Single-cell analysis of peptide expression and electrophysiology of right parietal neurons involved in male copulation behavior of a simultaneous hermaphrodite.

Male copulation is a complex behavior that requires coordinated communication between the nervous system and the peripheral reproductive organs involved in mating. In hermaphroditic animals, such as the freshwater snail Lymnaea stagnalis, this complexity increases since the animal can behave both as male and female. The performance of the sexual role as a male is coordinated via a neuronal communication regulated by many peptidergic neurons, clustered in the cerebral and pedal ganglia and dispersed in the pleural and parietal ganglia. By combining single-cell matrix-assisted laser mass spectrometry with retrograde staining and electrophysiology, we analyzed neuropeptide expression of single neurons of the right parietal ganglion and their axonal projections into the penial nerve. Based on the neuropeptide profile of these neurons, we were able to reconstruct a chemical map of the right parietal ganglion revealing a striking correlation with the earlier electrophysiological and neuroanatomical studies. Neurons can be divided into two main groups: (i) neurons that express heptapeptides and (ii) neurons that do not. The neuronal projection of the different neurons into the penial nerve reveals a pattern where (spontaneous) activity is related to branching pattern. This heterogeneity in both neurochemical anatomy and branching pattern of the parietal neurons reflects the complexity of the peptidergic neurotransmission involved in the regulation of male mating behavior in this simultaneous hermaphrodite.

The distinction between retractor and protractor muscles of the freshwater snail's male organ has no physiological basis.

Many animals are equipped with organs that can be everted, a notable example being male copulatory organs. The ability to protrude or evert an organ generally requires protractor and retractor muscles. Male copulatory behaviour of the pond snail Lymnaea stagnalis (L.) involves eversion (protraction) and retraction of the relatively large penis-carrying organ. For this preputium, protractor and retractor muscle bands have been defined, which implies eversion and retraction through the activity of these muscle bands. However, no physiological data are available that confirm that the terms protractor and retractor are appropriate. To test whether eversion and retraction are possible without protractor and/or retractor muscle bands, lesion experiments were performed. The results show that with either one or several muscle bands lesioned, snails were still capable of everting their preputium and using it for copulation. However, the majority of animals that had six or more muscle bands lesioned were unable to retract its preputium. Hence, retractor muscle bands serve their designated function whereas protractor muscle bands do not. We therefore suggest that a different terminology is used in which all muscle bands are retractors and, based on their location, are either called distal or proximal retractors. The findings furthermore indicate that the preputium muscle bands are normally contracted, possibly in a catch state, retaining the organ inside without high-energy expenditure.

Spatio-temporal dynamics of the egg-laying-inducing peptides during an egg-laying cycle: a semiquantitative matrix-assisted laser desorption/ionization mass spectrometry approach.

The activity-dependent release of peptides from the neuro-endocrine caudodorsal cell (CDC) system of the freshwater snail Lymnaea stagnalis regulates egg laying and related behaviors. In this study, we optimized a mass spectrometry-based approach to study the spatio-temporal dynamics of peptides that are largely derived from the CDC hormone precursor during an egg-laying cycle and a CDC discharge in vitro. Semi-quantitative peptide mass profiling using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) indicated a massive depletion of peptides from the neurohemal area in the cerebral commissure (COM) during egg laying and the existence of a reserve pool of peptides in the CDC somata that were transported to the COM to restore peptide levels. The depletion of CDC peptides from the COM was correlated to their release during an induced electrical discharge in vitro. Moreover, MALDI-MS of the releasate revealed extensive truncation of the carboxyl terminal peptide. Finally, two novel peptides of 1788 and 5895 Da, not encoded by the CDC hormone precursor, also exhibited temporal quantitative changes similar to those of CDC peptides. Sequencing of the peptide of 1788 Da by tandem mass spectrometry yielded the novel sequence HF(FH)FYGPYDVFQRDVamide. Together, this implicates a more complex set of CDC peptides for the regulation of egg laying than previously anticipated.

