Biomedical subjects

P R Benjamin

Publications and source records attributed to P R Benjamin.

At least 37 records · Page 2Linked to original sources

PubMed1998

Neural modulation of gut motility by myomodulin peptides and acetylcholine in the snail Lymnaea.

Families of peptide neuromodulators are believed to play important roles in neural networks that control behaviors. Here, we investigate the expression and role of one such group of modulators, the myomodulins, in the feeding system of Lymnaea stagnalis. Using a combination of in situ hybridization and antibody staining, expression of the myomodulin gene was confirmed in a number of identified behaviorally significant neuronal types, including the paired B2 motor neurons. The B2 cells were shown to project axons to the proesophagus, where they modulate foregut contractile activity. The presence of the five myomodulin peptide structures was confirmed in the B2 cells, the proesophagus, and the intervening nerve by mass spectrometry. Using a sensitive cell culture assay, evidence that the B2 cells are cholinergic also is presented. Application of four of the five myomodulin peptides to the isolated foregut increased both contraction frequency and tonus, whereas the main effect of acetylcholine (ACh) application was a large tonal contraction. The fifth myomodulin peptide (pQIPMLRLamide) appeared to have little or no effect on gut motility. Coapplication of all five myomodulin peptides gave a greater increase in tonus than that produced by the peptides applied individually, suggesting that corelease of the peptides onto the gut would produce an enhanced response. The combined effects that the myomodulin peptides and ACh have on foregut motility can mimic the main actions of B2 cell stimulation.

Acetylcholine↗

PubMed1998

Neurophysiological correlates of unconditioned and conditioned feeding behavior in the pond snail Lymnaea stagnalis.

We used a behavioral appetitive learning paradigm followed by electrophysiological analysis to investigate the neuronal expression of appetitive conditioning in Lymnaea. We first established the levels of unconditioned and conditioned feeding responses in intact animals. We then demonstrated that neuronal correlates of both unconditioned responses to touch and food and a conditioned response to touch could be found in semi-intact preparations of the same animals that had been subjected to behavioral tests and conditioning trials. In the conditioning experiments, the experimental animals received 15 trials in which touch to the lips, the conditioned stimulus (CS), was paired with sucrose, the unconditioned food stimulus (US). Control animals received 15 presentations of either CS or US, or both, applied in a random manner. After training, a strong conditioned response to touch was established in the experimental but not in the control groups. For subsequent electrophysiological analysis of posttraining neuronal responses to the touch CS, semi-intact preparations were set up from the same animals that had been behaviorally conditioned or subjected to control procedures. Intracellular recordings, made from previously identified motoneurons of the feeding system, allowed the fictive feeding response to the CS to be monitored. In experimental preparations, touch applied to the lips evoked significantly more fictive feeding cycles than in controls, and this demonstrated the existence of a neurophysiological correlate of the appetitively conditioned response observed in the whole animals.

Animals↗

PubMed1997

In vitro appetitive classical conditioning of the feeding response in the pond snail Lymnaea stagnalis.

In vitro appetitive classical conditioning of the feeding response in the pond snail Lymnaea stagnalis. J. Neurophysiol. 78: 2351-2362, 1997. An in vitro preparation was developed that allowed electrophysiological analysis of appetitive conditioning of feeding in the model molluscan system, Lymnaea. The network generating the feeding motor program (fictive feeding) is well characterized at the cellular level and consists of identified central pattern generator (CPG) interneurons, motor neurons, and modulatory interneurons. Activation of a modulatory interneuron, the slow oscillator (SO), evokes the three-phase fictive feeding rhythm in the same semi-intact preparations where tactile stimuli can be applied to the lips. By pairing touch as a conditioned stimulus (CS) with stimulation of the SO as an unconditioned stimulus (US), we established an effective in vitro paradigm for appetitive conditioning. Before training, touch to the lips evoked only brief and weak activity in the feeding interneurons and motor neurons. After 6-10 conditioning trials, there was a significant enhancement in the fictive feeding response to CS alone. This was not seen in controls (CS only, US only, random CS and US) and in preparations where there was no initial brief response to touch before conditioning. Direct recordings from the protraction phase N1M interneurons during in vitro conditioning indicated that the enhancement of the fictive feeding is due to an increased activation of these CPG cells by mechanosensory inputs from the lips. We also found that the conditioned response was not due to a facilitated activation of modulatory neurons in the feeding network, such as the SO or the cerebral giant cells (CGCs), because the activity of these cells remained unchanged after conditioning.

