SUMMARY During periods of high activity neurons are expected to swell due to the uptake of Cl – . To find out whether leech Retzius neurons possess swelling-activated Cl – channels that facilitate Cl – efflux and, hence, volume recovery, we exposed the cells to hypotonic solutions. In hypotonic solutions, the cells slowly swelled but did not undergo a regulatory volume decrease. However, the cell volume increased less than predicted for an ideal osmometer, suggesting the action of a compensatory mechanism. The cell swelling was paralleled by a marked decrease in the input resistance as well as by the activation of a membrane current with a reversal potential close to the Cl – equilibrium potential. This current was substantially diminished by removing bath Cl – , by applying the Cl – channel blocker DIDS, or by treating the cells with the tubulin polymerization inhibitor colchicine. Furthermore, in the presence of colchicine or vinblastine, the cell swelling was substantially increased. It is concluded that leech Retzius neurons possess swelling-activated Cl – channels that require an intact microtubule system for activation. The channels may help to restore cell volume after periods of high neuronal activity.

SUMMARY The biological mechanisms of behavioral selection, as it relates to locomotion, are far from understood, even in relatively simple invertebrate animals. In the medicinal leech, Hirudo medicinalis , the decision to swim is distributed across populations of swim-activating and swim-inactivating neurons descending from the subesophageal ganglion of the compound cephalic ganglion, i.e. the brain. In the present study, we demonstrate that the serotonergic LL and Retzius cells in the brain are excited by swim-initiating stimuli and during spontaneous swim episodes. This activity likely influences or resets the neuromodulatory state of neural circuits involved in the activation or subsequent termination of locomotion. When serotonin (5-HT) was perfused over the brain, multi-unit recordings from descending brain neurons revealed rapid and substantial alterations. Subsequent intracellular recordings from identified command-like brain interneurons demonstrated that 5-HT, especially in combination with octopamine, inhibited swim-triggering neuron Tr1, as well as swim-inactivating neurons Tr2 and SIN1. Although 5-HT inhibited elements of the swim-inactivation pathway, rather than promoting them, the indirect and net effect of the amine was a reliable and sustained reduction in the firing of the segmental swim-gating neuron 204. This modulation caused cell 204 to relinquish its excitatory drive to the swim central pattern generator. The activation pattern of serotonergic brain neurons that we observed during swimming and the 5-HT-immunoreactive staining pattern obtained, suggest that within the head brain 5-HT secretion is massive. Over time, 5-HT secretion may provide a homeostatic feedback mechanism to limit swimming activity at the level of the head brain.

SUMMARY Two general trends in the evolution of the nervous system have been toward centralization of neuronal somata and cephalization of the central nervous system (CNS). These organizational trends are apparent in the nervous system of annelid worms, including leeches. To determine if the anterior brain of the leech serves functions similar to those of the brains of more complex organisms, including vertebrates, we ablated one of the two major regions of the cephalic brain - the subesophageal ganglion (SubEG). For anatomical reasons, ablations were performed in embryos, rather than in adults. At the end of embryonic development, we observed the leeches' spontaneous behaviour and their responses to moderate touch. We observed that, although the midbody ganglia of the leech CNS display a high degree of local autonomy, the cephalic brain provides generalized excitation to the rest of the CNS, is a source of selective inhibition that modulates behaviour, integrates sensory information from the head with signals from the rest of the body, and plays an important role in organizing at least some complicated whole-body behaviours. These roles of the leech cephalic brain are common features of brain function in many organisms, and our results are consistent with the hypothesis that they arose early in evolution and have been conserved in complex nervous systems.