"Allohormones": a class of bioactive substances favoured by sexual selection.

During close bodily contact, many species transfer substances that influence the behaviour or physiology of conspecifics. Such transfer is especially common during courtship and copulation. When this is the case the involved bioactive substances are favoured by sexual selection because their effects include increased egg production, inhibited remating, and changed sperm transport or storage in the partner. The direct mode of action of these substances is fundamentally different from that of pheromones and nuptial gifts. Therefore, the term allohormone is introduced here. An allohormone is defined as a substance that is transferred from one individual to another free-living member of the same species and that induces a direct behavioural or physiological response, bypassing external sensory organs. Although we emphasise the importance of allohormones in reproductive processes, allohormones may also have important functions outside of copulation.

NPY in invertebrates: molecular answers to altered functions during evolution.

As in Lymnaea stagnalis NPY plays a key role in regulating energy flows but has no effect on food intake, two important questions arise: 1) How is the amount of food consumed related to energy storage? 2) Can we give a molecular explanation for this alteration in function of NPY during evolution? Recent data have shown that also in Lymnaea a leptin-like factor is produced by glycogen storing cells which inhibits food intake, a Lymnaea storage feedback factor (LySFF). So, food consumption seems in balance with the amount of energy stored in this animal. We suppose that NPY neurons in Lymnaea have receptors for LySFF so that their activity in regulating energy homeostasis reflects the amount of stored energy. By comparing the molecular structure of NPYs in invertebrates it became clear that only molluscan and arthropod NPY are synthesized from a prohormone similar to vertebrate NPYs and should be considered as real invertebrate homologs of NPY. Based on pharmacological data we suppose that the identified Lymnaea NPY receptor is a Y1 subtype. This might explain that LyNPY has no effect on food intake in Lymnaea as this function of NPY in mammals is regulated through the Y5 subtype receptor.

A conserved location for the central nervous system control of mating behaviour in gastropod molluscs: evidence from a terrestrial snail.

We have investigated the role of the right mesocerebrum in the expression of mating behaviour in the garden snail Helix aspersa. Using an in vivo stimulation and recording technique, we provide evidence for both sensory and motor functions in the mesocerebral neuronal population. Some neurones were specifically sensitive to tactile stimuli delivered to the skin on the superior tentacles and around the genital pore. Electrical stimulation of the right mesocerebrum evoked genital eversion and, in combination with tactile stimulation, dart-shooting and penial eversion. Genital eversions were also elicited by injections of APGWamide. During courtship, one recorded unit increased its activity only in correlation with penial eversion, while six other units increased their activity only during dart-shooting. Three additional units increased their activity during both types of behaviour. In addition, most of the recorded units showed increased neuronal activity during times of contact with a partner. Comparison of our results with available data from other molluscs leads us to conclude that the right anteromedial region of the cerebral ganglion is an evolutionarily conserved region of the gastropod brain specialised for the control of male mating behaviour. It is striking to find such functional conservation in the central nervous system of phylogenetically distant gastropods given the large differences in behaviour during mating.

Parasites flicking the NPY gene on the host's switchboard: why NPY?

It was investigated whether up-regulation of the NPY gene by the schistosome Trichobilharzia ocellata in its snail host Lymnaea stagnalis redirects the host's energy flows. We cloned the cDNA encoding Lymnaea NPY (LyNPY), purified and sequenced the peptide, and used synthesized peptide for physiological and morphological studies. Increasing the LyNPY titer in nonparasitized snails (mimicking parasitosis) by 1) implantation of slow-release pellets and 2) injections suppressed reproductive activity and reduced growth in a dose- and time-dependent manner without affecting food intake. When the LyNPY titer was back to normal, reproduction and growth were resumed, coinciding with a transient increase of food intake serving to replenish glycogen stores. Observations on double-immunostained whole mount preparations of brains support these data. A close association was found between LyNPY-positive axons and axons both from ovulation hormone-producing neurons and molluscan insulin-like peptide-producing neurons involved in regulation of growth. As no synaptic(-like) contacts were observed, it is supposed that LyNPY acts nonsynaptically. No morphological interaction was found between LyNPY-positive axons and motoneurons innervating the feeding apparatus. Our data explain why it is an advantageous strategy for endoparasites to up-regulate the highly conserved NPY gene in their host.