Animals↗

PubMed1997

Behavioral function of glutamatergic interneurons in the feeding system of Lymnaea: plateauing properties and synaptic connections with motor neurons.

Intracellular recording techniques were used to examine the electrical properties and behavioral function of a novel type of retraction phase interneuron, the N2 ventral (N2v) cells in the feeding network of the snail Lymnaea. The N2vs were compared with the previously identified N2 cells that now are renamed the N2 dorsal (N2d) cells. The N2vs are a bilaterally symmetrical pair of electrotonically coupled plateauing interneurons that are located on the ventral surfaces of the buccal ganglia. Their main axons project to the opposite buccal ganglion, but they have an additional fine process in the postbuccal nerve. N2v plateaus that outlast the duration of the stimulus can be triggered by depolarizing current pulses and prematurely terminated by applied hyperpolarizing pulses. Gradually increasing the amplitude of depolarizing pulses reveals a clear threshold for plateau initiation. N2v plateauing persists in a high Mg2+/nominally zero Ca2+ saline that blocks chemical synaptic connections, suggesting an endogenous mechanism for plateau generation. The N2vs fire sustained bursts of action potentials throughout the N2/rasp phase of the fictive feeding cycle and control the retraction phase feeding motor neurons. The N2vs excite the B3 and B9 feeding motor neurons to fire during the rasp phase of the feeding cycle. They also inhibit the B7 and B8 feeding motor neurons. The B8 cells recover from inhibition and fire during the following swallowing phase. These synaptic connections appear to be monosynaptic as they persist in high Mg2+/high Ca2+ (HiDi) saline that blocks polysynaptic pathways. Strong current-induced plateaus in the N2vs generate brief inhibitory postsynaptic responses in the B4CL rasp phase motor neurons, but this was due to the indirect N2v --> N2d --> B4CL pathway. The N2vs are coupled electrotonically to the N2d cells, and triggering plateau in a N2v usually induced one or two spikes in a N2d. Previous experiments showed that the N2ds generate plateau potentials during a fictive feeding cycle. Here we show that the main component of the "plateauing" waveform is due to the electrotonic coupling with the N2v cells. The differential synaptic connections of the N2v and N2d cells with retraction phase motor neurons results in a sequence of motor neuron burst activity B9 --> B4CL --> B8 that produces the full retraction (rasp --> swallow) movements of the feeding apparatus (buccal mass). We conclude that the N2v cells are an essential component of the interneuronal network required to produce feeding motor neuron activity.

Animals↗

PubMed1997

Glutamatergic N2v cells are central pattern generator interneurons of the lymnaea feeding system: new model for rhythm generation.

We aimed to show that the paired N2v (N2 ventral) plateauing cells of the buccal ganglia are important central pattern generator (CPG) interneurons of the Lymnaea feeding system. N2v plateauing is phase-locked to the rest of the CPG network in a slow oscillator (SO)-driven fictive feeding rhythm. The phase of the rhythm is reset by artificially evoked N2v bursts, a characteristic of CPG neurons. N2v cells have extensive input and output synaptic connections with the rest of the CPG network and the modulatory SO cell and cerebral giant cells (CGCs). Synaptic input from the protraction phase interneurons N1M (excitatory), N1L (inhibitory), and SO (inhibitory-excitatory) are likely to contribute to a ramp-shaped prepotential that triggers the N2v plateau. The prepotential has a highly complex waveform due to progressive changes in the amplitude of the component synaptic potentials. Most significant is the facilitation of the excitatory component of the SO --> N2v monosynaptic connection. None of the other CPG interneurons has the appropriate input synaptic connections to terminate the N2v plateaus. The modulatory function of acetylcholine (ACh), the transmitter of the SO and N1M/N1Ls, was examined. Focal application of ACh (50-ms pulses) onto the N2v cells reproduced the SO --> N2v biphasic synaptic response but also induced long-term plateauing (20-60 s). N2d cells show no endogenous ability to plateau, but this can be induced by focal applications of ACh. The N2v cells inhibit the N3 tonic (N3t) but not the N3 phasic (N3p) CPG interneurons. The N2v --> N3t inhibitory synaptic connection is important in timing N3t activity. The N3t cells recover from this inhibition and fire during the swallow phase of the feeding pattern. Feedback N2v inhibition to the SO, N1L protraction phase interneurons prevents them firing during the retraction phase of the feeding cycle. The N2v --> N1M synaptic connection was weak and only found in 50% of preparations. A weak N2v --> CGC inhibitory connection prevents the CGCs firing during the rasp (N2) phase of the feeding cycle. These data allowed a new model for the Lymnaea feeding CPG to be proposed. This emphasizes that each of the six types of CPG interneuron has a unique set of synaptic connections, all of which contribute to the generation of a full CPG pattern.

