Segmental specialization of neuronal connectivity in the leech

1. Every segmental ganglion of the leech Hirudo medicinalis contains two serotonergic Retzius cells. However, Retzius cells in the two segmental ganglia associated with reproductive function are morphologically distinct from Retzius cells elsewhere. This suggested that these Retzius cells might be physiologically distinct as well. 2. The degree of electrical coupling between Retzius cells distinguishes the reproductive Retzius cells; all Retzius cells are coupled in a non-rectifying manner, but reproductive Retzius cells are less strongly coupled. 3. Retzius cells in standard ganglia depolarize following swim motor pattern initiation or mechanosensory stimulation while Retzius cells in reproductive ganglia either do not respond or hyperpolarize. 4. In standard Retzius cells the depolarizing response caused by pressure mechanosensory neurons has fixed latency and one-to-one correspondence between the mechanosensory neuron action potentials and Retzius cell EPSPs. However, the latency is longer than for most known monosynaptic connections in the leech. 5. Raising the concentration of divalent cations in the bathing solution to increase thresholds abolishes the mechanosensory neuron-evoked EPSP in standard Retzius cells. This suggests that generation of action potentials in an interneuron is required for production of the EPSP, and therefore that the pathway from mechanosensory neuron to Retzius cell is polysynaptic. 6. P cells in reproductive segments have opposite effects on reproductive Retzius cells and standard Retzius cells in adjacent ganglia. Thus the difference in the pathway from P to Retzius is not localized specifically in the P cell, but elsewhere in the pathway, possibly in the type of receptor expressed by the Retzius cells.     

Postural interneurons in the abdominal nervous system of lobster

The nature of the synaptic relationship between 7 identified postural interneurons and 5 pairs of superficial motoneurons was examined by obtaining dual intracellular recordings from interneuron-motoneuron pairs in the lobster 2nd abdominal ganglion. For six different interneuron-motoneuron pairs EPSPs recorded from motoneurons occurred with a short (1 to 3 ms) fixed latency following each presynaptic spike recorded from the interneuron. This suggests that there is a monosynaptic relationship between these interneurons and motoneurons. Monosynaptic pathways accounted for 27% of all excitatory connections. Preliminary evidence indicates that the monosynaptic potentials are mediated by an excitatory chemical synapse since: all IPSPs occurred with latencies greater than 5 ms, there was no evidence for electrical coupling, and one of the interneurons produced facilitating PSPs. A majority of all monosynaptic connections were made by two of the flexion producing interneurons (FPIs), 201 and 301. The synaptic outputs of these FPIs were similar in that both made monosynaptic connections with a different bilaterally homologous pair of motoneurons. Both also produced larger EPSPs and more vigorous spiking in contralateral members of the bilateral motoneuron pairs. A previous study demonstrated that interneurons 201 and 301 are the only postural interneurons yet identified that express motor programs indistinguishable from command neurons. Taken together, these results suggest that certain intersegmental interneurons share properties with command neurons and driver neurons, and that there may not be a sharp morphological or functional distinction between these two cell types.     

Convergence of mechanosensory inputs onto neuromodulatory serotonergic neurons in the leech

By the frequency-dependent release of serotonin, Retzius neurons in the leech modulate diverse behavioral responses of the animal. However, little is known about how their firing pattern is produced. Here we have analyzed the effects of mechanical stimulation of the skin and intracellular stimulation of mechanosensory neurons on the electrical activity of Retzius neurons. We recorded the electrical activity of neurons in ganglia attached to their corresponding skin segment by segmental nerve roots, or in isolated ganglia. Mechanosensory stimulation of the skin induced excitatory synaptic potentials (EPSPs) and action potentials in both Retzius neurons in a ganglion. The frequency and duration of responses depended on the strength and duration of the skin stimulation. Retzius cells responded after T and P cells, but before N cells, and their sustained responses correlated with the activity of P cells. Trains of five impulses at 10 Hz in every individual T, P, or N cell in isolated ganglia produced EPSPs and action potentials in Retzius neurons. Responses to T cell stimulation appeared after the first impulse. In contrast, the responses to P or N cell stimulation appeared after two or more presynaptic impulses and facilitated afterward. The polysynaptic nature of all the synaptic inputs was shown by blocking them with a high calcium/magnesium external solution. The rise time distribution of EPSPs produced by the different mechanosensory neurons suggested that several interneurons participate in this pathway. Our results suggest that sensory stimulation provides a mechanism for regulating serotonin-mediated modulation in the leech.     

