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.

Re-irradiation and hyperthermia for recurrent breast cancer in the orbital region: a case report.

Based on the good results of re-irradiation plus hyperthermia in breast cancer recurrences on the chest wall, it was decided to offer similar treatment to a patient with recurrent metastatic breast cancer in the orbital region. A female patient was diagnosed in 1997 with breast cancer stage T4N0M0. She was treated with six neo-adjuvant chemotherapy courses and mastectomy, followed by hormonal treatment. In December 1998, she was diagnosed with metastatic disease in the medial upper quadrant of the left orbit. This was excised, followed by 40 Gy radiotherapy. Nine months later, the tumour had recurred in the left orbit at the margin of the radiotherapy field. This again was treated with surgery, followed by 30 Gy radiotherapy. Two months thereafter, the eyelid tumour progressed and hormonal therapy was changed, without an effect on the eyelid tumour. Screening gave no evidence of tumour activity elsewhere. The patient preferred treatment with re-irradiation plus hyperthermia to a surgical approach. Eight fractions of 4 Gy were given in 4 weeks, combined with once weekly hyperthermia. One week after treatment, the tumour had regressed completely. The patient died 22 months following treatment. Until last follow-up, a few weeks before death, the patient mentioned a dry left eye for which she used eyedrops, an unchanged vision and no further difficulties. On examination, there was epilation of the eyelids, a slight conjunctival oedema, no subcutaneous fibrosis and no evidence of tumour regrowth. For this patient, a surgical approach would have resulted in loss of the left eye. Toxicity of re-irradiation plus hyperthermia might lead to either a loss of vision or a delayed loss of her left eye due to treatment-induced toxicity. The chosen local treatment resulted in a very good palliative effect, which lasted for the patient's remaining lifetime of 22 months.

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.

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.

Locust adipokinetic hormones: carrier-independent transport and differential inactivation at physiological concentrations during rest and flight.

Since concomitant release of structurally related peptide hormones with apparently similar functions seems to be a general concept in endocrinology, we have studied the dynamics of the lifetime of the three known adipokinetic hormones (AKHs) of the migratory locust, which control flight-directed mobilization of carbohydrate and lipid from fat body stores. Although the structure of the first member of the AKHs has been known for 20 years, until now, reliable data on their inactivation and removal from the hemolymph are lacking, because measurement requires AKHs with high specific radioactivity. Employing tritiated AKHs with high specific radioactivity, obtained by catalytic reduction with tritium gas of the dehydroLeu2 analogues of the AKHs synthesized by the solid-phase procedure, studies with physiological doses of as low as 1.0 pmol per locust could be conducted. The AKHs appear to be transported in the hemolymph in their free forms and not associated with a carrier protein, despite their strong hydrophobicity. Application of AKHs in their free form in in vivo and in vitro studies therefore now has been justified. We have studied the degradation of the three AKHs during rest and flight. The first cleavage step by an endopeptidase is crucial, since the resulting degradation products lack any adipokinetic activity. Half-lives for AKH-I, -II and -III were 51, 40, and 5 min, respectively, for rest conditions and 35, 37, and 3 min, respectively, during flight. The rapid and differential degradation of structurally related hormones leads to changes in the ratio in which they are released and therefore will have important consequences for concerted hormone action at the level of the target organ or organs, suggesting that each of the known AKHs may play its own biological role in the overall syndrome of insect flight.

Atrophy and degeneration of peptidergic neurons and cessation of egg laying in the aging pond snail Lymnaea stagnalis.

The morphology of the neuroendocrine caudodorsal cells (CDCs), which are involved in the regulation of female reproduction in the pond snail Lymnaea stagnalis, was studied in young (200 to 234 days of age) and old (400 to 500 days) animals. Lucifer Yellow fills of ventral CDCs showed that in young animals ventral CDCs branch ipsilaterally as well as contralaterally in the cerebral commissure. In old animals these branches were reduced at different degrees and in some cases even lacking completely, leaving only an axon crossing the commissure. Immunocytochemical stainings with antibodies against CDC peptides (CDCH-I and alpha CDCP) corroborated the finding that ventral CDCs degenerate. Among the other types of CDCs (dorsal, lateral), degeneration was found as well. The immunocytochemical findings showed that in old animals the axon terminals of the CDCs were strongly stained, indicating that they are packed with secretory vesicles containing peptides. It was also found that these darkly stained, peptide-containing axon terminals protruded into the perineurium. These findings suggest that accumulation of peptides in the terminals of the CDCs of old animals may be due to the impaired release. The relationship between atrophy and degeneration of CDCs and cessation of egg-laying activity in Lymnaea is discussed.

