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.     

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.     

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.     

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).     

Blastomere ablation and the developmental origin of identified monoamine-containing neurons in the leech

Ablation of different identifiable blastomeres of the early embryo of the leech Helobdella triserialis was found to lead to the absence of different sets of segmentally iterated monoamine-containing neurons in subsequent development. Thus the ablation of one of the paired N ectoteloblasts leads to the absence of one member of each of the three bilateral pairs of serotonin-containing neurons (one of which is the Retzius cell) from each segmental ganglion. The ablation of one of the paired OP blastomeres (precursors of the paired O and P ectoteloblasts) leads to the absence of one member of each of the two bilateral pairs of lateral dopamine-containing neurons that lie in the body wall of each segment. And the ablation of one of the paired Q ectoteloblast leads to the absence of one member of the bilateral pair of medial dopamine-containing neurons that lie in the body wall of each segment. These results suggest that each of these sets of monoamine-containing neurons is derived from a particular blastomere. Upon ablation of that blastomere the set does not develop from any other source.     

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.     

Neuronal control of swimming in the medicinal leech

Summary The cell bodies and function of twelve neurons whose impulse pattern is clearly related to that of the swimming rhythm were identified in the segmental ganglion of the leech. These include excitatory and inhibitory motor neurons of the dorsal and ventral longitudinal muscles and the excitatory flattener motor neuron of the dorsoventral muscles. During swimming the membrane potential of these cells oscillates between a depolarized and a hyperpolarized phase. The activity of this ensemble of cells is sufficient to account for the contractile rhythm of the swimming animal. The following connections were found between these motor neurons. Electrotonic junctions link: (1) bilaterally homologous cells; (2) excitors of the dorsal longitudinal muscles; (3) excitors of the ventral longitudinal muscles; (4) inhibitors of both dorsal and ventral longitudinal muscles. The dorsal inhibitors project via an inhibitory pathway to the dorsal excitors, and the ventral inhibitor projects via an inhibitory pathway to the ventral excitors. The membrane potential oscillation of the excitors is at least partly attributable to the phasic inhibitory synaptic input which they receive from the inhibitors. The excitatory shortener motor neuron of the entire longitudinal musculature is maintained in an inactive state during swimming. This control is achieved by rectifying electrotonic junctions linking this neuron to the dorsal and ventral excitors. These junctions allow passage of only depolarizing current from the shortener to the dorsal and ventral excitors and of only hyperpolarizing current in the reverse direction. Furthermore, both dorsal and ventral inhibitors project via inhibitory pathways to the shortener neuron.We are greatly indebted to Ann Stuart for advice and help in this study, and for communicating to us some unpublished findings. We thank Elizabeth Mullenbach for excellent technical assistance.This research was supported by grant GB 31933 X from the National Science Foundation, and by Public Health Service Research grant GM 17866 and Training Grant GM 01389 from the Institute for General Medical Sciences.     

Neuronal control of heartbeat in the medicinal leech

Summary The leech heartbeat consists of a constriction-dilation rhythm of two lateral heart tubes extending over the length of the body. The beats of the segmental sections of these two tubes are coordinated in such a manner that the heart tube of one body side produces a frontward peristaltic wave while the heart tube on the other body side produces nearly concerted constrictions. This rhythm is metastable, in that left and right heart tubes alternate between peristaltic and concerted constriction modes, with a given mode lasting for tens or hundreds of beat cycles.The constriction-dilation cycles of the segmental heart tube sections are controlled by a set of rhythmically active motor neurons, the heart excitors, or HE cells. A bilateral pair of HE cells is located in all but the two frontmost and the two rearmost segmental ganglia of the ventral nerve cord. Each HE cell innervates via excitatory synapses the circular muscle fibers in the wall of the ipsilateral heart tube section. The activity cycle of the HE cells consists of an active phase, during which they are depolarized and produce a burst of impulses, and an inactive phase during which they are repolarized by a burst of inhibitory synaptic potentials. The intersegmentally coordinated activity cycles of the HE cell set are maintained in an isolated ventral nerve cord. Hence the generation of the heart excitor rhythm does not require sensory feedback.We are indepted to Amy Kelly and King-Wai Yau for advice on the use of the intracellular staining technique and to John Kretz for calling to our attention the existence of an afferent impulse burst rhythm emanating from denervated heart tube preparations. We thank Georgia Harper for excellent technical assistance. This research was supported by Grant GB 31933X from the National Science Foundation and NIH research grants GM17866 and Training Grant GM 01389 from the Institute of General Medical Sciences.     

