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.

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.

Recovery of song preferences after excitotoxic HVC lesion in female canaries.

The courtship solicitation display (CSD) of the female canary is a model to study estrogen dependent auditory preferences for male songs. The forebrain auditory-vocal nucleus, HVC, is part of the circuit that determines such preferences. To further develop this model we show that bilateral excitotoxic lesions of the medial part of HVC involving between 18-60% of the bilateral nucleus are behaviorally effective while complete unilateral lesions are not. Further, we show that animals recover their song preferences over a period of several months after the lesion. This functional recovery does not involve anatomical recovery of the HVC. Even 9 months after the lesion, the HVC size of these females was similar to that of females sacrificed 2 days after the lesion and thus was 40 +/- 8% smaller compared to normal females. Further, ipsilaterally, the lesion procedure transiently disturbed the neurochemistry, such as GAD-mRNA expression, in the part of HVC that did not undergo cell death. These results suggest that the integrity of the lateral part of at least one HVC is required to perform CSD in response to relevant auditory stimuli.

Pattern generation in the buccal system of freely behaving Lymnaea stagnalis.

Central pattern generators (CPGs) are neuronal circuits that drive active repeated movements such as walking or swimming. Although CPGs are, by definition, active in isolated central nervous systems, sensory input is thought play an important role in adjusting the output of the CPGs to meet specific behavioral requirements of intact animals. We investigated, in freely behaving snails (Lymnaea stagnalis), how the buccal CPG is used during two different behaviors, feeding and egg laying. Analysis of the relationship between unit activity recorded from buccal nerves and the movements of the buccal mass showed that electrical activity in laterobuccal/ventrobuccal (LB/VB) nerves was as predicted from in vitro data, but electrical activity in the posterior jugalis nerve was not. Autodensity and interval histograms showed that during feeding the CPG produces a much stronger rhythm than during egg laying. The phase relationship between electrical activity and buccal movement changed little between the two behaviors. Fitting the spike trains recorded during the two behaviors with a simple model revealed differences in the patterns of electrical activity produced by the buccal system during the two behaviors investigated. During egg laying the bursts contained less spikes, and the number of spikes per burst was significantly more variable than during feeding. The time between two bursts of in a spike train was longer during egg laying than during feeding. The data show what the qualitative and quantitative differences are between two motor patterns produced by the buccal system of freely behaving Lymnaea stagnalis.

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.

Bidirectional transmission in the cerebrobuccal connective of Aplysia during feeding.

The neuronal activity of the cerebrobuccal connective (CBC) of Aplysia was recorded, using 2 implanted electrodes, under three conditions; 1) in the absence of feeding behaviour, 2) during appetitive feeding behaviour and 3) during consummatory feeding behaviour. Cross-correlation analysis of the recordings was then performed to subdivide spikes on the basis of their direction and speed of propagation. This revealed differences in the neuronal activity during the 3 conditions. There was little activity in the CBC when animals were not feeding. During appetitive and consummatory feeding behaviour the activity in the CBC increased. Units travelling in each direction were present, but with differential activity during the 2 behavioural patterns.

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.

The role of cAMP in regulation of electrical activity of the neuroendocrine caudodorsal cells of Lymnaea stagnalis.

The neuroendocrine caudodorsal cells (CDCs) of the pond snail Lymnaea stagnalis release a number of peptides, including the ovulation hormone, caudodorsal cell hormone (CDCH), during a period of high electrical activity (the CDC-discharge). Earlier studies have shown that during the CDC-discharge adenylate cyclase activity is increased, and that the cyclic adenosine monophosphate (cAMP) analogue 8-chlorophenylthio (8-CPT)-cAMP induces exocytosis and release of peptides from the CDCs. Here, we have investigated the role of cAMP, adenylate cyclase and phosphodiesterase in determining the state of excitability of the CDCs. The cAMP analogue 8-CPT-cAMP induced long-lasting discharges in CDCs. Simultaneous inhibition of the phosphodiesterase by 3-isobutyl-1-methylxanthine (IBMX) and activation of the adenylate cyclase by forskolin gave similar results. These agents also induced discharges of CDCs in dissociated cell culture, indicating that the responses to an increase of cAMP were an endogenous property of the cells. The CDC-afterdischarge can be induced by a period of repetitive electrical stimulation. Inhibition of the phosphodiesterase-activity by IBMX did not change the resting membrane potential, but increased the proportion of preparations that responded to this stimulation with an afterdischarge by more than 200%. It is suggested that cAMP-regulating enzymes are involved in stimulus-response coupling of the afterdischarge in CDCs. The induction of an after discharge probably requires both a low phosphodiesterase-activity and the activation of the adenylate cyclase. The low excitability of the CDCs following an afterdischarge might originate from a refractoriness in the activation of the adenylate cyclase.