Complex behavior induced by egg-laying hormone in Aplysia

Previous studies have described a pattern of complex behavior that occurs in the marine mollusc Aplysia during egg laying. Egg laying and the behavior are initiated by a burst of impulse activity in the neuroendocrine bag cells of the abdominal ganglion or by injection of bag cell extract. To more precisely identify the factors responsible for inducing the behavior we injected animals with egg laying hormone (ELH), one of the neuropeptides secreted by the bag cells. We found that ELH causes a behavior pattern similar to what occurs during spontaneous egg laying. This includes a temporal pattern of head movements consisting of waves and undulations, followed near the beginning of egg deposition by a transition to head weaves and tamps and inhibition of locomotion. There was also a small decrease in respiratory pumping. Except for respiratory pumping, a similar pattern occurred in a second group of animals injected with atrial gland homogenate, which is presumed to induce bag cell activity, but not in controls. These results further implicate ELH in regulation of the behavior. We discuss possible sites of action of ELH and the neural mechanisms by which the behavior is controlled.Abbreviations ELH egg laying hormone - ASW artificial sea water     

Local hormonal modulation of neural activity in Aplysia

The abdominal ganglion of Aplysia provides a convenient experimental system for cellular studies on the roles of peptides as chemical messengers in the nervous system. There are indications that the bag cells, a group of neuroendocrine cells, synthesize and release egg laying hormone (ELH), a peptide with an apparent molecular weight of 6000. Our recent investigations indicate that a burst of impulse activity in the bag cells produces five types of long-lasting responses, some excitatory, others inhibitory, in 26 identified neurons and 2 identified cell clusters located near the bag cells in the abdominal ganglion. The responses have slow, smoothly graded onsets, and many of them result in modulation of neuronal activity for 3 hours or more. Physiological and ultrastructural data support the hypothesis that they are induced by a bag cell hormone (or hormones) that is released into vascular and interstitial spaces of the ganglion to act on the target neurons. Local application of purified ELH to one of the target neurons provides evidence that the bag cell effect is mediated by ELH. Many of the target neurons are known to be parts of neuronal circuits that control specific behavioral and homeostatic processes. Since egg laying is initiated by the bag cell discharge and is associated with a stereotyped behavior pattern lasting several hours, the actions of these peptide-secreting neurons on the central nervous system may serve to regulate certain elements of behavior and homeostasis during egg laying.     

Studies on the Neurosecretory Control of Egg Laying in Aplysia

Studies are reviewed on the electrophysiological and endocrinologicalfeatures of a group of neurosecretory cells (the big cells)in the abdominal ganglion of Aplysia. Electrophysiological studiessuggest that the bag cells are involved in the regulation ofa phasic behavioral function such as egg laying. Egg layingoccurs approximately one hour following the injection into thehemocele of a crude extract of the bag cells or of the perfusateof an abdominal ganglion in which a pleural abdominal connectiveis electrically stimulated. The bag cells appear to act as aunit, releasing a measured dose of egg laying hormone when theyare triggered into activity.     

Neuronal input contributes to sequence of Aplysia egg laying behaviors

Egg laying in Aplysia is controlled by the bag cell neuroendocrine system, which releases multiple peptides during a long-lasting electrical discharge. Following the discharge, a fixed sequence of head and neck movements is performed in which two phases can be distinguished: an appetitive or preparatory phase, in which the substrate is prepared, and a consummatory phase, when the egg string is deposited. During egg laying, feeding responses are suppressed. In this study, Aplysia fasciata was used. When the movement of the egg string through the genital groove was prevented by ligation, lesions of the nerve innervating the genital pore completely abolished the consummatory egg-laying behaviors. This shows that a nervous connection between the genital pore area and the central nervous system is important for the consummatory egg-laying behaviors.We found that suppression of feeding responses to seaweed occurred only during the consummatory phase of egg laying in controls, but animals with ligated genital grooves continued to show normal responses to food. It is hypothesized that a neuronal feedback, possibly together with the bag cell peptides, is critical for the temporal organization of egg-laying behavior in Aplysia.Abbreviations CNS central nervous system - ELH egg laying hormone     