Neuronal expression of a newly identified Drosophila melanogaster G protein alpha 0 subunit.

Guanine nucleotide-binding proteins (G proteins) mediate signals between activated cell-surface receptors and cellular effectors. A bovine G-protein alpha-subunit cDNA has been used to isolate similar sequences from Drosophila genomic and cDNA libraries. One class, which we call DG alpha 0, hybridized to position 47A on the second chromosome of Drosophila. The nucleotide sequence of the protein coding region of one cDNA has been determined, revealing an alpha subunit that is 81% identical with rat alpha 0. The cDNA hybridizes strongly to a 3.8 kb mRNA and weakly with a 5.3 kb message. Antibodies raised against a trp-E-DG alpha 0 fusion protein recognized a 39,000 Da protein in Drosophila extracts. In situ hybridization to adult Drosophila sections combined with immunohistochemical studies revealed expression throughout the optic lobes and central brain and in the thoracic and abdominal ganglia. DG alpha 0 message and protein were also detected in the antennae, oocytes, and ovarian nurse cells. The neuronal expression of this gene is similar to mammalian alpha 0, which is most abundantly expressed in the brain.     

Neuronal interactions between neuropeptide W- and orexin- or melanin-concentrating hormone-containing neurons in the rat hypothalamus

Neuropeptide W (NPW) was recently discovered as the endogenous ligand for GPR7 and GPR8, which are orphan G protein-coupled receptors isolated from the porcine brain. These receptors are assumed to be involved in feeding regulation and/or energy homeostasis. Recent anatomical studies have revealed that high levels of GPR7 mRNA are distributed in the brain, including the hypothalamus and amygdala. However immunohistochemical studies on the distribution and localization of NPW have revealed differing results concerning whether or not NPW-containing cell bodies and their processes are present in the hypothalamus. Only a few immunohistochemical reports have been published concerning the presence of NPW-containing neurons in the brains of rodents, while there have been no anatomical studies of the co-localization of this neuropeptide with other transmitters. On this basis, we used a specific antiserum against NPW to determine immunohistochemically the presence of NPW-containing neurons in the rat hypothalamus. Many NPW-like immunoreactive cell bodies and their processes could be detected in the caudal region of the lateral hypothalamus but not in its anterior or middle regions. Given this positive identification of NPW-containing neurons in the lateral hypothalamus, we further studied the nature of interaction between NPW-containing neurons and neurons containing feeding regulating peptides such as orexin- and melanin-concentrating hormone (MCH). Very close interactions between NPW-containing nerve processes and orexin- and MCH-containing neuronal cell bodies and processes could be observed. These morphological findings strongly suggest that NPW is involved in the regulation of feeding and/or sleep/arousal behavior through orexin- and/or MCH-mediated neuronal pathways.     

Differential expression of voltage-gated calcium channels in identified visual cortical neurons.

Using the whole-cell patch-clamp technique, Ca2+ channel currents were examined in three distinct types of neurons derived from rat primary visual cortex. Callosal-projecting and superior colliculus-projecting neurons were identified following in vivo retrograde labeling with fluorescent "beads." A subset of intrinsic GABAergic visual cortical neurons was identified with the monoclonal antibody VC1.1. Although high voltage-activated Ca2+ channel currents were measured in all three cell types, clear differences in the densities of these channels were observed. There were also marked variations in the relative amplitudes of the inactivating and noninactivating components of the high voltage-activated currents, suggesting that N- and L-type Ca2+ channels are differentially distributed. Although low voltage-activated or T-type currents were measured in subsets of both types of projection neurons, they were not observed in VC1.1-positive cells. These results provide a direct demonstration that voltage-gated Ca2+ channels are expressed in neurons of the mammalian visual cortex and reveal that the distribution and densities of different Ca2+ channel types in diverse classes of visual cortical neurons are distinct.     