Synaptic Integration in Electrically Coupled Neurons

Interactions among chemical and electrical synapses regulate the patterns of electrical activity of vertebrate and invertebrate neurons. In this investigation we studied how electrical coupling influences the integration of excitatory postsynaptic potentials (EPSPs). Pairs of Retzius neurons of the leech are coupled by a nonrectifying electrical synapse by which chemically induced synaptic currents flow from one neuron to the other. Results from electrophysiology and modeling suggest that chemical synaptic inputs are located on the coupled neurites, at 7.5 μm from the electrical synapses. We also showed that the space constant of the coupled neurites was 100 μm, approximately twice their length, allowing the efficient spread of synaptic currents all along both coupled neurites. Based on this cytoarchitecture, our main finding was that the degree of electrical coupling modulates the amplitude of EPSPs in the driving neurite by regulating the leak of synaptic current to the coupled neurite, so that the amplitude of EPSPs in the driving neurite was proportional to the value of the coupling resistance. In contrast, synaptic currents arriving at the coupled neurite through the electrical synapse produced EPSPs of constant amplitude. This was because the coupling resistance value had inverse effects on the amount of current arriving and on the impedance of the neurite. We propose that by modulating the amplitude of EPSPs, electrical synapses could regulate the firing frequency of neurons.     

Magnesium-resistant excitatory synaptic potentials in the leech Retzius cell.

Postsynaptic potentials (PSPs) recorded from leech Retzius cells in response to stimulation of interganglionic connective could not be reversed by soma depolarization or abolished by 40 mM Mg ion, nor could input resistance changes be detected during them. Alteration of external Cl and K over a tenfold range provided no clear evidence that the PSPs involved a conductance change to either ion. The method of extrapolation yielded an apparent PSP equilibrium potential of about -20 mV. The steep portion of the relationship between Retzius cell action potential amplitude and membrane potential extrapolated to an apparent reversal potential of -13 mV. It is likely that the connective-to-Retzius cell PSPs were principally electrical events. Their apparent reversal potentials could have been in the range associated with chemical synapses because they traversed an electrical synapse with a variable coupling resistance, or because the polarizing currents, passing "backwards" across electrical synapses, changed the amplitude of the presynaptic action potentials.     

Magnesium-resistant excitatory synaptic potentials in the leech retzius cell

Postsynaptic potentials (PSPs) recorded from leech Retzius cells in response to stimulation of interganglionic connective could not be reversed by soma depolarization or abolished by 40 mM Mg ion, nor could input resistance changes be detected during them. Alteration of external Cl and K over a tenfold range provided no clear evidence that the PSPs involved a conductance change to either ion. The method of extrapolation yielded an apparent PSP equilibrium potential of about ?20 mV. The steep portion of the relationship between Retzius cell action potential amplitude and membrane potential extrapolated to an apparent reversal potential of ?13 mV. It is likely that the connective-to-Retzius cell PSPs were principally electrical events. Their apparent reversal potentials could have been in the range associated with chemical synapses because they traversed an electrical synapse with a variable coupling resistance, or because the polarizing currents, passing “backwards” across electrical synapses, changed the amplitude of the presynaptic action potentials.     

Differential channeling of sensory stimuli onto a motor neuron in the leech

We studied a specific sensory-motor pathway in the isolated leech ganglia. Pressure-sensitive mechanosensory neurons were stimulated with trains of action potentials at 5–20 Hz while recording the responses of the annulus erector motorneurons that control annuli erection. The response of the annulus erector neurons was a succession of excitatory postsynaptic potentials followed by inhibitory postsynaptic potentials. The excitatory postsynaptic potentials had a brief time-course while the inhibitory postsynaptic potentials had a prolonged time-course that enabled their temporal summation. Thus, the net effect of pressure-sensitive neuron stimulation on the annulus erector neurons was inhibitory. Both phases of the response were mediated by chemical transmission; the excitatory postsynaptic potentials were transmitted via a monosynaptic pathway, and the inhibitory postsynaptic potentials via a polysynaptic one. The pattern of expression of this dual response depended on the field of innervation of the sensory neuron and it was under the influence of cell 151, a non-spiking interneuron, that could regulate the expression of the hyperpolarization. The interaction between pressure-sensitive neurons and annulus erector neuron reveals how sensory specificity, connectivity pattern and regulatory elements interplay in a specific sensory-motor network. Accepted: 6 November 1998     

Synaptic effects evoked by supraspinal and intraspinal stimulation in lamprey motoneurons

