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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)

In leech ganglia, an individual sensory cell that responds specifically to noxious mechanical stimulation of the skin (N cell) excites two different motoneurones. One raises the annuli of the skin into ridges (the AE cell), while the other innervates logitudinal muscles and thereby shortens the body segment (L cell). A comparison has been made of the way in which these two synapses behave when their common presynaptic cell is stimulated in various conditions.1. Using previously described criteria, N sensory cells have been shown to make monosynaptic chemical connexions with both the AE and L motoneurones (Nicholls & Purves, 1972). Following a single stimulus, the excitatory synaptic potential recorded in the AE motoneurone was only about one tenth the size of that in the L cell (approximately 0.5 mV compared to 5 mV). Trains of impulses in the same N sensory cell gave rise to synaptic potentials in the AE and the L motoneurones that underwent phases of facilitation and depression; the facilitation, however, was characteristically greater and longer lasting at synapses upon the AE motoneurone.2. The differences between the two synapses were accentuated in Ringer fluid containing increased concentrations of Ca and also in the cold. Under both of these conditions repetitive firing by the N sensory cell could give rise to synaptic potentials in the AE motoneurone which progressively increased in amplitude, while those in the L motoneurone became smaller.3. The results suggest that the differences in synaptic transmission can be accounted for by variations in the amount of transmitter released at the presynaptic N cell terminals, rather than by differences in the post-synaptic cells. The animal's behaviour corresponds to expectations from the physiology of the synapses.

The release of 5-hydroxytryptamine (5-HT) from presynaptic terminals has been studied by the voltage-clamp technique at synapses made by isolated Retzius and pressure (P) sensory neurones dissected from the leech C.N.S. and maintained in tissue culture. At these synapses facilitation, depression and modulation of release occur with action potentials and with voltage-clamp pulses. Depolarization of Retzius cells from a constant holding potential by steps of varying amplitude (5 ms in duration) caused graded release of 5-HT. The steep transfer function for release using these short test pulses resembled that seen at the giant synapse of the squid: synaptic potentials increased markedly with presynaptic depolarizations beyond -25 mV and decreased with large depolarizing pulses beyond +40 mV. When the steady holding potential of voltage-clamped Retzius cells was suddenly displaced to a new value within the range of -40 mV to -85 mV, there followed a slow but smaller change of the post-synaptic P-cell membrane potential in the same direction. After an initial delay of about 40 ms, the post-synaptic potential reached its new level with an exponential time course and a time constant of 0.7 s. Since Retzius and P cells are not electrically coupled, these effects can be accounted for by alterations in tonic release of transmitter. Changes of presynaptic holding potential to a more depolarized level resulted in an increase in voltage noise recorded in the P cell. Conversely, hyperpolarization from a depolarized level reduced noise. Noise analysis showed that these changes could be accounted for by quantal events with a mean amplitude of about 0.15 mV. This value is similar to that for spontaneous miniature potentials and quantal fluctuations observed at synapses between Retzius and P cells. Changes in steady holding potential also had marked effects upon the transfer function observed with brief depolarizing pulses of the Retzius cell. The post-synaptic responses evoked by depolarizations to 0 mV with pulses of 5 ms duration were reduced in amplitude as the holding potential of the Retzius cell was increased from the resting value of -45 to -75 mV. For example, depolarization to 0 mV starting from -45 mV evoked synaptic potentials as much as ten times larger than those evoked by depolarizations to 0 mV starting from -75 mV.(ABSTRACT TRUNCATED AT 400 WORDS)

1. The role of presynaptic Ca2+ entry in facilitation of transmitter release has been analysed by voltage-clamp measurements at synapses formed in culture by Retzius and P neurones isolated from the central nervous system (CNS) of the leech. The transmitter released by Retzius cells is serotonin. 2. Synaptic transmission persisted in solutions containing raised concentrations of divalent cations, reduced concentrations of Na+, and tetraethylammonium (TEA+) and 4-AP (to block K+ currents). Ca2+ and Sr2+ were more effective in promoting transmitter release than Ba2+, as assessed by the postsynaptic potentials in P cells. The degree and time course of facilitation in Ca2+- and Sr2+-containing solutions were similar to those observed for synapses bathed in normal L-15 medium. 3. Transmitter release depended upon the amplitude and the duration of presynaptic depolarization and inward Ca2+ current. Peak Ca2+ currents and postsynaptic potentials occurred with depolarizing steps to +15 mV. Frequent or prolonged pulses depressed the postsynaptic potentials. 4. Pairs of depolarizing pulses that caused facilitation were accompanied by identical inward Ca2+ currents. These results indicate that the mechanism responsible for facilitated serotonin release must occur following Ca2+ entry and that residual Ca2+ plays a role.

