Development of NMDA and non-NMDA receptor-mediated excitatory synaptic transmission in geniculocortical and corticocortical connections in organotypic coculture preparations

Development of N-methyl-D-aspartate (NMDA) and non-NMDA receptor-mediated excitatory synaptic transmission was studied in the visual cortex using organotypic slice cocultures. A slice of visual cortex (VC) dissected from newborn rats was cocultured with either a chunk of embryonic lateral geniculate nucleus (LGN) or another VC. During 7-38 days in vitro (DIV), geniculocortical monosynaptic excitatory postsynaptic potentials (EPSPs) were recorded from layer IV neurons in response to stimulation of the LGN in cocultures of the VC with the LGN. Similarly, corticocortical monosynaptic EPSPs were recorded from layers II/III and V/VI neurons in cocultures of two VCs when stimulating the partner VC. The initial slopes of the non-NMDA and NMDA receptor-mediated components of the EPSPs, which were dissociated pharmacologically, were assessed and compared among three different culture stages, early (7-11 DIV), middle (12-15 DIV) and late (17-38 DIV). With progression of the culture stage, the non-NMDA component tended to increase in both the geniculocortical and corticocortical connections. In contrast, the NMDA component exhibited distinct developmental changes. The NMDA component in layer IV neurons, which receive geniculate inputs, showed a transient increase in the middle stage. In the corticocortical connection, the magnitude of the NMDA component was large in the early stage and maintained through all culture stages in layer V/VI cells, whereas in layer II/III cells it decreased sharply by the late stage. Our results suggest that glutamatergic transmission in the visual cortex develops differently in the geniculocortical and corticocortical connections.     

一种改进的高密度大鼠皮层神经元培养方法

目的建立一种便于量化操作且纯度较高的大鼠皮层神经元培养方法,并观察其生长的形态学变化规律。方法采用精确控制胎龄的SD大鼠胚胎,在低温磷酸盐缓冲液（PBS）中彻底剥离脑膜组织后取双侧皮层,充分剪碎消化,以600～700细胞/mm^2的密度接种于预处理过的固相培养载体上,并对不同培养时间的神经元进行形态学观察和纯度分析。结果利用本方法成功培养出含杂质量较少且纯度很高的高密度皮层神经元,其在不同生长阶段具有典型的形态学特征。结论该方法可以顺利的培养出纯度很高的高密度生长的皮层神经元,便于量化控制操作步骤,减少操作误差,为今后相关研究奠定了方法学基础。     

Early regionalisation of the neocortex and the medial ganglionic eminence

The two major functional classes of neurons that build the cerebral cortex are generated in two distinct parts of the telencephalon. Excitatory long distance projecting neurons are produced dorsally in the pallium, whereas local inhibitory interneurons are mainly produced in the medial ridge of the ventral telencephalon. These two parts of the telencephalon are molecularly regionalized from early embryonic stages, but cellular indices of regionalisation are observed only at later stages of development. We have looked for cellular indices of regionalisation in the cortical anlage at early embryonic stages, when the first efferent cortical neurons are generated. Similarly, we have looked for functional regionalisation of the medial ganglionic eminence at the same stages, when the future cortical interneurones are generated. Here, we summarize data showing that two regions in the mouse cortex embryo, the lateral and dorsal cortex, differ strongly in their early neurogenesis. Moreover, the two domains differ in their capacity to produce GABAergic neurons in vitro; this capacity is only observed in the dorsal cortex. The differentiation of the two domains appears to be independent of the laterorostral to mediocaudal gradient of maturation of the cortex. In the basal telencephalon too, the capacity to differentiate GABAergic neurons is not uniformly distributed across the medial ganglionic eminence. The neurogenesis of future cortical interneurons is seen to be highly active in a small area located in the rostral MGE, at mid dorso-ventral level.     

Development of binding sites for excitatory amino acids in cultured cerebral cortex neurons

