Leech Neurons In Culture Research Articles

Real-Time Measurements of Synaptic Autoinhibition Produced by Serotonin Release in Cultured Leech Neurons

We studied autoinhibition produced immediately after synaptic serotonin (5-HT) release in identified leech Retzius neurons, cultured singly or forming synapses onto pressure-sensitive neurons. Cultured Retzius neurons are isopotential, thus allowing accurate recordings of synaptic events using intracellular microelectrodes. The effects of autoinhibition on distant neuropilar presynaptic endings were predicted from model simulations. Following action potentials (APs), cultured neurons produced a slow hyperpolarization with a rise time of 85.4 +/- 5.2 ms and a half-decay time of 252 +/- 17.4 ms. These inhibitory postpotentials were reproduced by the iontophoretic application of 5-HT and became depolarizing after inverting the transmembranal chloride gradient by using microelectrodes filled with potassium chloride. The inhibitory postpotentials were reversibly abolished in the absence of extracellular calcium and absent in reserpine-treated neurons, suggesting an autoinhibition due to 5-HT acting on autoreceptors coupled to chloride channels. The autoinhibitory responses increased the membrane conductance and decreased subsequent excitability. Increasing 5-HT release by stimulating with trains of ten pulses at 10 or 30 Hz produced 23 +/- 6 and 47 +/- 2% of AP failures, respectively. These failures were reversibly abolished by the serotonergic antagonist methysergide (140 muM). Moreover, reserpine-treated neurons had only 5 +/- 4% of failures during trains at 10 Hz. This percentage was increased to 35 +/- 4% by iontophoretic application of 5-HT. Increases in AP failures correlated with smaller postsynaptic currents. Model simulations predicted that the autoinhibitory chloride conductance reduces the amplitude of APs arriving at neuropilar presynaptic endings. Altogether, our results suggest that 5-HT autoinhibits its subsequent release by decreasing the excitability of presynaptic endings within the same neuron.

Extracellular matrix glycoproteins inhibit neurite outgrowth of different types of identified leech neurons in culture

We explored the contribution of inhibitory peanut-binding extracellular matrix glycoproteins to the regeneration of characteristic outgrowth patterns by different types of identified neurons. Adult leech neurons were isolated one by one and plated in culture on a substrate that consisted of the capsules that encase the CNS ganglia. On the inside surface of this substrate, a combination of growth-promoting and -inhibiting extracellular matrix glycoproteins regulates the regeneration of distinctive outgrowth patterns by different neuron types. The role of inhibitory glycoproteins that bind to peanut lectin was studied by perturbation experiments in which peanut lectin was added to the culture medium. The effects of peanut lectin on the outgrowth patterns depended on the specific cell type that was tested. Anterior pagoda neurons, which on capsules produce a bipolar outgrowth pattern, in the presence of the lectin multiplied the number of primary neurites and the total neurite length and also lost their bipolarity. Annulus erector motoneurons, which on capsules grow poorly, in the presence of peanut lectin sprouted 70% more neurites and duplicated their total neurite length. By contrast, Retzius neurons which grow profusely on ganglion capsules, in the presence of peanut lectin increased the number of primary neurites without increasing their total neurite length or branch points. When neurons were plated on plastic, peanut lectin added to the culture medium did not affect the growth of neurons, thus showing that the effects of peanut lectin were induced by blocking the binding of neurons to inhibitory glycoproteins on the capsules. These results show that regeneration of different neuron types has different regulation by inhibitory extracellular matrix molecules.

Extracellular matrix glycoproteins inhibit neurite production by cultured neurons.

We have analyzed the role of extracellular matrix glycoproteins in the formation of a bipolar outgrowth pattern of identified leech neurons in culture. Adult anterior pagoda (AP) neurons cultured on the inner surface of the ganglion capsules that surround central nervous system, generate two processes oriented in opposite directions. This pattern differs from those produced by these neurons cultured on other substrates, and is similar to the pattern of developing AP neurons at embryonic day 10. We used different lectins to identify subsets of glycoproteins in the extracellular matrix (ECM) of the capsules and to study their contribution to the formation of the bipolar outgrowth pattern. ECM glycoproteins binding to peanut agglutinin (PNA) or Galanthus nivalis aglutinin (GNA) lectins were detected in ganglion capsules and in ganglion extracts that had been separated by electrophoresis and blotted to nitrocellulose membranes. Four protein bands bound to PNA lectin and six other bands, including laminin subunits, bound to GNA lectin. Other lectins failed to recognize any of the proteins. For AP neurons cultured on capsules, addition of PNA lectin to the culture medium produced a dose-dependent increase in the number of primary neurites without affecting their shape, length or number of branch points. However, PNA lectin used as substrate did not affect sprouting of AP neurons. Our results suggest that PNA-binding extracellular matrix glycoproteins regulate the formation of the bipolar pattern of AP neurons by inhibiting the formation of neurites.