After injury, neurones in the central nervous system (CNS) of the leech regenerate with a high degree of specificity. The aim of our experiments has been to study the sequential steps involved in neurite growth and synapse formation using isolated identified neurones in culture. An important requirement for sprouting of leech neurones is the substrate. Neurites grow only slowly and sparsely on polylysine or vertebrate laminin. The extracellular matrix of leech ganglion capsules contains a protease-sensitive factor which can be extracted with urea. With this material as substrate, growth proceeds rapidly in defined medium. Another neurite-promoting substrate is provided by the plant lectin concanavalin A (Con A). The activity of Con A, but not of the capsule matrix factor, is blocked by the Con A-specific hapten methyl alpha-D-mannoside. The morphology and branching pattern of the neurites in culture depend on the specific substrate and on the type of neurone. During stimulation, less Ca2+ uptake occurs into growth cones than in cell bodies. The mechanism of neurite growth seems not to depend on activity-mediated Ca2+ influx or on interactions between neuronal cell surfaces. However, even without profuse outgrowth, electrical and chemical synapses develop between neighbouring neurones. The type of synapse depends predictably on the types of neurones within the cell pair. Since the development of a synapse can be followed with time in culture, the sequential events can each be studied separately for this multi-step process.

1. Chemical synaptic transmission develops between individual identified neurones dissected from leech ganglia and maintained in culture. Impulses in Retzius cells give rise to hyperpolarizing synaptic potentials in pressure (P) sensory cells. In suitable medium the potentials develop by 3 days and can be observed for more than 3 weeks. 2. The synaptic potentials occur after a synaptic delay, exhibit facilitation and depression and are reversed by hyperpolarization. The blocking effects of reduced calcium and raised magnesium concentrations in the bathing fluid provide additional evidence for the chemical nature of transmission. 3. An increase in chloride conductance is involved in the generation of the synaptic potential in the P cell. With high intracellular Cl in the post-synaptic cell, the synaptic potentials become reversed and amplified. The amplitudes of these reversed responses range from 1 to 20 mV with a falling phase lasting for seconds. 4. Changes in the membrane potential of the presynaptic cell that modify the amplitude and duration of the action potential influence the efficacy of transmission. In addition, impulses in Retzius cells initiated from hyperpolarized values of membrane potential evoke smaller synaptic potentials in the P cells than impulses arising from a depolarized level. 5. With neurones placed directly next to one another in the dish, maintained depolarization of the presynaptic Retzius cell in the absence of conducted action potentials gives rise to slow synaptic potentials in the P cells. In some pairs, the response in the P cell consists of a marked increase in 'noise'. 6. Injection of horseradish peroxidase into the Retzius cell reveals neurites with distinctive varicosities growing over the P cell.

We have examined changes that develop in the synaptic interactions of sensory and motor nerve cells following surgical lesions to the central nervous system of the leech. In one type of operation an individual ganglion was isolated from the rest of the nervous system by severing all the incoming and outgoing fibres. During the next few weeks, marked changes appeared in synaptic interactions.1. In chronically isolated ganglia inhibitory potentials were recorded in the motoneurone which raises the skin into ridges (the AE cell) following impulses in sensory neurones that respond to pressure (P) or noxious (N) stimuli. In contrast the same AE cell in ganglia taken from normal animals shows excitatory synaptic potentials when the P or N sensory cells are stimulated.2. Another altered synaptic interaction in ganglia isolated by lesions was that between sensory cells responding to touch and a motoneurone that supplies longitudinal muscles (L cell). Instead of the pure, electrical coupling potential seen normally, a large, additional chemically mediated excitatory potential was also apparent.3. Some of the changes in synaptic interactions were not restricted to synapses within the isolated ganglion, but appeared gradually over the following year in successive ganglia along the length of the ventral nerve cord.4. Indirect evidence suggests that the altered synaptic potentials that became conspicuous after operations are also present but smaller and obscured in normal animals.5. It is concluded that some synapses in the leech nervous system are more readily changed than others by cutting the connectives. Furthermore, these changes influence in a predictable manner the way in which the animal behaves in response to mechanical stimuli.