Transmitter identification in neurons involved in male copulation behavior in Lymnaea stagnalis.

In this paper, we have mapped the cellular localization of various transmitters onto the central neurons which are involved in male copulation behavior in Lymnaea stagnalis, by combining retrograde tracing with immunocytochemistry and in situ hybridization. Evidence is provided that neurons which were backfilled from the penis nerve, the sole nerve to innervate the male copulatory organ, synthesize a multitude of neuropeptides (APGWamide, Lymnaea neuropeptide tyrosin [LNPY], conopressin, pedal peptide, SEEPLY, DEILSR, myomodulin, and Lymnaea inhibitory peptide [LIP]) as well as the classical neurotransmitter, serotonin. In the anterior lobe, the backfilled neurons mainly contain the tetrapeptide APGWamide and conopressin, and not LNPY or pedal peptide. The results suggest a central role in the regulation of copulation activity for the anterior lobe neurons that produce APGWamide and conopressin. Immunostainings of backfilled nervous systems revealed immunopositive axons originating from these neurons to form varicosities on the cell somata of neurons in the other clusters contributing to the innervation of the male sexual system. Neurons from the right parietal ganglion projecting into the penis nerve were electrophysiologically and morphologically identified by simultaneously recording from the cell body intracellularly and the penis nerve extracellularly and subsequently filling them with an anterograde tracer and subjecting them to immunocytochemistry. This method has provided links between morphology, physiology, and the transmitter contents of these neurons.

Functional role of peptidergic anterior lobe neurons in male sexual behavior of the snail Lymnaea stagnalis.

A morphologically defined group of peptidergic neurons in the CNS of the hermaphroditic snail, Lymnaea stagnalis, is concerned with the control of a very specific element of male sexual behavior. These neurons are located in the anterior lobe of the right cerebral ganglion (rAL). By using chronically implanted electrodes, we show that the rAL neurons are selectively active during eversion of the penis-carrying structure, the preputium. The preputium is normally contained inside the body cavity and is everted during copulation in the male role. Electrical stimulation of the rAL neurons through the implanted electrodes, induced eversion of the preputium in vivo. Injection of APGWamide (Ala-Pro-Gly-Try-NH2), a small neuropeptide that is present in all rAL neurons, induced eversion of the preputium. Application of APGWamide to in vitro preparations of the preputium caused relaxation of this organ. In contrast, injection of the neuropeptide conopressin, which is co-localized with APGWamide in 60% of the rAL neurons, did not induce any behavior associated with male sexual activities. These results show that the neurons of the rAL can induce an eversion of the preputium as occurs during male copulation by release of APGWamide during a period of electrical activity.

Role of neuropeptides encoded on CDCH-1 gene in the organization of egg-laying behavior in the pond snail, Lymnaea stagnalis.

