Perspectives on developmental neurobiology最新文献

GABA is formed primarily from decarboxylation of glutamate by a family of cytosolic and membrane-bound GAD enzymes. In the adult, GAD-derived GABA sustains the vitality of the central nervous system (CNS), since blockage of GAD rapidly leads to convulsions and death. In plants, cytosolic GAD synthesizes GABA in response to hormones and environmental stress. Since decarboxylation involves protonation, secretion of GABA serves to buffer cytosolic pH in plant cells. Families of GAD and GABAA receptor/Cl- channel transcripts and encoded proteins emerge early and seemingly everywhere during CNS development, with their abundance closely paralleling neurogenesis and peaking before birth. Micromolar GABA acts at receptor/Cl-channels to depolarize progenitor cells in the cortical neuroepithelium; it also elevates their cytosolic Ca2+ (Cac2+) levels. In some way, these effects decrease proliferation. GABA directs the migration of postmitotic neuroblasts at femtomolar concentrations and stimulates their random motility at micromolar concentrations via Ca2+ signaling mechanisms. Activation of GABAA receptors by micromolar GABA may limit motility via membrane depolarization and elevated Cac2+. These results indicate that in vitro GABA can affect embryogenesis of the CNS through effects on cell proliferation and migration. As neurons differentiate postnatally, Cl(-)-dependent depolarization disappears together with GABAergic Cac2+ signals. Physiologically occurring GABAergic signals at Cl-channels exist in tonic and transient forms. Since the former are found on progenitor cells while both are present in postmitotic neurons, mechanisms to generate transients differentiate in the latter. Surprisingly, tonic and transient forms of GABAergic signaling at Cl-channels are rapidly and smoothly interconvertible and seem to be derived from online GABA synthesis in a surface-accessible compartment of the membrane.

Non-NMDA ionotropic receptor channels were longtime thought to be impermeable to calcium. There is now increasing evidence that this is not a general feature. Neural cells bearing non-NMDA receptors permeable to divalent cations can be found not only in the adult CNS, but also at surprisingly early stages of development (well before the onset of synaptogenesis). Since modulation of cytosolic calcium is known to trigger numerous transcription, translation, and post-translation mechanisms, molecules acting at non-NMDA ionotropic receptors may profoundly affect the fate of individual cells, brain regions, and finally the whole nervous system. However, present knowledge of transduction mechanisms and possible roles (ranging from transcription of immediate early genes to regulation of cell survival) is still very fragmentary. These receptors can be activated and modulated by endogenous molecules, but also by exogenous naturally occurring or pharmaceutical substances. If significant amounts of these substances can pass the human placental barrier, their consumption or accidental intake during pregnancy may constitute a risk for the developing embryonic and foetal CNS.

The correct establishment and function of synapses depend on a variety of factors, such as guidance of pre- and postsynaptic neurons as well as receptor development and localization. gamma-Aminobutyric acid (GABA) has a pronounced effect on these events and elicits differentiation of neurons; that is, GABA acts as a trophic signal. Accordingly, activating preexisting GABA receptors, a trophic GABA signal enhances the growth rate of neuronal processes, facilitates synapse formation, and promotes synthesis of specific proteins. Transcription and de novo synthesis are initiated by the GABA signal, but the intracellular link between GABA receptor activation and DNA transcription is largely unknown. GABA also controls the induction and development of functionally and pharmacologically different GABAA receptor subtypes. The induced receptors are likely to be inserted only into the synaptic membrane domain. However, this ability to target the induced GABAA receptors is probably coupled to the maturation of neurons and not to the action of GABA per se. The induced GABAA receptors apparently mediate a pronounced inhibition of neurotransmitter release, whereas other subtypes of GABAA receptors may be modulatory rather than inhibitory.

Brain contains at least two pools of gamma-aminobutyric acid (GABA), the transmitter pool and the so-called metabolic pool. To a large extent these pools may reflect the presence of GABA in different intracellular compartments, as immunocytochemical studies show that GABA is not localized mainly in terminals but is distributed throughout neurons. An interesting issue is the extent to which the two major forms of glutamate decarboxylase (GAD65 and GAD67) are specialized to synthesize GABA for these pools. Although GAD65 and GAD67 differ significantly in several characteristics, they also have substantial similarities and interactions, and the presence of individual forms of GAD in certain cell types is consistent with the idea that GAD65 and GAD67 can each synthesize GABA for both pools. Substantial progress has been made in understanding the regulatory properties of GAD, but the available data provide little indication of how differences between the forms might enable each to serve the demands for GABA synthesis in a specific pool.