Binding of [3H]glutamate, [3H]AMPA (RS-alpha-amino-3-hydroxy-5-methyl-4-isoxazolo-propionate) and [3H]kainate was investigated in membranes prepared from cerebral cortex of 4-day-old and adult mice and from cerebral cortex neurons cultured for different periods of time (2, 4, 8 and 14 days). For all ligands, the number of binding sites increased as a function of development both in vivo and in culture. A significant number of binding sites for the ligands could be demonstrated on the cultured neurons already after 2 days in culture. Scatchard analysis of the binding data showed a single population of binding sites for glutamate (KD approximately 200 nM) and kainate (approximately 6 nM) regardless of the developmental stage in vivo or in culture. In case of [3H] AMPA binding two binding sites with KD values of approximately 6 nM and 100-200 nM could be demonstrated both in vivo and in culture. Binding of [3H]glutamate to cultured neurons could be displaced by N-methyl-D-aspartate (100 microM) and quisqualate (3 microM) in an additive manner but D,L-4-aminophosphonobutyrate (100 microM) had no effect. AMPA binding to cultured neurons was much more (40-fold) sensitive than kainate binding to the newly developed AMPA selective antagonist NBQX (2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline) indicating that kainate and AMPA bind to independent binding sites. Monitoring membrane potentials in the cultured neurons using the lipophilic cation TPP+ (tetraphenylphosphonium) it was demonstrated that potassium (55 mM) as well as glutamate, AMPA and kainate (100 microM) could depolarize the neurons both at early (2 days) and late (9 days) developmental stages in culture. The demonstration of functionally active receptors for the 3 excitatory amino acids in both immature (2 days in culture) and mature (8-9 days in culture) neurons is discussed in the light of previous studies of the development as a function of the culture period of effects of excitatory amino acids in neurons. It is concluded that no simple correlation exists between expression of binding sites for the excitatory amino acids and their ability to induce cytotoxicity and neurotransmitter release.     

Neurons in monkey visual cortex detect lines defined by coherent motion of dots

Form perception from coherent motion is an important aspect of vision. Representations of one-, two- and three-dimensional forms have been found at various stages of cortical processing using random-dot stimuli, whereas representations of biological objects like a walking human being concentrate at higher stages of processing. The perception of biological objects can be induced by sparse dot stimuli that consist of a few dots that mark the joints of the human body [G. Johansson (1973) Percept. Psychophys., 14, 201-211]. In the present study, we aimed to investigate whether neurons in early visual areas that respond to bars and edges defined by luminance contrast also signal bar-like objects from sparse dot stimuli. We studied single neurons with rows of 3-24 dots that were either collinear or scattered within a rectangular form. These dots were moved coherently on a uniform or dotted background, and human observers perceived them as rigid rods or other bar-like objects. We found neurons in the visual cortex of the awake, behaving monkey that responded to these stimuli and were sensitive to the orientation of these objects as for conventional bars or edges. Stimulus conditions that failed to induce these percepts in human observers also evoked weaker responses or none in these neurons. We found these neurons with increasing frequency in areas V1, V2 and V3/V3A. The results suggest that the visual cortex not only detects biological objects, but also lines and other bar-like objects from sparse dot stimuli, and that this function evolves at an early stage of processing.     

Appearance of putative amino acid neurotransmitters during differentiation of neurons in embryonic turtle cerebral cortex.

Pyramidal and nonpyramidal neurons can be recognized early in the development of the cerebral cortex in both reptiles and mammals, and the neurotransmitters likely utilized by these cells, glutamate and gamma-aminobutyric acid, or GABA, have been suggested to play critical developmental roles. Information concerning the timing and topography of neurotransmitter synthesis by specific classes of cortical neurons is important for understanding developmental roles of neurotransmitters and for identifying potential zones of neurotransmitter action in the developing brain. We therefore analyzed the appearance of GABA and glutamate in the cerebral cortex of embryonic turtles using polyclonal antisera raised against GABA and glutamate. Neuronal subtypes become immunoreactive for the putative amino acid neurotransmitters GABA and glutamate early in the embryonic development of turtle cerebral cortex, with nonpyramidal cells immunoreactive for GABA and pyramidal cells immunoreactive for glutamate. The results of controls strongly suggest that the immunocytochemical staining in tissue sections by the GABA and glutamate antisera corresponds to fixed endogenous GABA and glutamate. Horizontally oriented cells in the early marginal zone (stages 15-16) that are GABA-immunoreactive (GABA-IR) resemble nonpyramidal cells in morphology and distribution. GABA-IR neurons exhibit increasingly diverse morphologies and become distributed in all cortical layers as the cortex matures. Glutamate-immunoreactive (Glu-IR) cells dominate the cellular layer throughout development and are also common in the subcellular layer at early stages, a distribution like that of pyramidal neurons and distinct from that of GABA-IR nonpyramidal cells. The early organization of embryonic turtle cortex in reptiles resembles that of embryonic mammalian cortex, and the immunocytochemical results underline several shared as well as distinguishing features. Early GABA-IR nonpyramidal cells flank the developing cortical plate, composed primarily of pyramidal cells, shown here to be Glu-IR. The earliest GABA-IR cells in turtles likely correspond to Cajal-Retzius cells, a ubiquitous and precocious cell type in vertebrate cortex. Glutamate-IR projection neurons in vertebrates may also be related. The distinctly different topographies of GABA and glutamate containing cells in reptiles and mammals indicate that even if the basic amino acid transmitter-containing cell types are conserved in higher vertebrates, the local interactions mediated by these transmitters may differ. The potential role of GABA and glutamate in nonsynaptic interactions early in cortical development is reinforced by the precocious expression of these neurotransmitters in turtles, well before they are required for synaptic transmission.(ABSTRACT TRUNCATED AT 400 WORDS)     