Steps in the formation of neurites and synapses studied in cultured leech neurons

Leech neurons in culture have provided novel insights into the steps in the formation of neurite outgrowth patterns, target recognition and synapse formation. Identified adult neurons from the central nervous system of the leech can be removed individually and plated in culture under well-controlled conditions, where they retain their characteristic physiological properties, grow neurites and form specific chemical or electrical synapses. Different identified neurons develop distinctive outgrowth patterns that depend on their identities and on the molecular composition of the substrate. On native substrates, the patterns displayed by these neurons reproduce characteristics from the adult or the developing neurons. In addition, the substrate may induce selective directed growth between pairs of neurons that normally make contact in the ganglion. Upon contact, pairs of cultured leech neurons form chemical or electrical synapses, or both types depending on the neuronal identities. Anterograde and retrograde signals during membrane contact and synapse formation modify the distribution of synaptic terminals, calcium currents, and responses to 5-hydroxytryptamine.

Cable Properties of Dendrites in Hippocampal Neurons of the Rat Mapped by a Voltage‐sensitive Dye

Dendrites of pyramidal neurons from embryonic rat hippocampus are investigated in culture using a voltage-sensitive fluorescent dye. The electrical response to somatic stimulation is observed as a time-resolved map with a resolution of 0.9 microm at a time constant of 0.4 ms without signal averaging. The data are interpreted in terms of a tapering cable with Hodgkin-Huxley parametrization. The spread of short hyperpolarizing transients is damped by capacitive shunting. The invasion of an action potential is boosted by voltage-gated conductances of a low density. No irregularity is observed at a bifurcation. The passive cable parameters of internal resistance and membrane resistance at resting voltage are Ri = 300 omega cm and Rm = 40 (k)omega cm2 respectively, at a maximum sodium conductance of approximately 4.4 mS/cm2. The electrotonic length constant and the dynamic length constant at 1 kHz are 580 and 90 microm respectively. These results are compatible with electrophysiological data of dendrites in slices of adult hippocampus and with optical data of narrow processes of leech neurons in culture. The functional implications of boosting an action potential by voltage-gated channels of low density are considered.

Outgrowth patterns and directed growth of identified neurons induced by native substrates in culture.

In this study, we have tested how various identified leech neurons in culture grow on surfaces that they normally contact in situ. Neurons were cultured either on ganglion capsules from which neurons had been removed or on skin. On these substrates, outgrowth patterns were characteristic for each cell type. Retzius cells plated on capsules extended bundles of thick, fasciculated processes with few branching points and in the opposite direction a tangle of fine neurites. Anterior pagoda (AP) neurons plated on capsules extended two single processes in opposite directions but failed to grow on skin. Sensory P and N neurons on capsules extended multiple processes. On skin, P neurons extended only two long branches in opposite directions over the superficial body wall. N neurons on skin extended multiple processes. Varicosities were common in the processes of P and N neurons on capsules or skin. The branching patterns described here bore closer resemblance to those in the developing or adult nervous system than to those on Concanavalin A or laminin-enriched extract. Pairs of Retzius or AP neurons plated at a distance on the same capsule extended neurites from one neuron toward the other and formed contacts. Such directed growth failed in hybrid pairs of Retzius and AP neurons or in pairs plated on laminin-enriched extract or Concanavalin A. Our results suggest that multiple growth-promoting molecules anchored to the extracellular matrix may cooperate in regulating the branching pattern of neurons, fasciculation, and direction of growth.

Different determinants on growth and synapse formation in cultured neurons.

