Differential action of tetrodotoxin on identified leech neurons

1. In leech segmental ganglia, the maximum rate of depolarization of action potentials was found to depend largely on Na in the Retzius (R) cell, the mechanosensory P, N and T cells and an identifiable neuron of unknown function, the X cell. 2. Tetrodotoxin (TTX) 15 100 mumol/l had little or no effect on R and X cells. In contrast, membrane excitation in N, P and T cells was depressed in dose- and use-dependent fashion. 3. The data imply the existence of two kinds of Na channels in normal, fully differentiated leech neurons. Correlation of such differences should lead to a better understanding of how particular neurons perform different functions.     

Effect of colchicine and vinblastine on identified leech neurons

An identified neuron of the leech central nervous system is affected by the application of colchicine or vinblastine to its axon. It develops characteristic changes of membrane electrical properties, which are similar to those observed after surgical axotomy. The ionic mechanisms associated with the impulses induced by axotomy and colchicine treatment are not equivalent.     

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 pressuresensitive (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. © 1995 John Wiley & Sons, Inc.     

Properties of action potentials carried by divalent cations in identified leech neurons

Properties of divalent cation potentials carried by either Sr2+ or Ca2+ ions in Na+-free, TEA-Ringer solution were characterized in identified neurons of two species of leeches (Macrobdella and Haementeria). In Macrobdella, the overshoot of the potentials varied logarithmically with [Sr2+]0 (28.5 mV per 10-fold change). The overshoot, Vmax, and duration of the potentials increased with increasing divalent cation concentration and saturated at about 20 to 30 mM [Sr2+]0. The Vmax, amplitude, and duration of the potentials were reversibly blocked by Co2+ and Mn2+. The block by Mn2+ could be well-fitted by a reverse Langmuir-curve with an apparent KI of 100 micromolar. The local anesthetic procaine also reversibly inhibited the Vmax and duration of the potentials. The inhibition was greater at alkaline pH suggesting that procaine blocks the calcium channel from inside the membrane. The identified leech neurons examined in Macrobdella varied considerably in their ability to sustain somatic divalent cation potentials. Stimulation of T cells and most motoneurons produced no or only weak potentials, whereas stimulation of Retzius, N, Nut, and AP cells evoked overshooting potentials of several seconds' duration. Stimulation of the ALG cell of Haementeria in normal Ringer solution evoked a slowly-rising, purely Ca2+-dependent potential of approximately 100 ms duration. This response was TTX-resistant, unaffected by complete removal of Na+ from the Ringer solution, and abolished by 1 mM Mn2+. The overshoot varied logarithmically with a slope of 28 mV/decade change in [Ca2+]0.     

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)     

Anatomical analysis of contacts between identified neurons that control heartbeat in the leech Hirudo medicinalis

Summary The rhythmic constriction of the heart tubes in the leech Hirudo medicinalis is controlled by an identified set of motor neurons (HE cells) and interneurons (HN cells) (reviewed by Calabrese and Peterson 1983). Electrophysiological recordings have indicated particular synaptic relationships among HE and HN cells. In the present study, the synaptic framework mediating the interactions among HE cells and HN cells was examined anatomically. Using light and electron microscopy of physiologically identified, HRP-injected cells, we have examined the zones of interaction and types of contacts between specific cells. HE cells, which have very fine, threadlike processes, interact with their contralateral homologues throughout most of the middle third of the ganglionic neuropil. When HE-cell neuntes come together, the apposed plasma membranes are rigidly parallel, separated by an intercellular gap of 6 nm, for up to 6 m. These specializations must form the structural basis for the strong electrical coupling observed (Peterson 1983) between HE-cell pairs. HE cells also emit from the main neurite a series of extremely fine processes that extend dorsally. These appear in the light microscope to contact processes of the ipsilateral HN cell of the same ganglion, and are also in a position to make contact with the axons of more anterior HN cells. The intraganglionic processes of HN cells, which are studded with large varicosities, ramify in part of the region of neuropil occupied by HE-cell processes, as well as more posteriorly. Contacts between HE and HN cells, which are known to be mostly inhibitory synaptic contacts, are seen in the electron microscope to be formed between medium-diameter HN processes, which are filled with clear round synaptic vesicles, and multiple fine tendrils of the HE cell that surround the HN process. Certain HN cells form reciprocal inhibitory synapses with their contralateral homologues. These contacts occur near the midline, sometimes in the major mass of neuropil and sometimes embedded in the extracellular material that ensheathes the neuropil. The contacts are between medium-and small-diameter profiles that are both filled with synaptic vesicles. Our findings indicate that various classes of physiological interactions among HE and HN cells are mediated by anatomically distinct types of contacts and, at least in some cases, are segregated from each other on the neuritic trees of the cells.     