The role of serotonin in early cortical development

The cerebral cortex is widely innervated by serotonin (5-HT)-containing axons originating from neurons in the raphe nuclei. The early development of this monoamine system in the cortex prompted speculation long ago that it has important functions in cortical maturation and plasticity. Here we review evidence, derived from a plethora of studies and from our recent unpublished work, that supports an important role for 5-HT in a number of major events in the developing cortex, especially at the early stages. This evidence points to a regulatory role for 5-HT in neuronal proliferation, migration and differentiation, and in preventing apoptotic cell death.     

In utero cocaine-induced dysfunction of dopamine D1 receptor signaling and abnormal differentiation of cerebral cortical neurons.

Monoamines modulate neuronal differentiation, and alteration of monoamine neurotransmission during development produces specific changes in neuronal structure, function, and pattern formation. We have previously observed that prenatal exposure to cocaine in a clinically relevant animal model produces increased length of pyramidal neuron dendrites in the anterior cingulate cortex (ACC) postnatally. We now report that cocaine administered intravenously to pregnant rabbits at gestational stages preceding and during cortical histogenesis results in the early onset of hypertrophic dendritic outgrowth in the embryonic ACC. Confocal microscopy of DiI-labeled neurons revealed that the atypical, tortuous dendritic profiles seen postnatally in ACC-cocaine neurons already are apparent in utero. No defects in neuronal growth were observed in visual cortex (VC), a region lacking prominent dopamine innervation. In striking correlation with our in vivo results, in vitro experiments revealed a significant enhancement of spontaneous process outgrowth of ACC neurons isolated from cocaine-exposed fetuses but no changes in neurons derived from visual cortex. The onset of modified growth in vivo is paralleled by reduced D(1A) receptor coupling to its G-protein. These data suggest that the dynamic growth of neurons can be regulated by early neurotransmitter signaling in a selective fashion. Prenatal onset of defects in dopamine receptor signaling contributes to abnormal circuit formation and may underlie specific cognitive and behavioral dysfunction.     

Method for serum-free culture of late fetal and early postnatal rat brainstem neurons

Primary culture of postnatal brainstem neurons in defined medium has not been described in the literature. Successful primary culture of brainstem neurons is typically restricted to embryonic ages E14-E18. This study describes a method for culture of late fetal and early postnatal brainstem neurons using a serum-free culture medium. The culture system is based on Neurobasal medium supplemented with antioxidant-rich B27 (Life Technologies). Neuron survival was optimized by replacing glutamine with GlutaMaxI, by matching osmolality with neuronal age, and by using Hibernate medium to increase neuron survival during tissue dissociation. This paper describes the first reliable method for culturing brainstem neurons from late fetal and early postnatal stages of the rat for up to 6 days postpartum.     

Development of inhibitory circuitry in visual and auditory cortex of postnatal ferrets: immunocytochemical localization of GABAergic neurons.

The goal of this study was to describe the development of gamma-aminobutyric acid (GABA)-containing neurons in visual and auditory cortex of ferrets. The laminar and tangential distribution of neurons containing excitatory, inhibitory, and neuromodulatory substances constrain the potential circuits which can form during development. Ferrets are born at an early stage of brain development, allowing examination of inhibitory circuit formation in cerebral cortex prior to thalamocortical ingrowth and cortical plate differentiation. Immunocytochemically labelled nonpyramidal GABA neurons were present from postnatal day 1 throughout development, in all cortical layers, and generally followed the inside-out pattern of neuronal migration into the cortical plate. Prior to postnatal day 14, pyramidal neurons with transient GABA immunoreactivity were also observed. The density of Nissl-stained and GABA-immunoreactive neurons was high early in development, declined markedly by postnatal day 20, then remained relatively constant until adulthood. However, examination of the proportion of GABA neurons revealed an unexpected late peak at postnatal day 60, then a decrease in adulthood. Visual and auditory cortex were similar in most respects, but the peak at postnatal day 60 and the final proportion of GABA neurons was higher in auditory cortex. The late peak suggests that inhibitory circuitry is stabilized relatively late in sensory cortical development, and thus that GABA neurons could provide an important substrate for experience-dependent plasticity at late stages of development.     