In experiments onLampetra fluviatilis in response to electrical stimulation of bulbar reticulospinal neurons and descending fibers the postsynaptic potentials of segmental motoneurons and action potentials of single intraspinal axons were recorded intracellularly and the cord dorsum potentials were recorded by a surface electrode. Fast-conducting reticulospinal axons (Müller's axons) were shown to excite spinal motoneurons monosynaptically. Monosynaptic reticulo-motoneuronal EPSPs arise as the result of excitation of a limited number of descending fibers, they reproduce high frequencies of stimulation readily and, in some cases, they are divided into components of which the first may be attributed to an electrical, and the second to a chemical mechanism of transmission. Besides early monosynaptic EPSPs, late, probably polysynaptic, responses also are found.     

Paired-recordings from synaptically coupled cortical and hippocampal neurons in acute and cultured brain slices

Analysis of synaptic transmission, synaptic plasticity, axonal processing, synaptic timing or electrical coupling requires the simultaneous recording of both the pre- and postsynaptic compartments. Paired-recording technique of monosynaptically connected neurons is also an appropriate technique to probe the function of small molecules (calcium buffers, peptides or small proteins) at presynaptic terminals that are too small to allow direct whole-cell patch-clamp recording. We describe here a protocol for obtaining, in acute and cultured slices, synaptically connected pairs of cortical and hippocampal neurons, with a reasonably high probability. The protocol includes four main stages (acute/cultured slice preparation, visualization, recording and analysis) and can be completed in approximately 4 h.     

Effects of met-enkephalin on electrical coupling between identified neurons in the pulmonate snails,Helix and Lymnaea

1. The effects of met-enkephalin on electrical coupling between molluscan neurons have been investigated using the isolated brains of Helix pomatia and Lymnaea stagnalis.2. In the presence of both serotonin and met-enkephalin, non-rectifying electrical coupling is strongly facilitated between identified respiratory neurons in Helix, whilst coupling between putative, serotonin-containing, ciliomotoneurons in Lymnaea is facilitated by met-enkephalin alone.3. Facilitation of coupling by met-enkephalin is weaker in the strongly coupled neurons, VDl/RPaD2 of Lymnaea.4. These data suggest that met-enkephalin can modulate different groups of electrically coupled cells and may be involved in coordination of motor patterns.     

Anterograde and retrograde effects of synapse formation on calcium currents and neurite outgrowth in cultured leech neurons.

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)     

The A-like potassium current of a leech neuron increases with age in cell culture

Single leech neurons isolated and maintained in culture sprout and form electrical and chemical synapses, as they do in vivo, retaining most of the electrical properties of the intact membrane. However, some cells, such as Retzius, Anterior Pagoda (AP) cells and motoneurons, exhibit consistent changes of biophysical characteristics, which mimic those induced by axotomy in vivo and are reversed after reconnection. To improve our understanding of the mechanisms involved in these alterations and of their physiological significance, we investigated the early changes in outward currents developed by cultured AP neurons, using the patch-clamp technique in the whole-cell recording configuration. Different currents were isolated and a differential sensitivity to the time spent in culture and to internal calcium was observed. Three potassium currents were dissected: an A-like current, a delayed rectifier and a third unidentified component. The A-like potassium current was significantly increased with neuronal age in cell culture and was a function of the internal Ca²⁺ concentration, whereas the two other potassium currents remained unchanged. Intracellular recordings performed from axotomized neurons of cultured ganglia revealed clear-cut alterations in spike adaptation, which might be due to changes of the A-like current. Accepted: 24 September 1998     

Calcium spikes in a leech nonspiking neuron

The NS neurons are nonspiking cells, present as pairs in each midbody ganglion of the leech nervous system, which display a very extensive arborization. They were shown to regulate the coactivation of motoneurons. Here we have investigated the electrophysiological properties of these neurons under the hypothesis that transmission along the extensive neurites requires the aid of voltage-dependent conductances. The results indicate that NS neurons respond to electrical stimulation with a spike-like event, which was not an all-or-none but rather a graded phenomenon that depended on the intensity and duration of the electrical stimulus. The spike-like response was activated at a membrane potential of approximately −50 mV; its amplitude was a logarithmic function of the extracellular Ca²⁺ concentration and was unaffected by a broad range of changes in the extracellular Na⁺ concentration; intracellular application of tetraethylammonium (TEA) caused a large increase in its amplitude and duration. These data indicate that NS neurons bear voltage-dependent low-threshold Ca²⁺ and TEA-sensitive K⁺ conductances that could contribute to shaping synaptic signals, or transmission along the extensive neuritic tree.     

