The outgrowth of neurites by single identified leech neurons in culture is markedly influenced by the substrate, Extensive sprouting occurs within a few hours on the plant lectin Con A. In contrast, the same neurons grow far more slowly or not at all when plated on vertebrate extracellular matrix proteins, other lectins, or poly(L-lysine). Sprouting on Con A, unlike that on poly(L-lysine), is inhibited by the Con A-specific hapten sugar methyl a-D-mannoside. Another substrate, promoting even more extensive sprouting of leech neurons, is cell-free extraceflular matrix obtained from leech ganglion capsules. Urea extracts of extracellular matrix retain full neurite-promoting activity when dialyzed and used to coat culture dishes. Soluble growth factors are not required, since sprouting occurs in medium without macromolecules. These results show that sprouting depends not simply on attachment to the substrate but, critically, on its molecular composition; moreover, the pattern of outgrowth is characteristic and distinguishable for each type of neuron, Single identified neurons with known functions can be isolated from ganglia of the leech central nervous system and grown in culture (1). Such preparations have proved useful for studying the formation and function of electrical and chemical synapses (2). For example, an inhibitory serotonergic synapse develops from the Retzius cell onto the P (pressure-sensitive) cell when two such neurons are plated in close proximity on a poly(L-lysine)-coated culture dish (3-6). Although the cells connected in culture grow processes over each others' surfaces and form synapses, neurite outgrowth on the poly(L-lysine) substrate occurs only slowly and after a delay (3). This absence oflong sprouts on poly(L-lysine), an advantage for analyzing synaptic transmission or synaptic structure (5, 6), does not permit analysis of processes requiring neurite growth.A major aim of our studies has been to develop a culture system suitable for investigating how single identified leech neurons connect with targets over long distances, since developing and regenerating neurons are known to sprout extensively along the connective tissue and glia (7-10). As a first step, we have analyzed the conditions required for target-independent growth. It will be shown that cultured leech neurons grow rapidly and extensively on specific substrates. Thus, the plant lectin Con A (11) and components extracted from leech extracellular matrix (ECM) promote growth, whereas other lectins and vertebrate ECM proteins do not. The outgrowth of neurites can occur in defined, protein-free medium in the absence of other cells. MATERIALS AND METHODSChemicals. Unless stated otherwise, chemicals were of analytical grade and were purchased from Merck (Darmstadt, F.R.G.).Culture of Leech Neurons. Identified single neurons isolated from the ventral nerve cord of the leech Hirudo medicinalis were cultured as described previously (6). Leech ganglia were pinned in a Sylgard coated dish in Leibovitz L-15 medium (GIBCO) supplem...

The cell cycle kinetics of individual clones of cells in the embryonic cortex were studied to determine the amount of heterogeneity among cortical progenitors. This kinetic heterogeneity may be the first sign of heterogeneous differentiation in the proliferating cortical germinal zone. Retroviral lineage tracing of clonal progeny in the embryonic day 16 (E16) rat cortex (48 hr after the retroviral infection and [3H]thymidine labeling of proliferating cells) permitted a description of the formation of the preplate in the medial cortex and the formation of the subventricular zone (SVZ) in the lateral cortex. Forty percent of the retrovirally tagged clones were double‐labeled with a pulse of [3H]thymidine, corresponding to the 40% of ventricular zone cells in S‐phase at the time of [3H]thymidine injection. Most of clones had cell numbers with powers of 2 (2, 4, and 8 cells), suggesting synchronous modes of division. Nevertheless, 15% of the retrovirally tagged clones that were double‐labeled with [3H]thymidine at the time of [3H]thymidine injection showed asynchronous mode of division. Clonally related cells in the ventricular zone showed considerable variability in cell cycle times: 56% of the clones (8‐cell clones) were composed of faster cycling cells with cell cycle times of 12 hr, and 25% of the clones (4‐cell clones) represented slower cycling cells with cell cycle times of 16 hr. The clones migrating outside the ventricular zone differed in size and spatial distribution in the lateral versus medial cortex. In the lateral cortex, half the migrating clones were large proliferating 8‐cell clones with all their members contained within the forming SVZ. In the medial cortex, the majority of the migrating clones were 2‐cell and 4‐cell clones. Given that the medial cortex matures later than the lateral neocortex and that no SVZ has formed in the rat medial cortex by E16, we suggest that the majority of cells that leave the medial cortical VZ by E16 are cells destined to form the neuronal populations of the preplate. The early embryonic cortical ventricular zone includes a mosaic of specialized progenitor cells. Dev. Dyn. 1997;210:328–343. © 1997 Wiley‐Liss, Inc.

A double-labeling technique, combining retroviral tagging of individual cell lines (one clone per brain hemisphere) with the simultaneous [3H]thymidine-labeling of dividing cells in S phase, was used to study proliferation characteristics of individual precursor cell lines in the germinal zone of the developing rat forebrain. The cortical germinal zone was found to be segregated into three spatially distinct horizontal populations of precursor cell lineages, which differed in cell cycle kinetics, amount of cell death, and synchronous versus asynchronous mode of proliferation. The striatal germinal zone demonstrated a similar heterogeneity in the cell cycle characteristics of proliferating clones, but did not show nearly as distinct a spatial segregation of these different populations. The results demonstrate the clonal heterogeneity among precursor populations in the telencephalon and the differential spatial organization of the cortical and the striatal germinal zones. This germinal zone heterogeneity may predict some of the differences found among cellular phenotypes in the adult forebrain.

A study has been made of the electrical connections between touch sensory (T) neurones in the leech central nervous system (CNS) which display remarkable double rectification: depolarization spreads in both directions although hyperpolarization spreads poorly. Tests were made to determine whether this double rectification was a property of the junctions themselves or whether it resulted from changes in the length constants of processes intervening between the cell body and the junctions. Following trains of action potentials, T cells and their fine processes within the neuropile became hyperpolarized through the activity of an electrogenie sodium pump. When any T cell was hyperpolarized by 25 mV by repetitive stimulation, hyperpolarization failed to spread to the T cells to which it was electrically coupled. Further evidence for double rectification of junctions linking T cells was provided by experiments in which Cl− was injected electrophoretically. Cl− injection into one T cell caused inhibitory potentials recorded in it to become reversed. After a delay, Cl− spread to reverse IPSPs in the coupled T cell. Movement of Cl−, like current flow, was dependent on membrane potential. When the T cell into which Cl− was injected was kept hyperpolarized, Cl− failed to move into the adjacent T cell. Upon release of the hyperpolarization in the injected T cell, Cl− moved and reversed IPSPs in the coupled T cell. Together these results indicate that the gating properties of channels linking T cells are voltage-dependent, such that depolarization of either cell allows channels to open whereas hyperpolarization causes them to close.