Light-microscopic immunocytochemical localization of tyrosine hydroxylase in prenatal rat brain. II. Late ontogeny

The immunocytochemical localization of tyrosine hydroxylase is examined at embryonic (E) days 18 and 21 in rat brain in order to determine changes in the distribution and cytology of neurons showing immunoreactivity for the enzyme during late prenatal development. As compared with earlier stages of development, the distribution and morphology of the tyrosine hydroxylase-containing neurons at E18 and E21 more closely resemble catecholaminergic neurons in the adult brain. The changes occurring from the early to the late prenatal stages of development appear to be the result of an increase in number of cells and continued aggregation and migration of the labeled neurons. The major differences in the distribution of labeled perikarya between E18 and E21 are in the olfactory bulb and cerebral cortex. In the olfactory bulb, tyrosine hydroxylase-containing neurons are not detected until E21. In contrast in the cerebral cortex, a few neurons are transiently labeled for the enzyme at E18, but are not detected at E21 and have not been reported in the adult brain. The most striking change in the tyrosine-hydroxylase labeled structures in the late prenatal period is the increase in detectable immunoreactivity in bundles of axons and in terminal aborizations. The orderly appearance of tyrosine hydroxylase-labeled axons in the neostriatum and cortex are discussed in relation to the formation of these two contrasting regions innervated by catecholaminergic neurons.     

Culture of neuronal cells from postnatal rat brain: application to the study of neurotrophic factors.

1. The authors developed a primary culture technique for neuronal cells from postnatal rat brains and studied the effects of neurotrophic factors on the naturally developed neurons. 2. We demonstrated changes in the neurotrophic role of nerve growth factor (NGF) during the developmental stages of the rat: NGF was shown to act as a differentiation factor in the early stages and as a survival factor later. 3. It appeared that interleukin-6 (IL-6) supported the survival of septal cholinergic neurons obtained from 10-day-old rats. IL-6, however, did not induce the differentiation of embryonic rat septal cholinergic neurons. IL-6 improved the survival of mesencephalic catecholaminergic neurons from postnatal and embryonic rat brains, which have known not to be response to NGF.     

Neurite outgrowth patterns in cerebellar microexplant cultures are affected by antibodies to the cell surface glycoprotein L1

To probe for the role of the L1 cell surface glycoprotein during neurite outgrowth and fasciculation in the early postnatal mouse cerebellar cortex, a microexplant culture system was used. Fasciculation of neurites was reduced in the presence of antigen-binding fragments (Fab) of poly- and monoclonal L1 antibodies, as compared to untreated controls. In addition, speed of neurite outgrowth was enhanced in the presence of antibodies. Migration of cell bodies of small neurons was also significantly increased. Very similar effects on these outgrowth parameters were observed with Fab fragments from poly- and monoclonal neural cell adhesion molecule (N-CAM) antibodies. Antibodies from preimmune sera had no effect. These findings suggest that L1 antigen not only plays a role in adhesion of isolated neural cell bodies and migration of granule cell neurons in the early postnatal mouse cerebellar cortex (Lindner et al., 1983; Rathjen and Schachner, 1984), but also in neurite outgrowth and fasciculation.     

Susceptibility to apoptosis varies with time in culture for murine neurons and astrocytes: changes in gene expression and activity

Apoptotic pathways in the brain may differ depending on cell type and developmental stage. To understand these differences, we studied several apoptotic proteins in the murine cortex and primary cultures of neurons and astrocytes of various ages in culture. We then induced apoptosis in our cultures using serum deprivation (SD) and observed changes in these apoptotic proteins. When analyzed by nuclear morphology and TUNEL staining, early cultures showed greater apoptotic injury compared with late cultures, and neuronal cultures showed greater apoptosis than astrocyte cultures. The decrease in apoptosis with development correlated best with a down-regulation of procaspase-3 and bax and decreasing caspase activation. Early culture astrocytes had higher caspase-11 levels compared with neurons. Mitogen-activated protein (MAP) kinases were also differentially expressed with activation of extracellular signal-regulated kinase (ERK) and p38 higher in early culture astrocytes and stress-activated protein kinase/C-jun N-terminal kinase (SAPK/JNK) greater in early culture neurons. However, caspase inhibitors, but not MAP kinase inhibitors reduced cell death. Our findings demonstrate that apoptosis regulatory proteins display cell type and developmentally specific expression and activation.     