Within hours after identified neurones have been isolated from the CNS of the leech, they begin to sprout and to form synapses. Electrical recordings made by loose-patch clamp show that the tip of the isolated neurone has distinct properties with a high density of sodium channels. Neurites grow out from this tip after about 30 min and continue to grow for the next few days. The extent of growth, the branching pattern and the distribution of calcium channels all depend critically upon the molecular composition of the substrate. The tip of the neurone also represents a preferred region for synapse formation. For example when the tips of two serotonin-containing neuromodulatory neurones, the Retzius cells, are placed in contact, chemical synapses develop within about 6 h. These chemical synapses are bidirectional and become stronger over the next 2 days. Electrical synapses between the two Retzius cells develop more slowly and appear only after about 20 h. When the tip of one Retzius cell is apposed to the soma of another, chemical transmission develops more slowly. When other regions of these same cells are placed in contact, electrical transmission can appear before chemical. Together these results show that specialized areas of neuronal membrane are involved in neurite extension and in the formation of specific synaptic connections.

Segmental ganglia of the central nervous system of the leech were maintained in culture medium outside the animal for several weeks in order to study the properties of synapses and regeneration by identified sensory and motor nerve cells. A variety of preparations were used, including single ganglia, chains of ganglia and ganglia connected to the areas of skin and muscle that they normally innervate in the animal. (1) For up to 10 weeks after removal from the animal, resting and action potentials recorded from sensory and motor neurons resembled those seen in normal ganglia. The same individual cell in a cultured ganglion could be recorded from with intracellular electrodes on a second occasion after an interval of a few days. (2) Sensory cells, identified as touch, pressure or nociceptive according to their morphology and electrical properties, continued to respond selectively to stimuli of the appropriate modality applied to their receptive fields in the skin; action potentials in motor cells caused contractions in the appropriate muscles. Culture of ganglia for more than 3 weeks caused the disappearance of synaptic potentials and a loss of transparency in ganglia. (3) Certain chemically mediated synaptic interactions between sensory and motor nerve cells became markedly changed in cultured ganglia. These changes appeared over the first 3 weeks and consisted of abnormally large excitatory and inhibitory synaptic potentials. The changes in synaptic transmission observed in culture were in many respects similar to those occurring in ganglia maintained within an animal after lesions have been made in the nervous system (Jansen et al. 1974). (4) The morphological appearances of sensory cells were compared in cultured and normal ganglia after injection of horseradish peroxidase. In cultured ganglia, the branching pattern appeared normal, but varicosities became more conspicuous. (5) When connectives linking cultured ganglia were crushed or cut, regeneration occurred. By 7 days, impulses propagated through the regenerated fibers and evoked synaptic potentials on cells within the next ganglion. The course taken by regenerating axons was observed in cells injected with horseradish peroxidase. The results again resembled those seen in animals with similar lesions. (6) The cultured ganglia provide preparations in which it is possible to analyze the mechanisms that underlie long-term changes similar to those seen in the leech central nervous system in situ.