Temperature dependence of egg laying inAplysia brasiliana andA. californica

Summary The temperature dependence of egg laying was examined in winter-caughtAplysia. Cold-waterAplysia californica and warm-waterA. brasiliana were individually housed in the same large aquarium for 16 days at 15°C, and then for 16 days at 20°C. Initially, the majority of theA. californica were not reproductively mature (as determined by injections of atrial gland extracts) whereas all of theA. brasiliana were reproductively mature. When the temperature was increased from 15 to 20°C, both species showed a marked increase in the frequency of egg laying. At both temperatures,A. brasiliana laid eggs more frequently but produced smaller egg masses thanA. californica. We conclude that increased egg laying inA. californica was attibutable both to facilitation of oogenesis in previously reproductively immature animals and to increased activity of the bag cells which release an egg-laying hormone. Increased egg laying inA. brasiliana was attributable primarily to increased bag cell activity.     

Detection of Transgenic and Endogenous Plant DNA Fragments and Proteins in the Digesta,Blood, Tissues,and Eggs of Laying Hens Fed with Phytase Transgenic Corn

The trials were conducted to assess the effects of long-term feeding with phytase transgenic corn (PTC) to hens on laying performance and egg quality, and investigate the fate of transgenic DNA and protein in digesta, blood, tissues, and eggs. Fifty-week old laying hens (n = 144) were fed with a diet containing 62.4% PTC or non-transgenic isogenic control corn (CC) for 16 weeks. We observed that feeding PTC to laying hens had no adverse effect on laying performance or egg quality (P>0.05) except on yolk color (P<0.05). Transgenic phyA2 gene and protein were rapidly degraded in the digestive tract and were not detected in blood, heart, liver, spleen, kidney, breast muscle, and eggs of laying hens fed with diet containing PTC. It was concluded that performance of hens fed diets containing PTC, as measured by egg production and egg quality, was similar to that of hens fed diets formulated with CC. There was no evidence of phyA2 gene or protein translocation to the blood, tissues, and eggs of laying hens.     

Egg laying inAplysia

1. Central pathways for bag cell activation were identified by examining the frequency of spontaneous egg laying episodes in animals with central connective lesions. Bilateral lesions of the cerebropleural (but not the cerebropedal) connectives abolished spontaneous egg laying. In contrast, bilateral lesions of all cerebral ganglion peripheral nerves did not abolish spontaneous egg laying, suggesting that sensory input to the cerebral ganglion is not necessary for activating the bag cells. 2. Backfilling either pleuroabdominal connective labelled cell bodies in the cerebral ganglia (via the ipsilateral cerebropleural connective) that could project to the bag cells. Focal extracellular stimulation of these stained clusters activated the bag cells in isolated brains. 3. Central pathways for initiating egg laying behaviors were identified by selectively eliciting bag cell discharges in animals with central connective lesions. Bilateral lesions of the cerebropedal (but not the cerebropleural) connectives completely abolished elicited egg laying behaviors. 4. Pathways for motor output during rhythmic head and neck movements were identified by eliciting bag cell discharges in animals with peripheral nerve lesions. Bilateral lesions of the four tegumentary nerves in combination with the anterior pedal nerve completely abolished elicited egg laying behaviors, indicating that these nerves are necessary for normal motor output. A normal pattern of egg laying behaviors occurred when the four tegumentary and the anterior pedal nerves were left intact and all other pedal ganglion nerves were lesioned bilaterally, indicating that these nerves are also sufficient for normal motor output.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Muscle Cells of the Follicle of the Ovotestis in Aplysia as the Probable Target Organ for Bag Cell Extract

It has recently been demonstrated that activity of the bag cellsan identified gioup of neurosecretory neurons in Aplysia, leleasesa substance that causes egg laying. A cytologic analysis ofthe ovotestis, before and after administration of the bag cellextract, suggests that one function of the bag cell substanceis to cause the small muscle cells that surround each follicleof the ovotestis to contract, thus expelling ripe oocytes fromthe ovotestis ind beginning them on then journey through theoviduet     