Development of spinal motor networks in the chick embryo.

We have examined the cellular and synaptic mechanisms underlying the genesis of alternating motor activity in the developing spinal cord of the chick embryo. Experiments were performed on the isolated lumbosacral cord maintained in vitro. Intracellular and whole cell patch clamp recordings obtained from sartorius (primarily a hip flexor) and femorotibialis (a knee extensor) motoneurons showed that both classes of cell are depolarized simultaneously during each cycle of motor activity. Sartorius motoneurons generally fire two bursts/cycle, whereas femorotibialis motoneurons discharge throughout their depolarization, with peak activity between the sartorius bursts. Voltage clamp recordings revealed that inhibitory and excitatory synaptic currents are responsible for the depolarization of sartorius motoneurons, whereas femorotibialis motoneurons are activated principally by excitatory currents. Early in development, the dominant synaptic currents in rhythmically active sartorius motoneurons appear to be inhibitory so that firing is restricted to a single, brief burst at the beginning of each cycle. In E7-E13 embryos, lumbosacral motor activity could be evoked following stimulation in the brainstem, even when the brachial and cervical cord was bathed in a reduced calcium solution to block chemical synaptic transmission. These findings suggest that functional descending connections from the brainstem to the lumbar cord are present by E7, although activation of ascending axons or electrical synapses cannot be eliminated. Ablation, optical, and immunocytochemical experiments were performed to characterize the interneuronal network responsible for the synaptic activation of motoneurons. Ablation experiments were used to show that the essential interneuronal elements required for the rhythmic alternation are in the ventral part of the cord. This observation was supported by real-time Fura-2 imaging of the neuronal calcium transients accompanying motor activity, which revealed that a high proportion of rhythmically active cells are located in the ventrolateral part of the cord and that activity could begin in this region. The fluorescence transients in the majority of neurons, including motoneurons, occurred in phase with ventral root or muscle nerve activity, implying synchronized neuronal action in the rhythm generating network. Immunocytochemical experiments were performed in E14-E16 embryos to localize putative inhibitory interneurons that might be involved in the genesis or patterning of motor activity. The results revealed a pattern similar to that seen in other vertebrates with the dorsal horn containing neurons with gamma-aminobutyric acid (GABA)-like immunoreactivity and the ventral and intermediate regions containing neurons with glycine-like immunoreactivity.     

Differential modulation of chemical and electrical components of mixed synapses in the lobster stomatogastric ganglion

1. Two pairs of neurons in the pyloric network of the spiny lobster, Panulirus interruptus, communicate through mixed graded chemical and rectifying electrical synapses. The anterior burster (AB) chemically inhibits and is electrically coupled to the ventricular dilator (VD); the lateral pyloric (LP) and pyloric (PY) neurons show reciprocal chemical inhibition and electrical coupling. We examined the effects of dopamine (DA), serotonin (5HT) and octopamine (Oct) on these mixed synapses to determine the plasticity possible with opposing modes of synaptic interaction.
2. Dopamine increased net inhibition at all three pyloric mixed synapses by both reducing electrical coupling and increasing chemical inhibition. This reversed the sign of the net synaptic interaction when electrotonic coupling dominated some mixed synapses, and activated silent chemical components of other mixed synapses.
3. Serofonin weakly enhanced LP → PY net inhibition, by reducing electrical coupling without altering chemical inhibition. Serotonin reduced AB→ VD electrical coupling, but variability in its effect on the chemical component made the net effect non-significant.
4. Octopamine enhanced LP→ PY and PY→ LP net inhibition by enhancing the chemical inhibitory component without altering electrical coupling.
5. Differential modulation of chemical and electrical components of mixed synapses markedly changes the net synaptic interactions. This contributes to the flexible outputs that modulators evoke from anatomically defined neural networks.

     

Locust primary neuronal culture for the study of synaptic transmission

We have designed a cell culture system for thoracic neurons of adult Locusta migratoria that enables the establishment of functional synapses in vitro. Patch-clamp recordings revealed three different neuron classes. About half of the neurons (47%) had unexcitable somata with outward and no inward conductance. The other half generated either single (37%) or multiple action potentials (18%) and differed mainly in lower outward conductance. Selectively stained motor neurons were analyzed to demonstrate varied physiological properties due to culture conditions. Using paired patch clamp recordings we demonstrate directly synaptic transmission in morphologically connected neurons in vitro. Presynaptic stimulation resulted in postsynaptic potentials in 42 pairs of neurons tested, independent of the type of neuron. According to pharmacological experiments most of these synapses were either glutamatergic or GABAergic. In addition to these chemical synapses, electrical synapses were found. With the demonstration of synapse formation in cell culture of adult locust neurons, this study provides the basis for the future analysis of more defined insect neuronal circuits in culture.     