The influence of substrate and target on growth and synapse formation was investigated in identified leech neurons in culture. Nociceptive neurons and annulus erector motoneurons were cultured on ganglion capsules, a substrate they encounter in the leech. Within a few hours, single nociceptive cells sprouted profusely, whereas annulus erector cells failed to grow. When annulus erector neurons were plated near a nociceptive cell on the same capsule both neurons grew, made contact and formed an electrical synapse, different from the chemical synapse they form in the ganglion, but identical to that formed when plated on Concanavalin A. These results suggest that substrate and target have complementary effects regulating growth, but fail to define the type of synapse formed.

Synapse formation and function: insights from identified leech neurons in culture.

Identified leech neurons in culture are providing novel insights to the signals underlying synapse formation and function. Identified neurons from the central nervous system of the leech can be removed individually and plated in culture, where they retain their characteristic physiological properties, grow neurites, and form specific synapses that are directly accessible by a variety of approaches. Synapses between cultured neurons can be chemical or electrical (either rectifying or not) or may not form, depending on the neuronal identities. Furthermore, the characteristics of these synapses depend on the regions of the cells that come into contact. The formation and physiology of synapses between the Retzius cell and its partners have been well characterized. Retzius cells form purely chemical, inhibitory synapses with pressure-sensitive (P) cells where serotonin (5-HT) is the transmitter. Retzius cells synthesize 5-HT, which is stored in vesicles that recycle after 5-HT is secreted on stimulation. The release of 5-HT is quantal, calcium-dependent, and shows activity-dependent facilitation and depression. Anterograde and retrograde signals during synapse formation modify calcium currents, responses to 5-HT, and neurite outgrowth. The nature of these synaptogenic signals is being elucidated. For example, contact specifically with Retzius cells induces a localized selection of transmitter responses in postsynaptic P cells. This effect is signaled by tyrosine phosphorylation prior to synapse formation.

Defined neuronal arborizations by guided outgrowth of leech neurons in culture.

Identified neurons of Hirudo medicinalis were cultivated on a protein extract of the extracellular matrix (ECM) of the leech. Microscopic patterns of active ECM protein were prepared by UV photolithography using aluminium masks. The shape of the patterns was visualized by a colour pattern formed in a dye-polymer substrate. The neurons were explanted on the root of branched ECM patterns. The patterns guided the outgrowth of neurites along linear lanes and they induced a bifurcation of the neurites under certain conditions. Neurons with a reproducible, regular shape of arborization were obtained within 1-2 days.

Anterograde and retrograde effects of synapse formation on calcium currents and neurite outgrowth in cultured leech neurons.

The aim of our experiments has been to analyse how formation of chemical synapses affects the distribution of calcium (Ca2+) currents and neurite outgrowth of leech Retzius cells. Previous results showed that Ca2+ currents measured in the initial process or 'stump' of postsynaptic cells were significantly smaller than those in corresponding sites on presynaptic neurons. In the present experiments, neurons were plated together in close apposition as pairs or as triads, with the tip of one Retzius cell touching the soma of another. Ca2+ currents from selected areas of the neuronal surfaces were measured by loose-patch recording before and after the formation of chemically mediated synaptic connections, which developed in about 8 h. With three cells arranged in a row, the last of the series, which was purely postsynaptic (i.e. with no target), also showed a dramatic reduction in Ca2+ currents in its initial segment, compared with the currents seen in either the first cell (purely presynaptic) or the second cell of the chain (which was both postsynaptic to the first cell and presynaptic to the third). This suggests that retrograde as well as anterograde effects on Ca2+ currents occurred as a result of synapse formation: the Ca2+ currents in the middle cell did not decrease although a synapse had been formed on it. To test for additional consequences of synapse formation, neurite outgrowth was measured in postsynaptic cells and in single cells plated on an extract of extracellular matrix containing laminin (ECM-laminin). After 48 h, the total length of neuritic outgrowth in postsynaptic cells was only about one third of that in single cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Synaptogenesis and calcium current distribution in cultured leech neurons

The distribution of calcium (Ca$^{+}$) currents was studied by loose-patch technique in Retzius cells isolated from the leech central nervous system. Ca$^{+}$ tail currents were recorded in patches held at the equilibrium potential for potassium (E$_{\text{K}}$). Measurements were made from single cells and paired cells that had formed chemical or electrical synapses in culture. In single Retzius cells, Ca$^{+}$ currents in the stump of the initial process were three times larger than in the middle part of the soma; intermediate amplitudes were found in the polar region of the soma. After the formation of chemical synapses, Ca$^{+}$ currents at the stumps of postsynaptic cells and in the polar region of the somas of presynaptic cells were reduced. The formation of electrical synapses did not affect Ca$^{+}$ current distribution.