Neuronal control of leech swimming movements: interactions between cell 60 and previously described oscillator neurons

Summary A recently discovered member of the neuronal oscillator underlying swimming movements in the medicinal leech,Hirudo medicinalis, is described. This interneuron, named cell 60, exhibits membrane potential oscillations that are phase-locked to the swim oscillations observed in other oscillator neurons (phase angle, approximately 220°) and, when depolarized, acts to shift the phase of the swim oscillations. The soma of cell 60 lies near the posterior-lateral margin on the ventral aspect of most (and possibly all) segmental ganglia. The neurite crosses the midline, then turns anteriorly and projects into the lateral intersegmental connective. Cell 60 is connected to cell 28, a previously described dorsal swim oscillator neuron, via an electrically rectifying junction.Two interactions link cell 60 with cell 208, a swim oscillator neuron found on the ventral aspect of segmental ganglia: a short-latency, fatiguing inhibitory synapse and a powerful electrical interaction. The electrical interaction acts as a diode, in that current can pass from cell 60 to cell 208, but not in the reverse direction. The coupling coefficient in the forward direction is about 0.5 and is independent of the membrane potential difference between cells 60 and 208 provided that the diode connection is forward biased.The rectifying junction acts as a switch which is off during swimming activity because cell 208 oscillations are superimposed on a tonic depolarization of about 10–15 mV. This tonic potential reverse biases the electrical diode connection between cell 60 and cell 208, leaving the inhibitory synapse as the only effective interaction between these cells. The diode switch is on when cell 208 is hyperpolarized. In this circumstance, the dominant connection is electrical; therefore induced potential oscillations in cell 60 induce in-phase oscillations in cell 208.Abbreviations PIR Postinhibitory rebound - NM Neuromime     

The spatial pattern of light determines the kinetics and modulates backpropagation of optogenetic action potentials

Optogenetics offers an unprecedented ability to spatially target neuronal stimulations. This study investigated via simulation, for the first time, how the spatial pattern of excitation affects the response of channelrhodopsin-2 (ChR2) expressing neurons. First we described a methodology for modeling ChR2 in the NEURON simulation platform. Then, we compared four most commonly considered illumination strategies (somatic, dendritic, axonal and whole cell) in a paradigmatic model of a cortical layer V pyramidal cell. We show that the spatial pattern of illumination has an important impact on the efficiency of stimulation and the kinetics of the spiking output. Whole cell illumination synchronizes the depolarization of the dendritic tree and the soma and evokes spiking characteristics with a distinct pattern including an increased bursting rate and enhanced back propagation of action potentials (bAPs). This type of illumination is the most efficient as a given irradiance threshold was achievable with only 6 % of ChR2 density needed in the case of somatic illumination. Targeting only the axon initial segment requires a high ChR2 density to achieve a given threshold irradiance and a prolonged illumination does not yield sustained spiking. We also show that patterned illumination can be used to modulate the bAPs and hence spatially modulate the direction and amplitude of spike time dependent plasticity protocols. We further found the irradiance threshold to increase in proportion to the demyelination level of an axon, suggesting that measurements of the irradiance threshold (for example relative to the soma) could be used to remotely probe a loss of neural myelin sheath, which is a hallmark of several neurodegenerative diseases.     

Angiotensin-converting enzyme inhibition studies by natural leech inhibitors by capillary electrophoresis and competition assay.

A protocol to follow the processing of angiotensin I into angiotensin II by rabbit angiotensin-converting enzyme (ACE) and its inhibition by a novel natural antagonist, the leech osmoregulator factor (LORF) using capillary zonal electrophoresis is described. The experiment was carried out using the Beckman PACE system and steps were taken to determine (a) the migration profiles of angiotensin and its yielded peptides, (b) the minimal amount of angiotensin II detected, (c) the use of different electrolytes and (d) the concentration of inhibitor. We demonstrated that LORF (IPEPYVWD), a neuropeptide previously found in leech brain, is able to inhibit rabbit ACE with an IC(50) of 19.8 micro m. Interestingly, its cleavage product, IPEP exhibits an IC(50) of 11.5 micro m. A competition assay using p-benzoylglycylglycylglycine and insect ACE established that LORF and IPEP fragments are natural inhibitors for invertebrate ACE. Fifty-four percent of insect ACE activity is inhibited with 50 micro m IPEP and 35% inhibition with LORF (25 mm). Extending the peptide at both N- and C-terminus (GWEIPEPYVWDES) and the cleavage of IPEP in IP abolished the inhibitory activity of both peptides. Immunocytochemical data obtained with antisera raised against LORF and leech ACE showed a colocalization between the enzyme and its inhibitor in the same neurons. These results showed that capillary zonal electrophoresis is a useful technique for following enzymatic processes with small amounts of products and constitutes the first evidence of a natural ACE inhibitor in invertebrates.