Serum-free culture of rat post-natal and fetal brainstem neurons

Serum-free medium is essential for cell culture studies in which complete control of the environment is required. Primary culture of post-natal brainstem neurons in defined medium has not been described in the literature, and successful culture of primary brainstem neurons is typically restricted to embryonic ages E14-E18. This study describes a method for culture of fetal and post-natal brainstem neurons using a serum-free culture medium. The culture system is based on Neurobasal medium supplemented with antioxidant-rich B27. Media and supplements are commercially available products from Life Technologies. Neuron survival was optimized by replacing glutamine with GlutaMaxI, by matching osmolality with neuronal age, and by using Hibernate medium to increase neuron survival during tissue dissociation. Fetal E14, E16, E20, and post-natal P3 and P6 cultures were examined after 4, 7, and 9 days in culture. Neuron and glial cells present in the cultures were identified using immunocytochemistry with antibodies raised against microtubule-associated protein 2 (MAP2) and glial fibrillary acidic protein (GFAP), respectively. Fetal E14 cultures had more bipolar neurons than multipolar neurons compared with developmentally older P6 cultures. Early fetal cultures had a higher percentage of neurons than late fetal and early post-natal cultures. Neuron survival was similar between 4 and 9 days in culture for all age groups tested. This is the first reliable, defined culture medium that supports brainstem neurons from late fetal and early post-natal stages of the rat for up to 6 days post-partum.     

Survival and differentiation of dopaminergic neurons can be regulated by soluble factors from cortex in vitro

The transplantation of dopaminergic (DA) neurons is used for treating Parkinson's disease. However, their actual application is restricted by a limited source of DA cells. Here we report that DA cells can be increased 5- to 10-fold in vitro by the soluble factors from cortex in early developmental stages, which is much more than any previously identified growth factors such as BDNF, GDNF and NT3. We also show that the effect of the soluble factors from cortex is stronger than those of midbrain at embryonic early developmental ages. In contrast, at middle ages the soluble factors from midbrain present a much stronger effect. These findings suggest that the development of DA cells may be regulated by growth factors in a complex spatial and temporal network.     

Development of inhibitory circuitry in visual and auditory cortex of postnatal ferrets: immunocytochemical localization of calbindin- and parvalbumin-containing neurons

The inhibitory neurotransmitter gamma-aminobutyric acid (GABA) is thought to play an important role in activity-dependent stages of brain development. Previous studies have shown that different functional subclasses of cortical GABA-containing neurons can be distinguished by antibodies to the calcium-binding proteins parvalbumin and calbindin. Thus insight into the development of distinct subsets of inhibitory cortical circuits can be gained by studying the development of these calcium-binding protein-containing neurons. Previous studies in several mammalian species have suggested that calcium-binding proteins are upregulated in sensory cortex when thalamocortical afferents arrive. In ferrets, the ingrowth of thalamic axons into cortex occurs well into postnatal development, allowing access to early stages of cortical development and calcium-binding protein expression. We find in ferrets that both parvalbumin- and calbindin-immunoreactivity are present in primary visual and primary auditory cortex long before thalamocortical synapse formation, but that there is a sharp decline in immunoreactivity by postnatal day 20. Day 20 in ferrets corresponds to postnatal day 1 in cats, and thus previous studies in postnatal cats would have missed this early pattern of calcium-binding protein distribution. Another surprising finding is that the proportion of parvalbumin- and calbindin-immunoreactive neurons peaks secondarily late in development, between P60 and adulthood. This result suggests that the parvalbumin- and calbindin-containing subclasses of nonpyramidal neurons remain immature until late in the critical period for cortical plasticity, and that they are positioned to play an important role in experience-dependent modification of cortical circuits.     

The comparative characteristics of the neuronal responses of the rat somatic cortex during active and passive movements of the vibrissae

Responses of rat barrel-field cortex neurons were recorded during vibrissae deflection, voluntary movements of whiskers and vibrissae movements produced by stimulation of the motor cortex and facial muscles. Changes in the patterns of responses of the three groups of neurons were revealed during the performance of active vibrissae movements: neurons with short-latency responses, neurons with long-latency responses and neurons with changes in the activity occurred early before the movements onset. It is suggested that somatosensory cortical neurons receive peripheral (from the whiskers' follicles and facial muscles) as well as central (from the motor cortex) inputs that modulate their activity.