SUMMARY It is widely appreciated that the selection and modulation of locomotor circuits are dependent on the actions of higher-order projection neurons. In the leech, Hirudo medicinalis, locomotion is modulated by a number of cephalic projection neurons that descend from the subesophageal ganglion in the head. Specifically, descending brain interneuron Tr2 functions as a command-like neuron that can terminate or sometimes trigger fictive swimming. In this study, we demonstrate that Tr2 is dye coupled to the dopaminergic neural network distributed in the head brain. These findings represent the first anatomical evidence in support of dopamine (DA) playing a role in the modulation of locomotion in the leech. In addition, we have determined that bath application of DA to the brain and entire nerve cord reliably and rapidly terminates swimming in all preparations exhibiting fictive swimming. By contrast, DA application to nerve cords expressing ongoing fictive crawling does not inhibit this motor rhythm. Furthermore, we show that Tr2 receives rhythmic feedback from the crawl central pattern generator. For example, Tr2 receives inhibitory post-synaptic potentials during the elongation phase of each crawl cycle. When crawling is not expressed, spontaneous inhibitory post-synaptic potentials in Tr2 correlate in time with spontaneous excitatory post-synaptic potentials in the CV motor neuron, a circular muscle excitor that bursts during the elongation phase of crawling. Our data are consistent with the idea that DA biases the nervous system to produce locomotion in the form of crawling.

SUMMARY Little is known about the long-term regulation of epithelial ion transport in invertebrates and the specific mediators involved. For some years, we have been investigating the short-term regulation of transepithelial ion transport across the dorsal integument of the leech Hirudo medicinalis , and we have established a model of Na + uptake. In the present study, we investigated the effect of long-term acclimation on transintegumental ion transport by adapting leeches to high-salinity conditions. We dissected segments of dorsal integument and measured ion currents in Ussing chamber experiments. Electrophysiological variables, such as transepithelial potential( V T ) and short-circuit-current ( I sc ),were profoundly affected by adaptation to high-salinity conditions. The total transepithelial Na + current ( I Na ) decreased from 7.66±0.82 to 4.6±0.54 μA cm -2 in preparations adapted to high salinity. The involvement of epithelial Na + channels was determined as current inhibition ( I ami ) by apical application of amiloride; Na + channels were equally active in control epithelia and epithelia from leeches adapted to high salinity. Removal of Ca 2+ from the apical solutions, which is believed to reduce intracellular Ca 2+ concentrations, equalized transepithelial variables between high-salt-adapted integuments and control integuments. Extracellular purines regulate transepithelial Cl - secretion and Na + absorption. In a variety of tissues we tested ATP and adenosine for their effects on epithelial transport. Examination of integuments from pondwater- and high-salinity-adapted leeches revealed different sensitivities for these purines. Apical and basolateral application of ATP both stimulated transepithelial Na + uptake and I ami . Adenosine upregulated non-Na + currents and acted from the basolateral side only. Apical Ca 2+ -free conditions attenuated these effects of purines on transepithelial currents. Extracellular UTP had no effect on ion transport.

The fibre type composition of obliquely striated muscle of adult Hirudo medicinalis was investigated by enzyme histochemistry, by immunohistochemistry and by SDS-PAGE. The oxidative capacity of the fibres, assessed by succinate dehydrogenase activity, was similar in all three layers of body wall muscle (longitudinal, oblique and circular) and in dorsoventral muscles. Histochemical localisation of Mg 2+ -activated actomyosin ATPase activity gave stronger staining in the longitudinal muscle than in other layers. As muscle shortening speed is directly related to this form of ATPase activity, this suggests that the longitudinal layer fibres are faster contracting than the circular, oblique or dorsoventral muscles. Results with polyclonal antibodies specific for vertebrate myosins were consistent with the ATPase results, i.e. fibres with the lowest actomyosin ATPase activity reacted preferentially with an antibody for a slower myosin. Thus, anti-T2, selective for vertebrate tonic fibre myosin, bound preferentially to fibres in oblique, circular and dorsoventral muscles, whereas anti-S, selective for vertebrate slow twitch fibre myosin (faster than vertebrate tonic fibre myosin), bound preferentially to the bulk of longitudinal layer fibres. Whereas most of the longitudinal layer stained uniformly with the anti-S antibody, some fibres in the outermost bundles were negative for the anti-S antibody and were, therefore, different from the main mass of longitudinal fibres. SDS-PAGE analysis of contractile protein preparations from body wall muscle also revealed a differencein the composition of the oblique, circular and dorsoventral muscles compared to the longitudinal layer, supporting the conclusion that leech body wall muscle contains two fibre types.