Guided outgrowth of leech neurons in culture

Sensory neurons of Hirudo medicinalis were cultivated on patterns of extracellular matrix (ECM) protein which were prepared by UV irradiation using copper grid masks. The relation of the patterns and of the outgrowth of neurites was observed by scanning electronmicroscopy after gold decoration. The neurites were guided by narrow (10 μM) lanes of ECM protein. Branching of neurites was induced by branched lanes. Bent neurites were pulled off bent lanes at some distance behind the growth cone such that their length was reduced. Regularly spaced sites of adhesion became visible which remained connected to the neurites by extended filaments.

Intrinsic and extrinsic factors influencing properties and growth patterns of identified leech neurons in culture

An analysis has been made of intrinsic mechanisms influencing growth patterns of 2 identified leech interneurons in tissue culture. These small cells (known as DL and VL) display unusual arborizations in the ganglion. The distinctive branching patterns resemble the letter "T" for VL and the letter "Y" for DL. DL and VL cells contain serotonin and can be identified in situ with Neutral red. 1. DL and VL cells were isolated and cultured on 2 homogeneous substrates, concanavalin A (Con A) and leech extracellular matrix extract (ECM). Individual DL and VL cells in culture retained their ability to fire in spontaneous, rhythmical bursts. The synaptic connections formed in culture were specific and differed from those made by other serotonergic neurons. 2. On both Con A and ECM the neurons sprouted to reproduce their characteristic patterns. Every DL or VL cell that grew processes on ECM developed the appropriate Y- or T-shaped pattern. On Con A, most but not all of the cells showed specific Y or T patterns. The probability of sprouting was higher on Con A than on ECM. 3. Disruption of microtubular assemblies in freshly isolated DL and VL cells by treatment with nocodazole did not affect the later outgrowth of specific patterns in culture. 4. These results provide evidence that adult DL and VL leech neurons retain intrinsic information for determining a variety of their properties in culture: In particular, they reproduce type-specific neuritic patterns in the absence of extrinsic guidance cues. Moreover, the intrinsic pattern determining mechanism is not lost after the destruction of microtubular arrays within the cytoskeleton.

Local influence of substrate molecules in determining distinctive growth patterns of identified neurons in culture.

The growth and branching patterns of individual identified leech neurons in culture depend upon the molecular composition of the substrate. These differences in morphology have been analyzed quantitatively for nerve cells growing on the plant lectin Con A, on extracellular matrix extract (ECM) containing laminin, and on patterned substrates. The total length of neurite outgrowth was about four times greater, and the number of branching points per unit length was three times smaller on ECM laminin extract than on Con A. Single cells placed on a sharp well-defined border separating Con A and ECM-laminin extract sprouted neurites rapidly on both sides of the border without showing preference for either substrate. An individual nerve cell produced neurites with markedly different structure on the two substrates--curved and thick, with a higher branching frequency on Con A, but straight and slender with a lower branching frequency on ECM-laminin substrate. Similar changes were seen in individual neurites that crossed from one substrate to the other. These results show that local contact of the neuron with a particular substrate induces a particular pattern of local outgrowth. Hence, molecules anchored in extracellular matrix could provide information for regenerating or developing axons regarding the type of branching pattern to be produced in that region.

Contact-mediated loss of the nonsynaptic response to transmitter during reinnervation of an identified leech neuron in culture

We have examined the modification of responses to transmitter during the reformation of a specific synapse by identified leech neurons in culture. Single pressure-sensitive mechanosensory (P) neurons in culture and the soma of this cell in vivo have 2 conductances that are activated by application of 5-HT: a Cl conductance and a monovalent cation conductance (gCations); synaptic release of 5-HT by a serotoninergic Retzius cell in vivo and in culture activates only the Cl conductance and not gCations in the P cell. We have characterized the loss of gCations in P cells by manipulating the culture conditions. When 5-HT was applied from a pipette, innervated P cells and P cells paired with Retzius cells that had not formed synapses had a gCations that was markedly reduced compared with gCations in the single P cell. When 5-HT was included in the culture medium in which single P cells were grown, gCations was not reduced. When Retzius-P cell pairs were treated with reserpine, which was shown to deplete the presynaptic neuron of 5-HT, gCations in the P cell was as low as in untreated pairs. Pairing aldehyde-fixed Retzius cells with untreated P cells also resulted in the loss of gCations. The Retzius cell had both types of receptors, but synapse formation did not affect gCations in the presynaptic neuron. The results demonstrate that the loss of the nonsynaptic response of the P cell to transmitter is due to contact between the neurons rather than to the release of transmitter or trophic factors.(ABSTRACT TRUNCATED AT 250 WORDS)