Egg laying inAplysia

Aplysia egg laying is a complex sequence of head and neck movements initiated by the release of ovulatory and neuroactive hormones from the neurosecretory bag cells. This behavioral pattern is difficult to study in reduced preparations, because they do not show ovulation or egg laying behaviors. This paper describes the use of chronically implanted electrodes to elicit normal neurosecretory activity and provides an analysis of egg laying behaviors and the underlying muscle activity in intact, freely behaving A. californica and A. brasiliana. 1. Bag cell discharges elicited with a fine wire electrode implanted in the connective tissue sheath above the cell bodies were typically without noxious behavioral side effects. 2. Following selectively elicited bag cell discharges, egg laying consisted of four rhythmic head and neck movements that were separated functionally into appetitive behaviors ('waves' and 'undulations') used to explore and prepare the substrate and consummatory behaviors ('weaves' and 'tamps') used to distribute and attach the egg string. The amount of time an animal performed consummatory behaviors was positively related to the amount of eggs deposited. By contrast, the appetitive phase of egg laying was independent of the size of the egg mass. 3. The individual behaviors and their temporal sequence were similar following selectively elicited bag cell discharges, spontaneous discharges of animals with implanted electrodes and during normal egg laying of unoperated animals. 4. Three longitudinal muscle systems occurred within the head and neck. Following a selectively elicited bag cell discharge, spatially and temporally coordinated patterns of EJP bursts of different durations were recorded chronically from each muscle group. These EJP patterns were characteristic for specific head and neck movements used in appetitive and consummatory egg laying behaviors.     

Isolation and partial characterization of neuropeptides that mimic prolonged inhibition produced by bag cell neurons in Aplysia

The bag cell neurons of the marine mollusk Aplysia are part of a neural system that utilizes four neuropeptides as neurotansmitters. The peptides, derived from the egglaying hormone/bag cell peptide (ELH/BCP) precursor protein, are released during a 20-min burst discharge of the bag cells and produce several types of responses in various abdominal ganglion neurons. In the identified neurons L3 and L6, bag cell activity produces prolonged inhibition that lasts for more than 2 h. One of the bag cell peptides, alpha-BCP, mediates an early component of the inhibition in these neurons. To identify the co-transmitter mediating the prolonged component of inhibition, we purified material from an acid extract of abdominal ganglia using molecular sizing high-pressure liquid chromatography (HPLC) on TSK 250-125 followed by two steps of reverse-phase HPLC on C4 or C18. We isolated three inhibitory factors that mimic the prolonged component of inhibition. Mass spectroscopy and partial amino acid sequence analysis indicate one factor is ELH [2-36], that is, ELH that lacks the first, N-terminal amino acid. This inhibitory activity was similar in potency to that of ELH and is the first to be described for an ELH related peptide. The two other factors were approximately 3,300 and 4,700 Da and were effective at 10- and 50-fold lower concentration, respectively, than ELH or its fragment. Amino acid composition analysis suggests that they are not derived from the ELH/BCP precursor protein. The 4,700 Da factor is effective at the lowest concentration and produces an effect that lasts as long as 100 min. Therefore, it is the best candidate for the true inhibitory transmitter. Because the inhibited neurons in nervate the kidney, the function of prolonged inhibition may be to regulate kidney function during egg laying.     

Role of bag cells in egg deposition ofAplysia brasiliana

Summary Egg deposition behaviors are analyzed from time-lapse recordings during which spontaneous discharges of the neuroendocrine bag cells are recorded with chronically implanted cuff electrodes. In the laboratory,Aplysia brasiliana normally deposit long egg cordons on the substrate in a characteristic figure 8 pattern similar to the configuration of egg masses observed in the natural environment. The overt behaviors associated with egg deposition are rhythmic head movements consisting of three components that overlap with characteristic relative latencies: up-and-down undulations, side-to-side weaves and in-and-out tamps. The characteristics of the three behaviors and their time courses relative to the appearance of eggs on the substrate suggest that undulations prepare the substrate, weaves distribute the egg cordon and tamps attach the cordon to the substrate. The same rhythmic head movements are also elicited by injections of homogenized abdominal ganglia (HAG) containing bag cell clusters, with comparable relative latencies and maximum frequencies but for shorter total durations. The overt behaviors begin earlier for normal than for triggered egg laying, often before the spontaneous release of bag cell hormones. This suggests that the head oscillations in intact animals are not normally initiated by bag cell activity. The mean latency to the appearance of the egg cordon on the substrate is the same (about 34 min) following either HAG injections or spontaneous bag cell discharges, confirming previous suggestions that the bag cell discharge triggers ovulation. Furthermore, the head movements appear to terminate at the same time following release or injection of hormone. The accompanying paper demonstrates that the full expression of the behavioral effects of bag cell injections depend upon normal movement of eggs in the reproductive tract.Abbreviations CPG central pattern generator - ELH egg laying hormone - HAG homogenized abdominal ganglia     