Egg laying in the pond snail Lymnaea stagnalis is triggered by a discharge of the neuroendocrine caudodorsal cells (CDCs). The CDCs expresses three different caudorsal cell hormone (CDCH) genes. This gene family expresses, in total, 11 different peptides among which is the ovulation hormone. Besides the CDCs, the CDCH gene family is expressed in other central and peripheral neurons. In this study, we investigated the roles the different CDCH peptides play in the organization of egg-laying behavior. Egg-laying behavior is a sequence of stereotyped movements in which three phases can be distinguished: resting, turning, and oviposition. We have used the excitation of right pedal N (RPeN) motor neurons as a simple analogue of shell-turning behavior, one of the elements of egg-laying behavior. RPeN motor neurons were inhibited during the resting phase of egg laying but were subsequently excited at the onset of and during the turning phase. The excitatory effect could be evoked by application of beta3-CDCP on RPeN motor neurons in the CNS as well as in isolation but not by the ovulation hormone, alpha-CDCP or Calfluxin, the other CDCH-1 peptides tested. The ovulation hormone itself caused inhibition of RPeN motor neurons. Anti-CDCH-1 positive fiber tracts were found close to the cell bodies and axons of the RPeN motor neurons. Electrical stimulation of a nerve that contains these fibers resulted in excitation of the RPeN motor neurons. The effects of injection of CDCH-1 peptides into intact animals correlated well with the effects of these peptides on RPeN motor neurons. Injection of beta3-CDCP or alpha-CDCP into intact animals resulted in immediate turning behavior in the absence of egg laying itself. The ovulation hormone and Calfluxin had no immediate effect on the behavior. Furthermore, our data indicate that the individual CDCH-1 peptides act on different targets.

Behavior-dependent activities of a central pattern generator in freely behaving Lymnaea stagnalis.

Cyclic or repeated movements are thought to be driven by networks of neurons (central pattern generators) that are dynamic in their connectivity. During two unrelated behaviors (feeding and egg laying), we investigated the behavioral output of the buccal pattern generator as well as the electrical activity of a pair of identified interneurons that have been shown to be involved in setting the level of activity of this pattern generator (PG). Analysis of the quantile plots of the parameters that describe the behavior (movements of the buccal mass) reveals that during egg laying, the behavioral output of the PG is different compared with that during feeding. Comparison of the average durations of the different parts of the buccal movements showed that during egg laying, the duration of one specific part of buccal movement is increased. Correlated with these changes in the behavioral output of the PG were changes in the firing rate of the cerebral giant neurons (CGC), a pair of interneurons that have been shown to modulate the activity of the PG by means of multiple synaptic contacts with neurons in the buccal ganglion. Interval- and autocorrelation histograms of the behavioral output and CGC spiking show that both the PG output and the spiking properties of the CGCs are different when comparing egg-laying animals with feeding animals. Analysis of the timing relations between the CGCs and the behavioral output of the PG showed that both during feeding and egg laying, the electrical activity of the CGCs is largely in phase with the PG output, although small changes occur. We discuss how these results lead to specific predictions about the kinds of changes that are likely to occur when the animal switches the PG from feeding to egg laying and how the hormones that cause egg laying are likely to be involved.

Spontaneous switching between ortho- and antidromic spiking as the normal mode of firing in the cerebral giant neurons of freely behaving Lymnaea stagnalis.

1. Action-potential generation at sites remote from the cell body leads to antidromic firing and occurs in a wide variety of animals and experimental circumstances. Remote sites of spike generation may play a role in the functional subdivision of the axonal branches of a neuron and are also thought to play a role in synaptic integration. 2. Spontaneous ortho- and antidromic firing was investigated by recording the electrical activity of somata and axons of a pair of identified giant neurons [cerebral giant cells (CGCs)] in freely behaving animals. 3. At the soma of each CGC, the shape of the extracellular action potential was not constant but jumped between two well-defined levels. Subsequent recordings of synchronous firing in both cell bodies showed that the shape of the extracellular action potential depended on the firing sequence of the two CGCs. 4. Simultaneous recordings of the cell body and the main axon of a single CGC showed that spontaneous changes in the direction of spike conduction (orthodromic or antidromic) occurred. These changes in the direction of spike conduction coincided with the changes in the shapes of the extracellular action potentials recorded from the somata. 5. These results show that, under physiological conditions, spontaneous switching occurs between ortho- and antidromic spiking in the CGCs, and that action-potential generation at sites remote from the cell body is a physiologically relevant mechanism.