Acetylcholine↗

PubMed1997

Glutamate is the transmitter for N2v retraction phase interneurons of the Lymnaea feeding system.

Electrophysiological and pharmacological methods were used to examine the role of glutamate in mediating the excitatory and inhibitory responses produced by the N2v rasp phase neurons on postsynaptic cells of the Lymnaea feeding network. The N2v --> B3 motor neuron excitatory synaptic response could be mimicked by focal or bath application of -glutamate at concentrations of >/=10(-3) M. Quisqualate and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) were potent agonists for the B3 excitatory glutamate receptor (10(-3) M), whereas kainate only produced very weak responses at the same concentration. This suggested that non-N-methyl--aspartate (NMDA), AMPA/quisqualate receptors were present on the B3 cell. The specific non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 10(-5) M) blocked 85% of the excitatory effects on the B3 cell produced by focal application of glutamate (10(-3) M), confirming the presence of non-NMDA receptors. CNQX also blocked the major part of the excitatory postsynaptic potentials on the B3 cell produced by spontaneous or current-evoked bursts of spikes in the N2v cell. As with focal application of glutamate, a small delayed component remained that was CNQX insensitive. This provided direct evidence that glutamate acting via receptors of the non-NMDA, AMPA/quisqualate type were responsible for mediating the main N2v --> B3 cell excitatory response. NMDA at 10(-2) M also excited the B3 cell, but the effects were much more variable in size and absent in one-third of the 25 B3 cells tested. NMDA effects on B3 cells were not enhanced by bath application of glycine at 10(-4) M or reduction of Mg2+ concentration in the saline to zero, suggesting the absence of typical NMDA receptors. The variability of the B3 cell responses to NMDA suggested these receptors were unlikely to be the main receptor type involved with N2v --> B3 excitation. Quisqualate and AMPA at 10(-3) M also mimicked N2v inhibitory effects on the B7 and B8 feeding motor neurons and the modulatory slow oscillator (SO) interneuron, providing further evidence for the role of AMPA/quisqualate receptors. Similar effects were seen with glutamate at the same concentration. However, CNQX could not block either glutamate or N2v inhibitory postsynaptic responses on the B7, B8, or SO cells, suggesting a different glutamate receptor subtype for inhibitory responses compared with those responsible for N2v --> B3 excitation. We conclude that glutamate is a strong candidate transmitter for the N2v cells and that AMPA/quisquate receptors of different subtypes are likely to be responsible for the excitatory and inhibitory postsynaptic responses.

6-Cyano-7-nitroquinoxaline-2,3-dione↗

PubMed1996

Myomodulin gene of Lymnaea: structure, expression, and analysis of neuropeptides.

The myomodulin family of neuropeptides is an important group of neural cotransmitters in molluscs and is known to be present in the neural network that controls feeding behavior in the snail Lymnaea. Here we show that a single gene encodes five structurally similar forms of myomodulin: GLQMLRLamide, QIPMLRLamide, SMSMLRLamide, SLSMLRLamide, and PMSMLRLamide, the latter being present in nine copies. Analysis of the organization of the gene indicates that it is transcribed as a single spliced transcript from an upstream promoter region that contains multiple cAMP-responsive elements, as well as putative elements with homology to tissue-specific promoter-binding sites. The presence in nervous tissue of two of the peptides, GLQMLRLamide and PMSMLRLamide, is confirmed by mass spectrometry. In situ hybridization analysis indicates that the gene is expressed in specific cells in all ganglia of the CNS of Lymnaea, which will allow physiological analysis of the function of myomodulins at the level of single identified neurons.