Steps in the development of chemical and electrical synapses by pairs of identified leech neurons in culture

Experiments have been made to follow the development of chemical and electrical transmission between pairs of leech neurons in culture. 1. The cell bodies of identified neurons were isolated from the CNS by suction after mild enzyme treatment, together with a length of the initial segment (or 'stump'). The neurons tested were Retzius cells (R), annulus erector motoneurons (AE), Anterior pagoda cells (AP) and pressure sensory cells (P). Pairs of cells were placed together in various configurations, with different sites on their surfaces making contact. 2. When pairs of Retzius cells were apposed with their stumps touching, serotonergic, chemically mediated synaptic transmission became apparent before electrical transmission. By 2.5 h impulses in either of the two Retzius cells produced hyperpolarizing inhibitory potentials in the other. These potentials were reversed by raised intracellular Cl and showed clear facilitation. The strength of chemical transmission between Retzius cells increased over the next 72 h. 3. After chemical transmission had been established, weak non-rectifying electrical transmission became apparent between Retzius cells at about 24-72 h. By 4 days coupling became stronger and tended to obscure chemically evoked synaptic potentials. 4. When pairs of Retzius cells were aligned in culture with the tip of one cell stump touching the soma of the other, chemical transmission also developed rapidly. Transmission was, however, in one direction, from stump to soma. At later stages non-rectifying electrical coupling developed as with stump-stump configuration. With the cell bodies of two Retzius cells apposed, electrical coupling developed after several days, before chemical transmission could be observed. 5. When Retzius and P cells were cultured with their stumps in contact, inhibitory chemical synaptic transmission developed within 24 h. Transmission was always in one direction, from Retzius to P cell. Electrical coupling of Retzius and P cells never occurred whatever the spatial relations of the cells to one another. 6. Annulus erector motoneurons, which contain ACh and a peptide resembling FMRFamide, first developed electrical coupling when the two stumps were in contact and then, later, bi-directional chemical transmission. Anterior Pagoda pairs placed stump-to-stump showed electrical connections. 7. Electronmicrographs revealed the presence of synaptic structures within 24 h after Retzius-Retzius, Retzius-P or AE-AE stumps were apposed. 8. The specificity of connections between cultured cells was similar to that observed in earlier experiments.(ABSTRACT TRUNCATED AT 400 WORDS)

Influence of substrate on the distribution of calcium channels in identified leech neurons in culture.

The Retzius neuron from the leech, growing in culture on the plant lectin concanavalin A as substrate, produces broad flat growth cones and thick bundles of processes. The same cell extends fine straight processes with numerous branches when grown on a laminin-like substrate extracted from leech central nervous system extracellular matrix, referred to as "leech laminin extract." Cells growing on these two different substrates also show marked differences in the pattern of Ca2+ entry following evoked impulses, as detected optically by local changes in absorbance of the Ca2+-sensitive dye arsenazo III. Ca2+ enters the soma and initial segment of Retzius cells grown on both substrates. However, detectable Ca2+ entry only occurs into the processes of cells growing on leech laminin but not of those growing on concanavalin A. Optical recordings of changes in membrane potential made with the voltage-sensitive dye RH 155 taken from cells growing on either substrate indicate that a depolarization initiated in the soma spreads to the most distant processes with little or no distortion in amplitude or time course. This implies that all voltage-sensitive Ca2+ channels in the cell membrane are equally activated by depolarizing stimuli. Therefore, the fact that impulses evoke Ca2+ entry into processes of Retzius cells grown on leech laminin extract but not of cells grown on concanavalin A shows that the nature of the growth substrate can affect the number and distribution of their functional Ca2+ channels.