Modulatory role for the serotonergic cerebral giant cells in the feeding system of the snail, Lymnaea. I. Fine wire recording in the intact animal and pharmacology.

1. The role of the paired serotonergic cerebral giant cells (CGCs) in the feeding system of Lymnaea was examined by electrophysiological and pharmacological techniques. 2. The firing characteristics of the CGCs were recorded by fine wires attached to their cell bodies in freely moving intact snails (in vivo recording) and their "physiological" rates of firing determined during feeding and other behaviors. 3. The mean CGC firing rates recorded in vivo varied between 1 and 20 spikes/min but never reached the average rates seen in the isolated CNS (60-120 spikes/min). Maximum rates of firing were seen during bouts of radula biting/rasping movements characteristic of the consummatory phase of feeding (15 +/- 1.69 spikes/min, mean +/- SE, range 7-20 spikes/min), with lower rates seen during locomotion (6.7 +/- 0.75 spikes/min; range 5-9 spikes/min. The cells were rarely active when the animal was quiescent (1.45 +/- 0.91 spikes/min; range 0-2 spikes/min). 4. In vivo recorded CGC firing was phase locked to the feeding movements of the animal, with spikes occurring just before the opening of the mouth, during the protraction phase of the feeding cycle. 5. Evoking firing rates on the CGCs in the isolated preparation similar to those seen in vivo during rasping movements (7-20 spikes/min) did not elicit a fictive feeding pattern in an inactive preparation. Neither did bath application of 10(-9) M serotonin (5-HT; the transmitter of the CGCs). 6. To allow the modulatory role of the CGCs to be examined during patterned activity, the fictive feeding pattern was evoked in the isolated preparation by injecting depolarizing current into a modulatory neuron, the slow oscillator (SO). 7. The tonic firing activity of the CGCs was accurately maintained by current injection in the isolated preparation at rates equivalent to that occurring during feeding, locomotion, and quiescence in the intact snail. This was possible where the CGCs became silent after 1-2 h. Only when the CGCs activity was maintained at a rate (approximately 15 spikes/min) similar to that occurring during rasping, was the SO able to drive a full, high-frequency fictive feeding pattern (15-20 cycles/min). At lower rates of CGC firing, the SO-driven rhythm was either of lower frequency or no rhythm occurred at all (CGCs silent). 8. In many isolated preparations (80%) the CGCs remained active, and it was difficult to maintain specific levels of tonic activity by current injection.(ABSTRACT TRUNCATED AT 400 WORDS)

The neuropeptide schistosomin and haemolymph from parasitized snails induce similar changes in excitability in neuroendocrine cells controlling reproduction and growth in a freshwater snail.

Infection of the snail Lymnaea stagnalis with the schistosome parasite Trichobilharzia ocellata results in inhibition of reproduction and in giant growth. Parasite-related effects on the neuroendocrine centres that control these processes were studied electrophysiologically. Haemolymph from infected snails reduced the excitability of the caudodorsal cells, which control egg laying. In contrast, the excitability of the growth-controlling Light Green Cells was increased under these conditions. The endogenous anti-gonadotropic neuropeptide schistosomin, the presence of which is strongly enhanced in parasitized snails, induced similar effects. Schistosomin apparently plays an important role in the balance between reproduction and growth in Lymnaea. This balance is severely disturbed during parasitic infection, probably as a result of the release of the peptide.

Automatic wave form classification of extracellular multineuron recordings.

A PC-based method for the reconstruction of individual spike trains from extracellular multineuron recordings is described. Starting with virtually no knowledge about the wave forms in a record, a fully automatic template-finding algorithm extracts templates using the entire data set. In a second step, individual spike trains are reconstructed.

Inhibitory modulation by FMRFamide of the voltage-gated sodium current in identified neurones in Lymnaea stagnalis.