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.

The reconstruction of individual spike trains from extracellular multineuron recordings using a neural network emulation program.

A method for the reconstruction of the individual spike trains from extracellular multineuron recordings is described. A neural network emulation program is trained to recognize a sample set of digitized spikes. The digitized spikes are fed into the neural network, and the network output is used to classify spikes in terms of the training set. The system runs on any PC and its speed makes is especially well suited for the analysis of large amounts of data.

Immediate versus delayed shoulder exercises after axillary lymph node dissection.

A total of 144 evaluable patients with breast cancer were enrolled in a multicenter, randomized, prospective study to establish the role of delayed shoulder exercises on wound drainage and shoulder function after axillary lymph node dissection. Patients in group 1 (n = 78) started active shoulder exercises 1 day postoperatively. Patients in group 2 (n = 66) started on the eight postoperative day, following 1 week of immobilization of the arm. Patients in group 2 had 14% less wound drainage volume than those in group 1 (600 +/- 436 mL versus 701 +/- 398 mL); this difference, however, was not significant. Also, no differences could be established between the two groups when duration and volume of wound drainage, number and volume of seroma aspirations, wound complication rates, and shoulder function were compared 6 months after surgery.

Assessment of insertion techniques and complication rates of dual lumen central venous catheters in patients with hematological malignancies.

One hundred and twenty-three dual lumen silicone rubber central venous catheters were inserted into 101 patients with hematological malignancies undergoing intensive treatment. There was a perioperative complication rate of 13%. Open and closed techniques for inserting the catheter were compared. The operating time needed for introducing the catheter by the closed technique (average, 51 minutes) was significantly shorter (p less than 0.001) than the time needed for the open technique (70 minutes), whereas complication rates were equal in both techniques. On average, the catheters functioned for 149 days. Complications leading to removal were observed in 29.3% of patients, most of which were catheter-related infections (20.4%). Thromboembolic complications leading to removal were less frequent (4.1%) and appeared significantly earlier (p less than 0.001). These data indicate that introduction of the catheter by direct puncture of the subclavian vein is a quick and safe technique, and that this type of catheter is suitable for long-term use, both for infusion and for blood sampling.

Temperature distribution and pH changes during hyperthermic regional isolation perfusion.

Hyperthermic perfusion was given as a palliative treatment in three patients with a bulky tumour in the leg. During the treatment, temperature and pH data were collected in both tumour and normal tissues. The hyperthermia dose administered was 2 h at 41.9-42.7 degrees C. It was found that the temperature distribution was far from homogeneous. In one case tumour pH could be monitored throughout the whole treatment procedure. The tumour pH showed a steep decrease during the initial phase of the perfusion, from a mean value of 7.11 to 5.94 at the start of the hyperthermic phase. Subcutis pH decreased only 0.29 units during the whole procedure. In all three patients considerable tumour regression was observed, without severe toxicity. Hyperthermic perfusion appeared to be an effective debulking treatment. The pH decrease in tumour tissue, immediately before hyperthermia, may be responsible for the remarkable effectiveness of the hyperthermic treatment.

Intractible cutaneous non-Hodgkin's lymphoma of the lower limb. Complete remission after sequential regional isolated hyperthermic perfusion and perfusion with 1-phenylalanine-mustard (melphalan, L-Pam).

A 61-year-old man with an intractible and progressively disabling cutaneous non-Hodgkin's lymphoma (NHL) in the lower limb was treated with sequential regional isolated perfusion at 10-day intervals. The first perfusion was hyperthermic (40.2-43.1 degrees C tumor-temperature); the second was at controlled normothermia with high-dose 1-phenylalanine mustard (melphalan, L-Pam; 11 mg/l perfused tissue). This treatment resulted in a complete remission in the perfused area of significant duration and has prevented amputation.

The neuropeptide egg-laying hormone modulates multiple ionic currents in single target neurons of the abdominal ganglion of Aplysia.