Amino Acid Sequence↗

PubMed1996

Met-enkephalin Arg-Phe-immunoreactive neurons in the central nervous system of the pond snail Lymnaea stagnalis.

The distribution of an opioid peptide related to YGGFMRF was determined in the CNS and other organs of the pond snail, Lymnaea stagnalis, by RIA and immunocytochemistry. RIA revealed the highest levels in the CNS (1 pmol/organ) and penis (400 fmol/organ). There were also significant levels in the haemolymph, most of which was not associated with haemocytes (580 fmol/ml). Both serial section and whole-mount immunocytochemistry of the CNS revealed immunoreactive cells in every ganglion with the majority in the cerebral and pedal ganglia. In the pedal ganglia some of the immunoreactive cells were close to the cells of the A-cluster, which are known to respond to opioids, and could innervate them. In the cerebral ganglia the immunoreactive cells included a group of neurosecretory cells, the caudo dorsal cells (CDCs) and the terminals of these cells in the cerebral commissure were also stained. The CDCs secrete peptides into the haemolymph and so could be the source of the YGGFMRF immunoreactivity. Immunoreactivity (including the CDCs) was observed in locations that correspond to those reported for other fragments of proenkephalin, such as Met- and Leu-enkephalin, suggesting that they may share a common precursor, a Lymnaea proenkephalin. A map of the 358 YGGFMRF-immunoreactive cells in the CNS is presented, many of which have not been previously identified.

Amino Acid Sequence↗

PubMed1996

Post-translational processing of the alternative neuropeptide precursor encoded by the FMRFamide gene in the pulmonate snail Lymnaea stagnalis.

The neuropeptide gene encoding FMRFamide-like peptides in the pulmonate mollusc Lymnaea is subject to alternative splicing that generates cell-specific expression of distinct sets of peptides in the CNS. In this paper, we analyse the post-translational processing of the alternative protein precursor encoded by the exon I, III-V transcript (type 2 transcript). We raised anti-peptide antisera specific to distinct segments of the precursor in order to address the pattern of endoproteolytic cleavages, specifically around the tetrabasic site RRKR. We first showed that not all peptides predicted by the precursor structure are generated as final steady-state products. We then identified a novel peptide by biochemical purification, amino acid sequencing and mass spectrometry- the 35 amino acid SDPFFRFGKQQVATDDSGELDDEILSRVSDDDKNI, which we termed the acidic peptide, previously not predicted on the basis of the precursor structure. This novel peptide, abundant in the snail brain (0.7 pmol per central nervous system), includes the N-terminal sequence SDPFFRF, which was previously considered to be a variant of the known heptapeptide SDPFLRFamide, also encoded within the same protein precursor. We showed by in situ hybridization and immunocytochemistry that the acidic peptide is produced in all cells that transcribe type 2 FMRFamide mRNA. We mapped the expression of this novel peptide in the CNS and localized it mainly in three identifiable neuronal clusters - the E, F and B groups of cells - and some additional neurons, all situated in three of the eleven central ganglia. Immunoreactive neurons included the single identifiable visceral white interneuron (VWI or VD4), a key cell of the cardiorespiratory network.

Amino Acid Sequence↗

PubMed1996

Central pattern generator interneurons are targets for the modulatory serotonergic cerebral giant cells in the feeding system of Lymnaea.