The past decade has seen great progress in understanding the key genes involved in GABAergic transmission. The genes for GAD, multiple subunits of the ionotropic GABA receptors, metabotropic GABA receptors, and GABA uptake proteins have been cloned. Analysis of the cloned genes has yielded a plethora of fundamental insights into the role of the corresponding proteins in mediating GABAergic signals (reviewed in Tobin et al. and Erlander and Tobin). Tools based on these new studies, ranging from monoclonal antibodies to gene probes, have also allowed detailed mapping of expression patterns in the central nervous system (CNS). These new studies reveal that some components of GABAergic transmission have a very wide distribution, being expressed by GABAergic neurons throughout the CNS. Others have a much more restricted pattern of expression. The highly specific expression of GABAergic genes poses a set of fundamental challenges to developmental neurobiology. What genetic mechanisms underlie these patterns of expression? How are complex structures such as receptors assembled? How do the components of a GABAergic synapse come to be localized in proximity to each other so as to make functional transmission possible? Cell lines that express GABAergic phenotypes play an important part in answering these and related questions. With appropriate cell lines it should be possible to manipulate genes related to the GABAergic phenotype in ways that shed light on these questions. Recently, work from several laboratories, including our own, has shown that two pluripotent cell lines from the mouse, the P19 embryonal carcinoma line and embryonic stem (ES) cells, are capable of differentiating into neuron-like cells with GABAergic phenotypes. Since these cell lines are highly suitable for genetic manipulation, they should be extremely useful for studying the relationship between GABA-related genes and the phenotypes they encode.

Intracellular and patch clamp recording techniques were used to investigate the role of GABA in immature CA3 hippocampal neurons. During the first postnatal week spontaneous GABA release was detected as spontaneous ongoing synaptic potentials (SPSPs) or giant depolarizing potentials (GDPs). GDPs were generated at regular intervals and regulated by ionotropic glutamate receptors (GluRs), whereas SPSPs occurred randomly and were unaffected by ionotropic GluRs. Both GDPs and SPSPs were positively modulated by metabotropic GluRs through cyclic AMP-dependent protein kinase. Moreover GABA controlled its own release through GABAA and GABAB receptors, probably localized on GABAergic nerve terminals. At this developmental stage, GABA depolarized CA3 pyramidal cells through two distinct classes of chloride-permeable receptors: bicuculline sensitive and insensitive, respectively. The bicuculline-insensitive responses were blocked by picrotoxin in a noncompetitive way. Whole-cell GABA currents, recorded in the presence of bicuculline, had a slower desensitization rate and faster recovery from desensitization. In excised outside-out patches, in the presence of bicuculline, GABA activated single-channel currents with conductances of 14, 22, and 31 pS. These values were similar to those obtained when GABA was applied in the absence of bicuculline. Interestingly, GABA responses obtained in the absence of bicuculline, were sensitive to the blocking effect of zinc, whereas bicuculline-resistant responses were almost unaffected by this divalent cation. Expression of different subunits in native receptors (particularly of the alpha and rho type) may account for the functional differences observed in the present experiments. Activation of bicuculline-insensitive receptors would strengthen and prolong the depolarizing action of GABA, thus favoring the entry of calcium through voltage-dependent calcium channels. This calcium signal may be essential in promoting stabilization of synaptic contacts during a critical period of postnatal development.

This review explores the role of individual opioid receptor types and signal transduction pathways on cell growth and differentiation. The findings reviewed herein provide suggestive evidence that while no single opioid receptor or peptide type exclusively regulates growth, depending on the cell type, the activation of all three (mu, delta, or kappa) opioid receptor types can affect maturation in a cell type-dependent manner. Specific developmental responses are determined primarily by how a particular opioid receptor type is coupled to intracellular signaling effectors. Moreover, the coupling of opioid receptors appears to be developmentally regulated, and these protein-protein interactions change during ontogeny. The diversity of opioid receptor types and intracellular effectors may be a mechanism by which individual cells discriminate among different opioid signals, and may permit diverse opioid signals to be translated into a unique developmental logic in distinct neuronal and glial subpopulations.

The advent of transgenic mouse technology and the harnessing of homologous recombination to eliminate genes in living animals have provided powerful new approaches to investigate some of the fundamental issues in developmental neurobiology. In this chapter we illustrate several examples of the use of these technologies to investigate the murine cerebellum. These range from using inert, marker transgenes to follow the organization strategy of the cerebellum, to eliminating specific cell populations and modifying the function and fate of Purkinje cells. Finally, we provide a perspective on future extensions of these transgenic approaches.

The first cells specified during CNS development of vertebrates and invertebrates are the cells located at the midline of the neuroepithelium. In Drosophila the development of these cells requires inductive signals from the mesoderm. Later in CNS development, the midline cells are in turn influencing the flanking neuroectoderm, contributing to the establishment of dorsoventral positional information. During axonal pattern formation the midline cells are required in guiding commissural growth cones towards and across the midline. The midline consists of only few, easily identifiable neuronal and glial cells per segment. The development of midline glial cells is relatively well understood. Their differentiation appears to be controlled by the concomitant expression of two different sets of transcription factors. Activation of glial differentiation mediated by the ETS transcription factor encoded by pointed (whose activity depends on EGF-receptor signalling) occurs in concert with repression of neuronal differentiation mediated by the Zn-finger transcription factor encoded by tramtrack.

After entering target regions, afferent growth cones grow among putative target cells, stop extending upon meeting target cells, and transit into a synaptic ending. During these events, signals are transmitted to and from target cells to stimulate programs of differentiation. Here we describe three approaches to unraveling mechanisms of these phases of synaptogenesis. First, dye-labeling in the intact cerebellum has revealed the orchestration of afferent ingrowth and contacts with target cells. Second, an in vitro model based on purified granule neurons has shed light on the role of target cells in the arrest of afferent extension. Third, coculture of purified granule neurons (parallel fiber afferents) with purified Purkinje cells has demonstrated facets of afferent regulation of target cell differentiation. These analyses have suggested molecular mechanisms that mediate maturation of afferents and their targets.