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).     

An electrically coupled network of skeletal muscle in zebrafish distributes synaptic current

Fast and slow skeletal muscle types are readily distinguished in larval zebrafish on the basis of differences in location and orientation. Additionally, both muscle types are compact, rendering them amenable to in vivo patch clamp study of synaptic function. Slow muscle mediates rhythmic swimming, but it does so purely through synaptic drive, as these cells are unable to generate action potentials. Our patch clamp recordings from muscle pairs of zebrafish reveal a network of electrical coupling in slow muscle that allows sharing of synaptic current within and between segmental boundaries of the tail. The synaptic current exhibits slow kinetics (tau(decay) approximately 4 ms), which further facilitates passage through the low pass filter, a consequence of the electrically coupled network. In contrast to slow muscle, fast skeletal muscle generates action potentials to mediate the initial rapid component of the escape response. The combination of very weak electrical coupling and synaptic kinetics (tau(decay) <1 ms) too fast for the network low pass filter minimizes intercellular sharing of synaptic current in fast muscle. These differences between muscle types provide insights into the physiological role(s) of electrical coupling in skeletal muscle. First, intrasegmental coupling among slow muscle cells allows effective transfer of synaptic currents within tail segments, thereby minimizing differences in synaptic depolarization. Second, a fixed intersegmental delay in synaptic current transit, resulting from the low pass filter properties of the slow muscle network, helps coordinate the rostral-caudal wave of contraction.     

Calcium influx into dendrites of the leech Retzius neuron evoked by 5-hydroxytryptamine

5-Hydroxytryptamine (5-HT) is a ubiquitous neurotransmitter and neuromodulator that affects neural circuits and behaviours in vertebrates and invertebrates. In the present study, we have investigated 5-HT-induced Ca(2+) transients in subcellular compartments of Retzius neurons in the leech central nervous system using confocal laser scanning microscopy, and studied the effect of 5-HT on the electrical coupling between the Retzius neurons. Bath application of 5-HT (50mM) induced a Ca(2+) transient in axon, dendrites and cell body of the Retzius neuron. This Ca(2+) transient was significantly faster and larger in dendrites than in axon and cell body, and was half-maximal at a 5-HT concentration of 5-12mM. The Ca(2+) transient was suppressed in the absence of extracellular Ca(2+) and by methysergide (100mM), a non-specific antagonist of metabotropic 5-HT receptors, and was strongly reduced by bath application of the Ca(2+) channel blocker Co(2+) (2mM). Injection of the non-hydrolysable GTP analogue GTPgammaS increased and prolonged the dendritic 5-HT-induced Ca(2+) transient. The non-selective protein kinase inhibitor H7 (100mM) and the adenylate cyclase inhibitor SQ22536 (500 mM) did not affect the Ca(2+) transient, and the membrane-permeable cAMP analogue dibutyryl-cAMP (500 mM) did not mimic the effect of 5-HT application. 5-HT reduced the apparent electrical coupling between the two Retzius neurons, whereas suppression of the Ca(2+) influx by removal of external Ca(2+) improved the transmission of action potentials at the electrical synapses which are located between the dendrites of the adjacent Retzius neurons. The results indicate that 5-HT induces a Ca(2+) influx through calcium channels located primarily in the dendrites, and presumably activated by a G protein-coupled 5-HT receptor. The dendritic Ca(2+) increase appears to modulate the excitability of, and the synchronization between, the two Retzius neurons.     

Effect of calcium deficiency and addition of calcium antagonists on motoneuron synaptic potentials of isolated Emys orbicularis turtle spinal cord

In experiments carried out on the isolated spinal cord of the tortoise Emys orbicularis postsynaptic potentials produced in spinal motoneurons by stimulation of the descending tracts and dorsal roots were investigated by means of the intracellular recording technique. Postsynaptic potentials were completely and reversibly blocked in Ca2+-free solutions containing 5.0 mM Mg2+ or 2.0 mM Mn2+. The amplitude and frequency of spontaneous synaptic potentials were also reduced under these conditions. The effect of Ca2+-free medium indicates that the synaptic transmission in these synapses is mediated by chemical mechanism.