1. The objective of the experiments was to explore the modulatory functions of the serotonergic cerebral giant cells (CGCs) of the Lymnaea feeding system by examining their synaptic connections with the central pattern generator (CPG) interneurons and the modulatory slow oscillator (SO) interneuron. 2. One type of modulatory function, "gating," requires that the CGCs fire tonically at a minimum of 7 spikes/min. Above this minimum level the CGCs control the frequency of CPG interneuron oscillation-- "frequency control," a second type of modulation. In an SO-driven fictive feeding rhythm, an increase in the frequency of the rhythm, with increased CGC firing rate, resulted from a reduction in the duration of the N1 (protraction) and N2 (rasp) phases of the feeding cycle with little effect on the N3 (swallow) phase. 3. The CGCs excited the N1 phase interneurons SO and N1M (N1 medial) cells but had no consistent effects on the N1 lateral cells. The CGC-->SO postsynaptic response was probably monosynaptic (< or = 200 ms in duration) with unitary 1:1 excitatory postsynaptic potentials (EPSPs) following each CGC spike. The CGC-->N1M excitatory response was slow and nonunitary, and a burst of CGC spikes evoked a depolarization of the N1M cells that lasted up to 10 s and triggered N1M cell bursts. Both CGC-->SO and CGC-->N1M excitatory responses could be mimicked by the focal application of serotonin (5-HT). 4. Both CGC-->SO and CGC-->N1M excitatory connections systematically increased the N1M cell firing rate within the CGCs' physiological firing range (0-40 spikes/min). This was due to both the direct (CGC-->N1M) and indirect (CGC-->SO-->N1M) excitatory synaptic pathways. The CGC-induced increase in N1M cell firing rate probably accounted for the reduced duration of the N1M cell feeding burst by causing a more rapid reversal of the feeding cycle from the N1 phase to the N2 phase. This phase reversal was due to the previously described recurrent inhibitory pathway (N1-->N2 excitation followed by N2-->N1 inhibition). 5. The CGCs' ability to provide a depolarizing drive to the N1M cells meant that this excitatory connection was also likely to be important for gating. 6. Activity in the CGCs produced nonunitary, long-lasting, excitatory postsynaptic responses on the N2 ventral (N2v) CPG interneurons, and these were likely to be involved in both the gating and the frequency control by the CGCs on the N2 phase of the feeding rhythm. Suppressing CGC tonic firing initially increased the duration of the N2v plateau (which determines the duration of the N2 phase of the feeding cycle, frequency function) but eventually led to a loss of N2v plateauing (gating function). 7. Nonunitary, weakly inhibitory CGC-->N2 dorsal responses were recorded that could be mimicked by the application of 5-HT. 8. Spikes in the CGCs evoked 1:1 monosynaptic EPSPs in the N3 tonic (N3t) CPG interneurons. This excitatory effect could be mimicked by the application of 5-HT. Within the physiological range of CGC firing, this excitation did not appear to influence the firing rate of the N3t cells. 9. N3 phasic (N3p) CPG interneurons showed biphasic (hyperpolarizing followed by depolarizing) unitary responses to spikes evoked in the CGCs. The inhibitory synaptic response was maintained in a high-Ca2+/high-Mg2+ (Hi-Di) saline and was mimicked by the focal application of 5-HT, indicating that it was probably monosynaptic. The excitatory component was, however, reduced in a Hi-Di saline, indicating that it was probably polysynaptic. Suppressing the CGCs during an SO-driven feeding rhythm caused the N3p cells to fire less, suggesting that the removal of the excitatory component of the response might be significant. 10. We conclude that the general depolarizing effects of the CGCs on a number of the CP

Animals↗

PubMed1995

Identification, distribution and physiological activity of three novel neuropeptides of Lymnaea: EFLRlamide and pQFYRlamide encoded by the FMRFamide gene, and a related peptide.

We are interested in analysing the detailed modulation of defined neuronal systems by multiple neuropeptides encoded in the FMRFamide locus of the snail Lymnaea. Cloning of the FMRFamide gene has predicted the existence of two novel peptides previously unknown from biochemical analysis, the pentapeptides EFLRlamide and QFYRlamide. These peptides may form part of a new family of peptides sharing the sequence motif -FXRlamide. In this paper we adopt a novel approach to first identify and characterize -FXRlamide-like peptides in extracts from the central nervous system of Lymnaea. By a combination of high-performance liquid chromatography (HPLC) and continuous-flow fast atom bombardment mass spectrometry, we identify three novel peptides: EFLRlamide, pQFYRlamide and pQFLRlamide. The first two are those predicted in exon II of the FMRFamide locus whereas the last is, interestingly, a product which cannot be derived from post-translational modification of the predicted peptides but must be encoded by as yet unidentified nucleotide sequences. A specific antibody raised to EFLRlamide, and immunoreactive to all three peptides, revealed EFLRlamide-like expression throughout the central nervous system in the same cells where exon II is transcribed and the peptide SEEPLY (a post-translational product of exon II) was localized. Additional cells, however, were also identified. Immunoreactivity was mapped in a number of identified neurons in the central nervous system, including two heart cardioexcitatory motoneurons, the Ehe cells (E heart excitors of the visceral ganglion) and penial motoneurons in the right cerebral ganglion. The peripheral tissues (heart and penial complex) that these respective classes of neurons innervate also exhibited EFLRlamide immunoreactivity. The central and peripheral localization of EFLRlamide-like immunoreactivity suggested that EFLRlamide/pQFYRlamide may have an important physiological role in both these peripheral systems as well as in the central nervous system. This was confirmed by physiological experiments that showed that EFLRlamide and pQFYRlamide inhibited many central neurons and in particular the Bgp neurons in the right parietal ganglion. EFLRlamide had complex biphasic effects on the frequency of heart-beat: an initial inhibitory response was followed by a long-lasting increase in the rate of beating. Taken together with earlier work, this study now completes the analysis and localization of the full set of post-translational products of the FMRFamide precursor in Lymnaea and supplies further evidence towards the characterization of the physiological systems which such peptides may modulate in concert.