Ca2+ influx into identified leech neurons induced by 5-hydroxytryptamine

The role of 5-hydroxytryptamine (5-HT, serotonin) in the control of leech behavior is well established and has been analyzed extensively on the cellular level; however, hitherto little is known about the effect of 5-HT on the cytosolic free calcium concentration ([Ca(2+)](i)) in leech neurons. As [Ca(2+)](i) plays a pivotal role in numerous cellular processes, we investigated the effect of 5-HT on [Ca(2+)](i) (measured by Fura-2) in identified leech neurons under different experimental conditions, such as changed extracellular ion composition and blockade of excitatory synaptic transmission. In pressure (P), lateral nociceptive (N1), and Leydig neurons, 5-HT induced a [Ca(2+)](i) increase which was predominantly due to Ca(2+) influx since it was abolished in Ca(2+)-free solution. The 5-HT-induced Ca(2+) influx occurred only if the cells depolarized sufficiently, indicating that it was mediated by voltage-dependent Ca(2+) channels. In P and N1 neurons, the membrane depolarization was due to Na(+) influx through cation channels coupled to 5-HT receptors, whereby the dose-dependency suggests an involvement in excitatory synaptic transmission. In Leydig neurons, 5-HT receptor-coupled cation channels seem to be absent. In these cells, the membrane depolarization activating the voltage-dependent Ca(2+) channels was evoked by 5-HT-triggered excitatory glutamatergic input. In Retzius, anterior pagoda (AP), annulus erector (AE), and median nociceptive (N2) neurons, 5-HT had no effect on [Ca(2+)](i).     

Protein expression patterns of identified neurons and of sprouting cells from the leech central nervous system

It has previously been shown that cephalic, segmental, and caudal ganglia from the medicinal leech show differences in their protein composition. Here we studied whether the neuronal reorganization that occurs in cultured segmental ganglia from the medicinal leech is accompanied by detectable changes in the protein expression pattern. Using silver-stained two-dimensional gels we showed that after 5 and 12 days in culture changes in the protein patterns can be detected in isolated ganglia. The changes observed in the two-dimensional gels occurred concomitantly with a sprouting of serotoninergic neurites and a decreased transmitter content of dopaminergic neurites as shown by using the glyoxylic acid condensation reaction. In addition, we present evidence that Retzius cells, which can be identified by their characteristic morphology and action potential waveform, exhibit biochemically unique properties with respect to their protein expression pattern.     

Cell surface contact mediates neuronal recognition and synapse formation between two identified leech neurons

An early event in the formation of the serotonergic synapse by the Retzius (R) onto the pressure-sensitive (P) neurons of the leech is the elimination of an extrasynaptic response to transmitter from sites of contact on the postsynaptic cell. This event during synapse formation is cell-specific in that it is elicited in vitro by contact with the presynaptic R cell but not with other neurons. In the study reported here, we investigated the nature of this interaction between R and P neurons. The loss of the extrasynaptic response of the P cell was elicited by contact with R cells fixed in a mild paraformaldehyde solution, but not by R cells treated with the proteolytic enzyme trypsin prior to fixation. As well, a variety of lectins were assayed for their ability to interfere with synapse formation. The transmitter responses of P cells plated on lectin-coated substrates were unaffected. However, exposure of the R cell to the lectin wheat germ agglutinin (WGA), but not to other lectins, prior to pairing prevented the loss of the extrasynaptic response in contacted P cells and blocked the formation of the R? P synapse in culture. We conclude that recognition by the P cell of the R cell during synapse formation may be mediated by an R cell-specific surface protein which binds wheat germ agglutinin. 1994 John Wiley & Sons, Inc.     

Neuronal competition determines the spatial pattern of neuropeptide expression by identified neurons of the leech.

Staining adult and embryonic leech ventral nerve cords with antibodies raised against the molluscan neuropeptides small cardioactive peptide B (SCP) and FMRFamide results in segment-specific and bilaterally asymmetric patterns of cell staining. One immunoreactive neuron, the RAS interneuron, is present in only four rostral segmental ganglia, while another, the CAS interneuron, is restricted to the four most caudal abdominal ganglia and tail. In addition to their segment-specific distributions, only one RAS or CAS cell is found in each segmental ganglion, and they alternate sides between adjacent ganglia (either L-R-L-R or R-L-R-L) with a fidelity of about 95%. This paper utilizes cell deletion techniques to investigate the determination of the asymmetric and alternating pattern of RAS and CAS neurons. We show that developmentally equivalent RAS and CAS homologs are present on both sides of the appropriate ganglia, and that within each ganglion one of the initially paired homologs loses the ability to assume the immunoreactive RAS or CAS fate 2-3 days after axonogenesis has begun. These experiments suggest that there is a competitive interaction between bilateral homologs which ensures that only one mature RAS/CAS neuron is formed per ganglion, and that contralateral RAS/CAS neurons are not required in the same or adjacent ganglia for the determination of the RAS or CAS developmental pathways. Nerve cord transections between ganglia in the CAS domain can alter the spatial pattern of CAS neuron determination, confirming that both bilateral homologs retain the ability to express neuropeptide until late embryonic stages, and suggesting that the alternating pattern of RAS/CAS cells requires communication between adjacent ganglia through the longitudinal connectives.     

