of the rat and human neural stem cells in the microenvironment of the recipient adult rat brain and the absence of immune reaction suggest that the transplantation into the rat brain may serve as a model for studying the developmental biology of the human stem cells.     

Disturbances in Formation of the New and Old Cortex at Changes of Conditions of Embryonic Development

Using a model of acute hypoxia during pregnancy of rats, changes in the development of old (hippocampus) and new (sensorimotor) cortex associated with disturbance of neuronogenesis have been revealed in the studied brain structures at the period of action of a pathological factor. It was found that in rats submitted to hypoxia at the 13–14th days of embryogenesis, the number of degenerating neurons (including the pyramidal ones) at various levels of chromatolysis increased since the 5th day after birth; the increase was present for the entire first month of postnatal development. In the cortex of rat pups submitted to prenatal hypoxia there were observed deformation of neuronal bodies, vacuoles in the cytoplasm, shrinkage of apical dendrites of pyramidal neurons and delayed development of the structure (time of the appearance of spikes, formation of structural elements and the size of the cells) of the nervous tissue of the brain of the rat pups exposed to prenatal hypoxia. The columnar structure of the cortex was disturbed. In hippocampus, the process of degeneration of neurons started by 2–3 days later than in the cortex; by two weeks of postnatal development a massive degeneration and death of a part of neurons were also revealed. The morphometrical analysis showed a decrease in the number of neurons and their total area in the sensorimotor cortex (the layer V) and an increase in the number of glial elements at the 10–17th days after birth. In the hippocampus a decrease in the area occupied by neurons and in their size was detected in adult animals. The adult rats submitted to prenatal hypoxia were found to have disturbances of memory and learning. A correlation was shown between the disturbances of the conditions of embryonic development and the changes in the ability of learning and storage of new skills in the offspring.     

幼年大鼠视皮层神经元对闪光刺激的反应特性

哺乳动物视觉系统的发育延续到出生后,大鼠出生后 3~5 周是视觉系统发育的关键期 . 在关键期中,视皮层的兴奋性和抑制性突触连接逐渐成熟,形成有效的皮层内回路 . 为了观察发育关键期大鼠视皮层神经元的反应特性与成年大鼠的异同,使用胞外单细胞记录的方法对比研究了幼年和成年大鼠对闪光刺激的视觉反应特性 . 结果显示：与成年大鼠相比较,幼年大鼠视皮层神经元对持续闪光刺激显示出更强的适应性,对光刺激的诱发放电频率更低,而在没有光刺激时的自发放电频率更高,从而导致信噪比更低 . 这一结果表明,幼年大鼠视皮层对连续刺激的反应能力下降,对信号的分辨能力也更弱,其原因可能是兴奋性突触和抑制性突触发育的不同步所致 .     

Gonadal sex differentiation in Alligator mississippiensis,a species with temperature-dependent sex determination

Temperature of egg incubation determines sex in Alligator mississippiensis hatchlings. To define the timing and morphology of sexual differentiation, alligator gonads were examined histologically and ultrastructurally throughout embryogenesis. At the male-producing temperature (33° C), the onset of testis differentiation occurred in most embryos during developmental stages 21–22, when a number of somatic cells in the medulla of the gonad became enlarged, forming presumptive Sertoli cells. Some enlarged somatic cells were also observed at the female-producing temperature (30° C) during gonadogenesis, but they were less widespread than at 33° C. Ovarian differentiation at 30° C began slighlty later, during stage 22–23, and was characterised by proliferation of germs cells in the cortex of the gonad. Testis formation in alligators may depend upon presumptive Sertoli cells differentiating prior to a critical event in embryogenesis, such as germ cell proliferation and meiosis. If follows that ovary formation occurs if this requirement is not met, as at lower incubation temperatures.     