Experiments have been made to study the synaptic connexions between sensory cells in the C.N.S. of the leech. Each segmental ganglion contains six neurones that respond specifically to light touch applied to the skin; each of these ;touch cells' innervates a discrete area on the surface of the body and has a characteristic set of properties by which it can be recognized. Using intracellular electrodes it has been shown that these sensory cells interact with one another through chemical and electrical synapses by way of a stereotyped set of pathways.1. Action potentials occurring in one touch cell gave rise to synaptic potentials in the five other touch cells in the same ganglion and also in the three ipsilateral touch cells in the adjacent ganglia. Thus, synaptic interactions took place between sensory cells whose receptive fields lay within the same segment and on the same side of adjacent segments.2. The post-synaptic potentials consisted of a short-latency coupling potential, followed by an excitatory potential and a number of inhibitory potentials. These delayed synaptic potentials occurred inconsistently and with a variable latency; they could also be recorded in the cell which had been stimulated. All of the touch cells appeared to be equally effective in initiating synaptic potentials.3. The short-latency coupling potential was shown to be mediated through an electrical synapse by observing a voltage change in one touch cell when current was injected into its neighbour. It was not abolished by high concentrations of Mg in the bathing fluid, which blocked chemical synapses in this ganglion. This electrical synapse displayed remarkable rectification; a depolarization could spread from cell to cell in both directions, while a hyperpolarization could spread in neither.4. The inhibitory potentials were reversed by injecting Cl into the cell. In Cl-free Ringer solution this effect was so marked that the reversed IPSPs caused long trains of impulses in touch cells, which tended to excite each other by a process of positive feed-back.5. Synaptic potentials evoked by activation of a touch cell did not usually reach threshold since excitation and inhibition tended to cancel. The connexions between touch cells that mediated the delayed excitatory and inhibitory potentials are polysynaptic; the interneurones have not yet been found but some of their connexions could be inferred from electrical recordings.6. Action potentials in sensory cells of a different modality (responding to pressure) also initiated synaptic potentials in the same family of touch cells.7. The possible significance for integration of these synaptic interactions between sensory cells is discussed.

In leech ganglia, three sensory cells of different modality converge on a motoneurone, where they form chemical and electrical synapses. Each of these synapses behaves in a characteristic manner and the nature of the transmission mechanism has significant functional consequences for the operation of the reflexes. An analysis has been made of the effects of trains of impulses on synaptic transmission through these pathways, using frequencies that correspond to natural firing.1. At the chemical synapse between the nociceptive sensory cell and the motoneurone, two opposing events occur: facilitation and depression. Thus, with trains of impulses, the synaptic potentials first increase in amplitude and then decrease. The two processes could be separated by altering the Mg and Ca content of the bathing fluid. In concentrations of Mg that reduced the amplitude of a single control chemical synaptic potential, pure facilitation occurred during a train. Depression predominated during brief trains in raised concentrations of Ca, although synaptic potentials were initially larger. These results suggest that changes in the amount of transmitter released by each presynaptic action potential can account for the changes observed in chemical synaptic transmission.2. In contrast, electrical transmission between the sensory cell responding to touch and the same motoneurone did not show facilitation or depression. The electrical coupling potential in the motoneurone was relatively constant when the touch cell fired at high or low frequencies in normal Ringer fluid, high Mg, or high Ca fluid.3. Further differences between chemical and electrical synapses were apparent when the preparation was cooled to 4 degrees C. In the cold the latency of chemically evoked synaptic potentials in the motoneurone increased and their amplitude declined drastically with repetitive stimulation, while electrical coupling potentials were unaffected.4. A brief hyperpolarization of the presynaptic cell by injected current produced a marked and prolonged increase in chemically evoked synaptic potentials, but did not influence electrical synaptic transmission.5. The synapses of the sensory cell responding to pressure, which are both chemical and electrical, behaved as expected: the chemical synaptic potentials showed facilitation and depression while electrical transmission remained relatively constant.6. These experiments emphasize the different functional consequences of electrical or chemical synapses in reflex pathways for the transmission of signals that arise as a result of natural sensory stimuli.

The central nervous system of the leech has been used for the study of the formation of new synaptic connections by regenerating neurons. In control leeches, individual nerve cells in adjacent ganglia are connected in an orderly and stereotyped manner, with only little variation. In the present experiments, a bundle of axons running between two of the segmental ganglia has been severed and allowed to regenerate. Subsequently, the axons reestablish synaptic connections between certain identified nerve cells in the adjacent ganglia, selectively and accurately. Thus, individual sensory cells in one ganglion show a high degree of neural specificity in reestablishing cell to cell connections with a motor cell in the next ganglion. The performance of the regenerated synapses, however, is significantly altered in a consistent manner. The normal balance between the effects of inhibitory and excitatory innervation in leeches with regenerated synapses is different from that seen in normal leeches, with marked overemphasis on inhibition. Similar alterations have also been seen in a series of ganglia at a distance from the site of the lesion. After the operation, therefore, a widespread modification of synapses occurs along the length of the nerve cord.