Ca2+ influx into identified leech neurons induced by 5‐hydroxytryptamine

The role of 5‐hydroxytryptamine (5‐HT, serotonin) in the control of leech behavior is well established and has been analyzed extensively on the cellular level; however, hitherto little is known about the effect of 5‐HT on the cytosolic free calcium concentration ([Ca²⁺]_i) in leech neurons. As [Ca²⁺]_i plays a pivotal role in numerous cellular processes, we investigated the effect of 5‐HT on [Ca²⁺]_i (measured by Fura‐2) in identified leech neurons under different experimental conditions, such as changed extracellular ion composition and blockade of excitatory synaptic transmission. In pressure (P), lateral nociceptive (N1), and Leydig neurons, 5‐HT induced a [Ca²⁺]_i increase which was predominantly due to Ca²⁺ influx since it was abolished in Ca²⁺‐free solution. The 5‐HT‐induced Ca²⁺ influx occurred only if the cells depolarized sufficiently, indicating that it was mediated by voltage‐dependent Ca²⁺ channels. In P and N1 neurons, the membrane depolarization was due to Na⁺ influx through cation channels coupled to 5‐HT receptors, whereby the dose‐dependency suggests an involvement in excitatory synaptic transmission. In Leydig neurons, 5‐HT receptor‐coupled cation channels seem to be absent. In these cells, the membrane depolarization activating the voltage‐dependent Ca²⁺ channels was evoked by 5‐HT‐triggered excitatory glutamatergic input. In Retzius, anterior pagoda (AP), annulus erector (AE), and median nociceptive (N2) neurons, 5‐HT had no effect on [Ca²⁺]_i. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2005     

Distribution of monoamine-containing neurons in the brain stem of the frog, Rana temporaria

The distribution of monoamine (catecholamine and 5-hydroxytryptamine)-containing nerve cell bodies in the brain stem and hypothalmus of the frog (Rana temporaria) was investigated with the help of the histofluorescence technique of Falck and Hillarp ('62). At the level of the hypothalmus of this amphibian brain, catecholamine-containing nerve cell bodies are found mainly within three areas of the periventricular gray substance, namely the peroptic recess organ, the paraventricular organ and the lateral recess region. On the other hand, most of the 5-hydroxytryptamine (serotonin)-containing nerve cell bodies of the brain stem of Rana temporaria appear to be concentrated within the midbrain tegmentum. This huge mesencephalic nerve cell collection can be subdivided into medial and lateral groups. More caudally, at the level of the isthmic tegmentum, another group of 5-hydroxytryptamine-containing perikarya located close to the midline, within the so-called raphae region, is clearly outlined. The latter group of neurons extends caudally as far as the level of the medulla oblongata. In addition, a small group of catecholamine-containing nerve cell bodies is also found in the ventromedial portion of the rostral midbrain tegmentum, whereas a few other catecholamine type neurons are scattered throughout the lower brain stem of the frog and more especially near the ependymal wall of the fourth ventricle. As a whole, the 5-hydroxytryptamine-containing neuronal systems of the brain stem of Rana temporaria are much more elaborated than the catecholamine neuronal systems of the same structure.     

Network interactions among sensory neurons in the leech

Interactions among mechanosensory neurons, sensitive to touch, pressure and nociceptive stimuli in the leech nervous system were studied in isolated ganglia and in body-wall preparations. Pairs of touch-pressure, touch-nociceptive and pressure-nociceptive neurons were tested by suprathreshold stimulation of one neuron while recording the response of the other, in both directions. Pressure and nociceptive stimulation evoked depolarizing and hyperpolarizing responses in touch cells, mediated by interneurons. The relative expression of these responses depended on the stimulus duration. One or two pressure cell spikes produced, predominantly, a depolarization of the touch cells, and increasing number of spikes evoked a hyperpolarization. Nociceptive cells produced primarily the hyperpolarization of touch cells at any stimulus duration. When touch cells were induced to fire by injection of positive current into the soma, stimulation of pressure cells inhibited touch cell activity. However, when touch cells were induced to fire by peripheral stimulation, pressure cell activation failed to inhibit touch cell firing. The results suggest that excitation of pressure and nociceptive cells would not limit the responses of touch cells to peripheral stimuli, but would inhibit the firing of touch cells evoked by their central connectivity network.