Formation of "caudate spindles" in the rabbit sensomotor cortex during ontogeny

Electrical activity of the sensomotor and visual areas of the neocortex during stimulation of the caudate nucleus was recorded in young rabbits aged 3–60 days and in adults. Single stimulation of the caudate nucleus was found to cause the appearance of characteristic bursts of spindle-like rhythmic activity ("caudate spindles"), described previously in cats and monkeys, in the adult rabbit cortex. The latent period of the caudate spindle was about 200 msec, its duration 1–3 sec, and the frequency of its oscillations of the order of 12 Hz. Caudate spindles were most marked in the sensomotor cortex of the ipsilateral hemisphere. In rabbits under 10 days old caudate spindles were not found even if the intensity of stimulation was increased many times. Starting from the age of 15 days bursts of rhythmic activity resembling caudate spindles, but with lower frequency (about 8 Hz), longer latent period (up to 350 msec), and also with a higher threshold, appeared in the sensomotor cortex. The definitive type of caudate spindles was established toward the end of the first month of postnatal life, corresponding to the time of formation and complication of conditioned-reflex activity in developing animals.Brain Institute, Academy of Medical Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 17, No. 1, pp. 11–15, March, 1985.     

Influence of neural grafting on rat brain with damaged temporal cortex

This study investigated the feasibility and certain aspects of grafting nerve tissue from 15- and 21-day embryo rats into the left temporal rat cortex damaged by a solution (1 µg/1 µliter) of kainic acid (KA). After unilateral damage induced in the temporal cortex by KA, extensive lesions were found in a number of brain structures (the hippocampus, thalamus, etc.) removed from the KA application site; asymmetry between hemispheres was also revealed from the areas of cross-sections of these structures. In the presence of temporal cortex from 21-day embryos grafted into the lesioned area and setting up axonal connections with the host brain, damage to brain structures removed from the lesioned site was either prevented or substantially reduced. Asymmetry between hemispheres as gauged from the area of their cross sections was no longer present in brain with such grafts; moreover, grafts from 15-day embryos transplanted into a cortex lesioned by KA projected out onto the brain surface, growing and compressing the latter. The damaging action of KA on the host brain extended in the presence of these grafts. A viable graft located within the damaged area is thought to inactivate excitatory transmitters accumulating due to KA action, probably fulfilling the function of the damaged cortex in some measure once connections with the host brain have been set up.Institute of Higher Nervous Activity and Neurophysiology, Academy of Sciences of the USSR, Moscow. Translated from Neirofiziologiya, Vol. 22, No. 5, pp. 586–595, October–September, 1990.     

Expression of α2A adrenoceptors during rat neocortical development

Norepinephrine has been suggested to play a neurotrophic role during development and is present in the brain as early as embryonic day (E) 12. We have recently demonstrated that the α_2A adrenoceptor subtype is widely expressed during times of neuronal migration and differentiation throughout the developing brain. Here, we report the temporal and spatial expression pattern of α_2A adrenoceptors in neocortex during late embryonic and early postnatal development using in situ hybridization and receptor autoradiography. Functional α₂ receptors in embryonic rat cortex were also detected using agonist stimulated [³⁵S]GTPγS autoradiography. Both α_2A mRNA and protein expression were strongly increased by E19 and E20, respectively. The increased expression was in the cortical plate and intermediate and subventricular zones, corresponding to tiers of migrating and differentiating neurons. This transient up‐regulation of α_2A adrenoceptors was restricted to the lateral neocortex. At E20, functional α₂ adrenoceptors were also detected in deep layers of lateral neocortex. During the first week of postnatal development, the expression of α_2A mRNA and protein changed markedly, giving rise to a more mature pattern of anatomical distribution. The temporal and spatial distribution of α_2A adrenoceptors in developing neocortex is consistent with expression of functional proteins on migrating and differentiating layer IV to II neurons. These findings suggest that α_2A receptors may mediate a neurotrophic effect of norepinephrine during fetal cortical development. The early delineation of the lateral neocortex, which will develop into somatosensory and auditory cortices, suggests an intrinsic regulation of α_2A mRNA expression. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 259–269, 1999     

The differentiation of the nerve and glial cells of neocortical transplants placed into the brain of adult rats

Embryonal neural tissue of 17-day-old rat embryos was transplanted into the brain of adult Wistar rats to test the differentiation of transplants with reference to the normal cerebral cortex development. The control and the experimental rats were decapitated 2, 5, 7, 10, 15, 20, 25, and 35 days after the transplantation. Differentiation of neural tissue was studied using monoclonal antibodies against neurofilaments as well as by counting the proportion of differentiated neurons. The glial differentiation was studied by immunohistochemical method using monoclonal antibodies against acid glial fibrillar protein and vimentin. The differentiation of neural cells of transplants proved to be synchronous with the normal ones while the differentiation of glial cells accelerates.