Voltage-dependent Na + and Ca 2+ inward currents underlying the action potential in cultured embryonic ganglion cells of the leech Hirudo medicinalis have been investigated using the gigaseal whole-cell current or voltage-clamp technique. Dissociated ganglion cells were isolated from 7- to 14-day-old embryos, and maintained in primary culture for up to 5 days. More than 95% of the cultured cells had voltage-dependent K + currents and about 75% of the cells had voltagedependent inward currents. Action potentials of 60mV amplitude and 4 ms duration, similar to those in embryonic nerve cells in vivo , could be recorded. Three types of inward currents occurred in these cells: (1) an initial Na + current, which activated and inactivated rapidly; (2) a second Na + current, which activated slowly and persisted during membrane depolarization, showing very little inactivation, and (3) a Ca 2+ -dependent inward current. Both types of Na + currents were resistant to tetrodotoxin (TTX, 0.2-5 μmol l −1 ). The Ca + current was also carried by Ba 2+ , and was blocked by cobalt and cadmium. The fast Na + current was first expressed in cells from 8-day-old embryos, 1 day earlier than the Ca 2+ current. Between days 8 and 14 the density of the fast Na + current increased from 22±3 to 51±6 μA cm −2 (±S.D., N =11), while the Ca 2+ current grew from 10 μA cm −1 ( N =2) to 15±4 μA cm −2 ( N =10) during this time.

Organic anion concentrations in the blood of Hirudo medicinalis were determined in samples from individual animals using reverse-phase high-pressure liquid chromatography (HPLC) and ultraviolet detection. Quantitatively important anions were malate, α-ketoglutarate, succinate, lactate and fumarate, constituting about 70% of total blood anions. Malate had the highest concentration, 34–68 mequiv I −1 blood, which could (depending on metabolic state) exceed the level of blood Cl − (approx. 36 mequiv I −1 ). Organic acid concentrations in the blood were considerably higher than in the tissue. Blood organic acid concentrations changed more with P O O2 than with temperature. They were unaffected by short periods of aerobic exercise, but stress due to handling and prolonged restraint led to a drastic increase of blood lactate and succinate levels, while malate, fumarate and a-ketoglutarate levels decreased. After feeding on hypertonic, Cl − -rich meals, the Cl − concentration in leech blood increased far more than the cation concentrations. This was not compensated by a decrease of organic acid levels. The regulatory mechanisms for inorganic and organic ion homeostasis function independently.

In the leech, Hirudo medicinalis , the functional properties of the peripheral nephridial nerve cells (NNCs) were investigated using long-term extracellular recordings from both intact and isolated preparations The NNCs respond selectively to changes in external Cl − concentration: their electrical activity is highest in fluid containing normal (i.e. low) extracellular [Cl − ]. Their sensitivity to Cl − is confined to the dendritic tree. Extracellular [Cl − ] is a control factor for ion homeostasis. It increases drastically after a blood meal, and after the animal has left the water or invaded brackish water. The NNCs continuously monitor the extracellular Cl − concentration: their burst rate changes, without adapting, by a factor of 4 in the physiological range between 40 and 90 mmoll −1 . Intracellular recordings from the NNC in isolated nephridial complexes suggest that a high Cl − conductance in combination with active transport of Cl − could be responsible for the observed Cl − sensitivitys

In the leech, the physiological significance of high-pressure phases (HIP) and lowpressure phases (LOP) of the lateral vessels was studied by intravascular pressure recordings and observation of blood flow in different parts of the circulatory system, and by measurements of the blood flow velocities in the dorsal vessel. Different shapes of the pressure pulses were found in the anterior lateral vessel segments during HIP and LOP phases, according to the different modes of diastolic filling in both phases. Pressure recordings in the lateral abdominal vessels showed the different action of the lateral abdominal sphincters in the HIP and LOP phases of the ipsilateral lateral vessel. The LOP contractions were responsible for the blood supply to the capillary networks of the organs and the body wall, with the possible exception of the intestine. The HIP contractions caused a forward bloodflow within the lateral vessel. In the dorsal vessel, the blood pressure was about 0.9-1.9kPa in different animals. The blood flow was discontinuous with velocities of 0.5-10 mms −1 . Average blood flow in the dorsal vessel was 22.9μl min −1 . An improved model of the circulation in the leech is presented.