Amino Acid Sequence↗

PubMed1995

Alternative RNA splicing generates diversity of neuropeptide expression in the brain of the snail Lymnaea: in situ analysis of mutually exclusive transcripts of the FMRFamide gene.

In the CNS of the snail Lymnaea stagnalis, Phe-Met-Arg-Phe-amide (FMRFamide)-like and additional novel neuropeptides are encoded by a common, multi-exon gene. This complex locus, comprising at least five exons, is subject to post-transcriptional regulation at the level of alternative RNA splicing. Our aim was first to analyse the pattern by which exons of this neuropeptide locus combine during splicing of the primary RNA transcript, and second to investigate the functional significance of splicing by mapping the expression and neuronal localization in the CNS of the alternative mRNA transcripts, in the context of defined neuronal networks and single identified neurons. The approach was a combination of comparative in situ hybridization and immunocytochemistry, using a battery of exon-specific oligonucleotides and anti-peptide antisera. The analysis illustrated that exons III, IV and V were always coexpressed and colocalized whereas the expression of exon II was always differential and mutually exclusive. Both sets of exons were, however, coexpressed with exon I: the total number of exon I-expressing neurons was equal to the combined number of neurons expressing exon III/IV/V and neurons expressing exon II. In addition, it was revealed that the extreme 5' of exon II, encoding a potential hydrophobic leader signal, was not expressed in the CNS of Lymnaea but was apparently spliced out during RNA processing. Both mRNA transcripts of the FMRFamide locus, type 1 (exons I/II) and type 2 (exons I/III/IV/V), were translated in the CNS and the resulting protein precursors were also expressed in a mutually exclusive fashion, as were their respective transcripts. The expression of alternative transcripts within identified networks or neuronal clusters was heterogeneous, as exemplified by the cardiorespiratory network. On the basis of this work and a previous cDNA analysis, we put forward a revised model of differential splicing and expression of the FMRFamide gene in the CNS of Lymnaea.

Amino Acid Sequence↗

PubMed1995

Novel interneuron having hybrid modulatory-central pattern generator properties in the feeding system of the snail, Lymnaea stagnalis.

1. We used intracellular recording techniques to examine the role of a novel type of protraction phase interneuron, the lateral N1 (N1L) in the feeding system of the snail Lymnaea stagnalis. 2. The N1Ls are a bilaterally symmetrical pair of electrotonically coupled interneurons located in the buccal ganglia. Each N1L sends a single axon to the contralateral buccal ganglia. Their neurite processes are confined to the buccal neuropile. 3. In the isolated CNS, depolarization of an N1L is capable of driving a full (N1-->N2-->N3), fast (1 cycle every 5 s) fictive feeding rhythm. This was unlike the previously described N1 medial (N1M) central pattern generator (CPG) interneurons that were only capable of driving a slow, irregular rhythm. Attempts to control the frequency of the fictive feeding rhythm by injecting varying amounts of steady current into the N1Ls were unsuccessful. This contrasts with a modulatory neuron, the slow oscillator (SO), that has very similar firing patterns to the N1Ls, but where the frequency of the rhythm depends on the level of injected current. 4. The N1Ls' ability to drive a fictive feeding rhythm in the isolated preparation was due to their strong, monosynaptic excitatory chemical connection with the N1M CPG interneurons. Bursts of spikes in the N1Ls generated summating excitatory postsynaptic potentials (EPSPs) in the N1Ms to drive them to firing. The SO excited the N1M cells in a similar way, but the EPSPs are strongly facilitatory, unlike the N1L-->N1M connection. 5. Fast (1 cycle every 5 s) fictive feeding rhythms driven by the N1L occurred in the absence of spike activity in the SO modulatory neuron. In contrast, the N1L was usually active in SO-driven rhythms. 6. The ability of the SO to drive the N1L was due to strong electrotonic coupling, SO-->N1L. The weaker coupling in the opposite direction, N1L-->SO, did not allow the N1L to drive the SO. 7. Experiments on semintact lip-brain preparations allowed fictive feeding to be evoked by application of 0.1 M sucrose to the lips (mimicking the normal sensory input) rather than by injection of depolarizing current. Rhythmic bursting, characteristic of fictive feeding, began in both the SO and N1L at exactly the same time, indicating that these two cell types are activated in "parallel" to drive the feeding rhythm. 8. The N1L is also part of the CPG network. It Excited the N2s and inhibited the N3 phasic (N3p) and N3 tonic (N3t) CPG interneurons like the N1Ms.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1994