1. The putative neurotransmitter FMRFa (Phe-Met-Arg-Phe-amide) caused an inhibitory modulation of the voltage-gated sodium current (INa) in central neurones, the peptidergic caudo dorsal cells (CDCs) of the mollusc Lymnaea stagnalis. FMRFa reduced INa at all command potentials tested (ranging from -35 to +20 mV), but the amplitude of the effect of FMRFa was voltage dependent, inhibition being stronger at more negative potentials (50 +/- 5% reduction at half-maximal INa activation versus 25 +/- 8% at the peak of the I-V curve). 2. INa current traces were well fitted by a Hodgkin & Huxley based model, using m3 activation kinetics and two time constants for inactivation. 3. The steady-state inactivation curve of INa was characterized by half-maximal inactivation at -42.5 +/- 1.81 mV and a slope factor of 4.6 +/- 0.28 mV. The fastest time constant of inactivation ran from 100 +/- 5 to 0.8 +/- 0.32 ms and the slower time constant from 505 +/- 45 to 4.8 +/- 1.40 ms in the range -40 to -5 mV. 4. FMRFa had no significant effect on either component of inactivation, nor on the voltage dependence of steady-state inactivation, nor on the maximal conductance. 5. FMRFa affected the activation of INa. The activation time constant was increased, ranging from 0.75 +/- 0.050 to 0.22 +/- 0.017 ms under control and from 0.91 +/- 0.043 to 0.31 +/- 0.038 ms with FMRFa in the voltage range -25 to +5 mV. The steady-state activation curve was shifted to less negative potentials: half-maximal activation occurred at -26.5 +/- 1.2 mV under control and at 23.6 +/- 1.4 mV with FMRFa; the slope factor (4.6 +/- 1.4 mV in control experiments) was not affected. The combination of slower activation kinetics and a shift in the voltage dependence of activation in the Hodgkin & Huxley based model, adequately explained the reduction of INa by FMRFa. 6. The physiological consequence is that the spiking threshold is increased, causing an arrest of on-going firing activity and a decrease in excitability.

Molluscan ovulation hormone containing neurons and age-related reproductive decline.

Behavioural and neurophysiological experiments were conducted to examine the state of the neurosecretory caudodorsal cells (CDCs) during female reproductive decline in the freshwater snail Lymnaea stagnalis. Old animals that had ceased egg laying do not respond to an oviposition-inducing stimulus. Female reproductive organs in such animals are still intact, though less sensitive to CDC-hormone. The CDC contents of old nonlaying snails are still effective in producing ovulation in young animals. The electrophysiological properties of the CDCs of old nonlaying snails only differ from those of egg-laying snails in one respect; the duration of the afterdischarge is longer. This difference is not directly related to age but to egg-laying activity of the snails since the afterdischarge in old layers did not differ significantly from that in young layers. It is concluded that the CDCs of old nonlaying animals are in principle still intact. Female reproductive decline and subsequent cessation of female reproduction in Lymnaea may result from impairment of input to the CDCs.

Inhibitory modulation of neuronal voltage-dependent sodium current by Phe-Met-Arg-Phe-amide.

The tetrodotoxin (TTX)-sensitive, voltage-gated Na(+)-current (INa) in a cluster of peptidergic neurons, involved in egg laying, in the CNS of the mollusc Lymnaea stagnalis, is modulated by the neuropeptide FMRFa (Phe-Met-Arg-Phe-NH2). Application of FMRFa reversibly reduced the isolated INa in a dose-dependent fashion. The physiological consequence is that the threshold for action potential generation is increased, causing an arrest of ongoing firing activity. The inhibitory action of FMRFa reported here is the first known example of modulation of the voltage-gated INa by a putative neurotransmitter in intact nerve cells. This finding underlines the importance of modulation of ionic currents as a mechanism of regulation of neuronal excitability and includes the voltage dependent Na current in the range of currents subject to transmitter modulation.