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.

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.

Crustaceans are attracted out of their burrows by dim light whereas they withdraw in response to light of high intensity. We have studied attraction and withdrawal responses in freely walking crayfish (Procambarus clarkii) and in animals tethered above a treadmill device. Electrophysiological recordings have been made of retinal and extraretinal visual units to determine their possible inputs Attraction was induced by light of 0.17-1.41x; the crayfish walked forward with the abdomen extended. Withdrawal was observed at intensities above 5.6lx; the crayfish walked backwards with a cyclic pattern of flexion and extension of the tail. A group of sustaining visual neurones of the optic nerve with low thresholds was found to respond to light in the range of intensities that produced attraction. Two groups of neurones responded in the range of intensities that produced withdrawal: sustaining visual neurones with high thresholds and the caudal photoreceptors. In animals in which the visual fields of the high-threshold fibres had been covered with black paint, the attraction response was not affected, while withdrawal was only observed in response to light stronger than 11 lx. When tested on a treadmill, the latency of the responses to light and the direction of walking of crayfish reflected attraction and withdrawal responses. Moreover, under these conditions, illumination of the eye induced forward walking at intensities that produce attraction and backward walking at intensities that produce withdrawal. In contrast, illumination of the tail induced only backward walking

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)

Light induces two contrasting behavioral responses in crayfish: attraction at low intensities and withdrawal at high intensities. The aim of our experiments has been to study whether screening pigments of the eye influence the selection of attraction or withdrawal responses. During illumination, screening pigments mask photoreceptor cells, reducing the gain of the visual system. Comparison of the time and light-intensity functions of pigment migration and of attraction and withdrawal responses suggest that pigment migration might influence the selection as well as the latency of the response.