Direct peptide profiling by mass spectrometry of single identified neurons reveals complex neuropeptide-processing pattern.

A novel strategy combining peptide fingerprinting of single neurons by matrix-assisted laser desorption ionization mass spectrometry, molecular cloning, peptide chemistry, and electrospray ionization mass spectrometry was used to study the intricate processing pattern of a preprohormone expressed in identified neurons, the neuroendocrine light yellow cells (LYCs) of the gastropod mollusc, Lymnaea stagnalis. The cDNA encoding the precursor, named prepro-LYCP (LYCPs, light yellow cell peptides), predicts a straightforward processing into three peptides, LYCP I, II, and III, at conventional dibasic processing sites flanking the peptide domains on the precursor. However, matrix-assisted laser desorption ionization mass spectrometry of single LYCs revealed trimmed variant peptides derived from LYCP I and II. The variants were much more abundant than the intact peptides, indicating that LYCP I and II serve as intermediates in a peptide-processing sequence. Using the molecular masses of the peptides as markers to guide their isolation by well established purification methods, the structural identities of the peptides could be confirmed by amino acid sequencing. Furthermore, matrix-assisted laser desorption ionization mass spectrometry could detect colocalization of a novel peptide with the LYCPs.

Amino Acid Sequence↗

PubMed1994

Neuropeptides myomodulin, small cardioactive peptide, and buccalin in the central nervous system of Lymnaea stagnalis: purification, immunoreactivity, and artifacts.

The neuropeptides myomodulin, small cardioactive peptide (SCP), and buccalin are widely distributed in the phylum Mollusca and have important physiological functions. Here, we describe the detailed distribution of each class of peptide in the central nervous system (CNS) of the snail Lymnaea stagnalis by the use of immunocytochemical techniques combined with dye-marking of electrophysiologically identified neurons. We report the isolation and structural characterization of a Lymnaea myomodulin, PMSMLRLamide, identical to myomodulin A of Aplysia californica. Myomodulin immunoreactivity was localized in all 11 ganglia, in their connectives, and in peripheral nerves. In many cases, myomodulin immunoreactivity appeared localized in neuronal clusters expressing FMRFamide-like peptides, but also in a large number of additional neurons. Double-labelling experiments demonstrated myomodulin immunoreactivity in the visceral white interneuron, involved in regulation of cardiorespiration. SCP-like immunoreactivity also appeared in all ganglia, and double-labelling experiments revealed that in many locations it was specifically associated with clusters expressing distinct exons of the FMRFamide gene that are differentially expressed in the CNS. Characterization of the two types of SCP-antisera used in this study, however, suggested that they cross-reacted with both FMRFamide and N-terminally extended FMRFamide-like peptides. Selective preadsorption with these cross-reacting peptides resulted in elimination of the widespread staining and retention of bona fide SCP immunoreactivity in the buccal and pedal ganglia only. Buccalin immunoreactivity was limited to the buccal and pedal ganglia. It did not coincide with the distribution of either myomodulin or SCP. Most immunoreactive clusters were found in the pedal ganglia.

Amino Acid Sequence↗

PubMed1994

Neural network controlling feeding in Lymnaea stagnalis: immunocytochemical localization of myomodulin, small cardioactive peptide, buccalin, and FMRFamide-related peptides.