Optical recording of calcium and voltage transients following impulses in cell bodies and processes of identified leech neurons in culture

Optical methods were used to examine the spread of electrical potentials and the distribution and time course of calcium transients in individual identified nerve cells isolated from the leech. A photodiode array detected voltage transients by measuring absorbance changes of cells stained with the voltage-sensitive dye RH155 added to the bath. Calcium transients were recorded by measuring absorbance changes of the dye arsenazo III, which had been injected into the cells. In addition, Lucifer yellow was injected to outline the some and processes. Calcium changes resulting from individual action potentials were recorded from N, P, and Retzius cells without averaging. Signals from T cells and anterior pagoda (AP) cells were weaker but could be detected with averaging. These results are in accord with previous studies on calcium contributions to action potentials in these cells. For all cells, larger or wider action potentials gave bigger signals. Calcium changes from each of a train of action potentials were of equal amplitude, showing no sign of facilitation. Calcium transients from Retzius cells that had formed chemical synapses with P cells had properties similar to those of isolated cells. We were also able to detect responses from prolonged subthreshold depolarizations to -40 mV from a hyperpolarized membrane potential (-65 mV). These signals rose throughout the duration of the pulse (1-2 sec). With the photodiode array we mapped the distribution of the calcium signals. The amplitudes from each pixel are proportional to the amount of calcium entering that element in response to the stimulating pulse, if the simplifying assumption is made that the calcium buffering of the cytoplasm is uniform throughout the cell. The largest signals were detected over the axon stump left from the cell isolation procedure. Large signals were also detected from the soma. Weak signals were detected from the processes of some cells. From many Retzius cells, no signals at all were detected from the newly formed processes. Using the photodiode array, we also recorded voltage transients from the cells. Signals were recorded from all over the arborization of the neuron, with no obvious variation in time course, showing that the entire cell, including fine slender processes and broad growth cones, was essentially isopotential. Combining these observations with the measured distribution of calcium transients in the same cell suggests that the density of calcium channels in most cells is less in the outgrowing processes than in the soma or axon stump.

Transmitter localization and vesicle turnover at a serotoninergic synapse between identified leech neurons in culture

An electron microscopic study has been made of chemical synapses that develop between identified nerve cells isolated from the CNS of the leech and maintained in culture. Structures resembling synapses were observed in pairs of Retzius cells and P sensory cells at which chemical transmission had been demonstrated by recording with microelectrodes. Vesicle recycling was shown by following the uptake of extracellular markers after stimulation. The membrane separation between the presynaptic Retzius cell (which is known to liberate serotonin) and the postsynaptic P cell was wider in synaptic than in extrasynaptic regions. The Retzius cell contained clusters of clear vesicles apposed to thickenings of the presynaptic membrane. These clear vesicle clusters were capped by a layer of dense core vesicles that did not contact the presynaptic membrane thickenings. Subsynaptic cisternae were found in the postsynaptic cell opposite the presynaptic membrane thickenings. Occasional slight postsynaptic membrane thickenings were seen. Extracellular material was observed within the synaptic cleft. Similar synaptic structures developed between pairs of Retzius cells in culture; even a single Retzius cell was able to form autapses upon itself. Structures resembling transmitter release sites were found in Retzius cells at a distance from any postsynaptic membranes. These are presumed to be locations for the diffuse release of transmitter. Presynaptic structures resembling release sites were never observed in P cells apposed to Retzius cells. Antibody to serotonin (5-HT) labelled with colloidal gold showed serotonin to be localized in the dense core vesicles in Retzius cells. Stimulation of pairs of Retzius and P cells by raised concentrations of K+ resulted in uptake of extracellular markers. Only Retzius cells became labelled. Ferritin was found in cisternae, in dense core vesicles, and in clear vesicles. HRP was found in cisternae and in clear vesicles. Colloidal gold was taken up by coated vesicles and was occasionally found in both clear and dense core vesicles. The uptake of extracellular markers following stimulation was blocked by high Mg++. These results show that structures develop between pairs of cells at which chemical transmission develops and that transmitter release leads to turnover of dense core and clear vesicles.

Attachment of Con A or extracellular matrix initiates rapid sprouting by cultured leech neurons.

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 alpha-D-mannoside. Another substrate, promoting even more extensive sprouting of leech neurons, is cell-free extracellular 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.