The bag cell neurons of the abdominal ganglion of Aplysia are a useful system for the study of peptidergic neurotransmission. A 20 min burst of impulse activity in the bag cells induces or augments repetitive firing in LB and LC neurons in the abdominal ganglion for up to several hours. Previous experiments have indicated that this effect is mediated by the putative bag cell transmitter egg-laying hormone (ELH). Using voltage-clamp analysis we found that bag cell bursts (BCBs) evoke long-lasting changes in membrane current in these neurons that are mimicked by the application of ELH. The combined ELH-evoked current is inward at all membrane potentials between -110 and -10 mV and consists of 3 separable currents persisting for 30-120 min. They include (1) a depolarizing current that is activated at membrane potentials above -40 mV. This current, termed ISI, is blocked by prolonged exposure to 10 mM Ni2+/0 mM Ca2+ and is not abolished by 0 mM Na+ or 100 mM TEA+/0 mM Na+ in the bathing medium. It is therefore a Ca2+-sensitive current and does not involve Na+ as a charge carrier. (2) There is a hyperpolarizing current that is activated at membrane potentials below approximately -70 mV. This current, termed IR, is blocked by external Rb+ (5 mM) and Cs+ (10 mM) and has a chord-conductance that shifts with the external [K+] according to the Nernst potential for potassium. It is therefore an inwardly rectifying K+ current. (3) There is a small, steady depolarizing current, termed Ix. This current is the only one that remains after prolonged exposure to 10 mM Ni2+/0 mM Ca2+-containing bathing medium. It is Na+ dependent and is associated with a small increase in membrane conductance that is largely independent of membrane voltage. All 3 currents are slow to inactivate; they appear to sum algebraically to produce the net BCB- or ELH-evoked current.

Chemically mediated positive feedback generates long-lasting afterdischarge in a molluscan neuroendocrine system.

The peptidergic neuroendocrine caudodorsal cells (CDCs) of Lymnaea stagnalis control egg laying. The CDC network consists of 100 electrotonically coupled neurons that form two clusters in the cerebral ganglia. Upon prolonged, repeated, intracellular stimulation of one CDC, excitation spreads over the network and leads to a 30-min period of spiking activity: the afterdischarge. During the afterdischarge a number of peptides, including the ovulation hormone, are released. When two ganglia rings from different animals were pinned down next to each other, an afterdischarge initiated in the CDCs of one CNS activated the CDCs of the other CNS, indicating that excitation spreads in the absence of physical contact between the CDCs. A single isolated intercerebral commissure (COM), the neurohaemal area of the CDCs, displayed the same discharge-inducing capability when brought in the vicinity of a second, intact, CNS. Other parts of the CNS did not possess this property. CDC afterdischarges could also induce repetitive spiking in adjacent isolated CDC somata showing that the effect can be directly on the CDCs themselves. The discharge-inducing factor was well separated from the ovulation hormone on a Bio-Gel P-6 column. The factor was pronase-degradable and inhibitors of proteolytic enzymes increased the factor's longevity. It is concluded that, contingent upon the CDC-discharge, a small (less than or equal to 1500 Da) excitatory peptide is released that acts directly on the CDCs. Its function is argued to be: (1) the spread of excitation from a subset of CDCs, receiving external input, over the entire CDC network; and (2) to provide a positive feedback to generate a maximum (all-or-none) response.

Ring neuron control of columellar motor neurons during egg-laying behavior in the pond snail.

Egg-laying in Lymnaea is characterized by the stereotyped egg-laying behavior (ELB), composed of foot contractions and shell movements. Egg-laying can be induced by a clean water stimulus, that triggers a discharge of the neuroendocrine caudo-dorsal cells (CDCs), which release the ovulation hormone into the blood. A part of the behavior is lost when egg-laying is triggered by hormone injection, indicating that during natural stimulus-induced or spontaneous egg-laying this part (the first phase) may be controlled by neuronal events in the CNS triggered by (a) factor(s) not released to the blood. The authors have identified an unpaired neuron, the ring neuron, that is excited during an in vitro afterdischarge of the CDCs, and which, by its numerous axonal branches in the pedal ganglia, modulates motorneurons of the columellar muscle, which controls shell movements. These motor-neurons, identified as such in reduced preparations by 1 for 1 muscle potentials and elements in the connecting nerve, all receive either excitatory or inhibitory input from the ring neuron, as well as from an unknown neuron which has common input of the ring neuron and the motorneurons. The action of the CDCs on the ring neuron cannot be mimicked by the ovulation hormone, and we therefore conclude that the first part of the ELB is probably caused by a nonhormonal local action of the CDCs on the ring neuron and possibly the common input neuron.