This paper investigates the distribution of four classes of neuropeptides, myomodulin, small cardioactive peptide (SCP), buccalin, and FMRFamide, in central neurons forming the network that underlies feeding behavior in the snail Lymnaea stagnalis. Intracellular dye-marking and immunocytochemical analysis, using antisera to the different classes of peptides, were applied to identified neurons of all three levels of the hierarchy of the circuitry: modulatory interneurons (cerebral giant cells, CGC; slow oscillator, SO), central pattern generator (CPG) interneurons (N1, N2, N3), motoneurons (B1-B10), and their peripheral target organs. Myomodulin immunoreactivity was detected in the CGC interneurons, in the SO, and in ventral N2-type CPG interneurons. Several large buccal motoneurons, the paired B1, B2, B3, B7, and neurons located in the dorsal posterior area (putative B4 cluster types) were also myomodulin immunoreactive. Target organs of buccal motoneurons, the buccal mass, salivary glands, and oesophagus contained myomodulin-immunopositive fibers. SCP appeared in N2-type interneurons and was found colocalized with myomodulin in the B1 and B2 motoneurons. SCP-containing neurons in the B4 cluster area were also detected. The buccal mass and salivary glands exhibited SCP-immunoreactive fibers. Buccalin immunoreactivity was scarce in the buccal ganglia and was identified only in N1-type interneurons and three pairs of dorsal posterior neurons. In the periphery, immunoreactive fibers were localized in the oesophagus only. None of the buccal neuronal types examined revealed immunoreactivity to SEQPDVDDYLRDVVLQSEEPLY ("SEEPLY"), a peptide encoded in the FMRFamide precursor protein of Lymnaea. SEEPLY immunoreactivity was confined to a pair of novel ventral neurons with projections to the laterobuccal nerve innervating the buccal mass. Immunoreactive fibers were also traced in this organ.

Amino Acid Sequence↗

PubMed1994

Alternative mRNA splicing of the FMRFamide gene and its role in neuropeptidergic signalling in a defined neural network.

Neuronal signalling involves multiple neuropeptides that are diverse in structure and function. Complex patterns of tissue-specific expression arise from alternate RNA splicing of neuropeptide-encoding gene transcripts. The pattern of expression and its role in cell signalling is difficult to study at the level of single neurons in the complex vertebrate brain. However, in the model molluscan system, Lymnaea, it is possible to show that alternate mRNA expression of the FMRFamide gene is specific to single identified neurons. Two different transcripts are expressed in a mutually exclusive manner in different neurons. Post-translational processing of the two precursor proteins leads to completely distinct sets of neuropeptide transmitters. The function of these transmitter cocktails, resulting from alternate mRNA splicing, was studied physiologically in identified neurons forming part of a behaviourally important network regulating heartbeat.

Alternative Splicing↗

PubMed1994

Functional morphology of the light yellow cell and yellow cell (sodium influx-stimulating peptide) neuroendocrine systems of the pond snail Lymnaea stagnalis.

Neuroendocrine light yellow cells of the pond snail Lymnaea stagnalis express a neuropeptide gene encoding three different peptides. The morphology of the cell system has been studied by in situ hybridization, using two synthetic oligonucleotides encoding parts of light yellow cell peptides I and III, and by immunocytochemistry with antisera to synthetic light yellow cell peptide II and to two fragments of light yellow cell peptide I. One large cluster of light yellow cells was observed in the ventro-lateral protrusion of the right parietal ganglion, smaller clusters lying in the posterior dorsal part of this ganglion and in the visceral ganglion. The cells had an extended central neurohaemal area. Immunopositive axons projected into all nerves of the ganglia of the visceral complex, into the superior cervical and the nuchal nerves, and into the connective tissue surrounding the central nervous system. Axon tracts ramified between the muscle cells of the walls of the anterior aorta and of smaller blood vessels. Peripheral innervation by the light yellow cell system was only found in muscular tissue of the ureter papilla. The antisera to the two peptide fragments of light yellow cell peptide I not only stained the light yellow cells, but also the identified yellow cells, which have previously been shown to produce the sodium influx-stimulating neuropeptide. The latter cells were negative to the in situ hybridization probes and antisera specific to the light yellow cell system. It is therefore unlikely that the yellow cells express the light yellow cell neuropeptide gene. Nevertheless, the cells contain a neuropeptide sharing antigenic determinants with light yellow cell peptide I.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