Role of vascular adrenergic mechanisms in the haemodynamic and PGI2 stimulating effects of angiotensin in diabetic dogs

In aware of the well-known altered vascular responsiveness in the diabetic vasculature, this study aimed to compare the haemodynamic and PGI₂ releasing effects of angiotensin in metabolically healthy (12) and alloxan-(560 umol/kg) diabetic (12) dogs as well as to analyze the role of vascular adrenoceptors in this. In vivo the effect of intracoronarially administered angiotensin (63-125-250-500-1000 pmol/kg/min) on coronary blood flow, mean arterial blood pressure, myocardial contractile force and heart rate was investigated without and with pretreatment of 2 umol/kg phentolamine. In vitro PGI₂ release by isolated coronary rings was induced by 50 nmol/1 angiotensin before and after pretreatment with 5 umol/1 phentolamine and measured by radioimmunoassay. Angiotensin enhances dose-dependently both the mean arterial blood pressure and coronary blood flow, while it provokes a considerable (p < 0.05) increase of PGI₂ formation by isolated coronary arterial rings. These alterations could be prevented by phentolamine administration both in vivo and in vitro, while this drug did not affect the angiotensin-induced enhancement of diabetic coronary blood flow. On the other hand the increase of blood pressure by angiotensin was found to be more (p < 0.05) expressed in diabetes and it could be further potentiated by phentolamine. PGI₂ synthesis by isolated diabetic coronary rings could not be modified either by angiotensin alone or in combination with phentolamine. On the basis of above data, the lack of stimulated vascular PGI₂ formation mediated by alpha-adrenergic mechanisms is supposed to causatively contribute to the diminished sensitivity of diabetic coronary arteries to vasodilation.     

Oviposition behavior and progeny allocation of the polyembryonic waspCopidosoma floridanum (Hymenoptera: Encyrtidae)

Oviposition behavior was used to determine the primary clutch size and sex ratio of the polyembryonic wasp Copidosoma floridanumAshmead (Hymenoptera: Encyrtidae) parasitizing Pseudoplusia includens(Walker) (Lepidoptera: Noctuidae). The laying of a female egg was associated with a pause in abdominal contractions during oviposition, while the laying of a male egg was associated with uninterrupted abdominal contractions. Although unmated females produced only male broods, they also displayed male and female egg oviposition movements. Wasps always laid a primary clutch of one or two eggs. For mated females if only one egg was laid, the emerging secondary clutch was all male or female, but if two eggs were laid a mixed brood of males and females was almost always produced. The secondary clutch of single sex broods was usually between 1000 and 1200 individuals, but the secondary clutch of mixed broods averaged 1143 females and 49 males. Thus, the primary sex ratio for mixed broods was 0.5 (frequency males), but the secondary sex ratio was 0.042. Manipulation of the sequence of male and female egg oviposition or of the primary clutch did not produce major alterations in the secondary clutch size or sex ratio.     

Functional and morphological evidence for the existence of neurites from abdominal ganglion bag cell neurons in the head-ring ganglia of Aplysia

Summary Three lines of evidence are presented indicating that axons of the Aplysia neuroendocrine bag cells extend into the head-ring ganglia of the CNS. When the abdominal ganglion was bisected longitudinally, separating the two bag cell clusters, an afterdischarge induced in one cluster generated an afterdischarge in the other via activity through the head-ring ganglia to which each half abdominal ganglion was attached by connective nerves. This suggests that some axons of bag cells in each cluster communicate through the head-ring ganglia. Retrograde labelling of bag cells occurred when rhodamine-onjugated latex microspheres were injected into the cerebral or either pleural ganglion, a direct demonstration that bag cell axons extend into these ganglia. Finally, cell LP1 in the left pleural ganglion was inhibited during a bag cell afterdischarge, an action mimicked by application of alpha-bag cell peptide (BCP). Since BCP can act only close to its site of release due to susceptibility to peptidase activity, it is likely that LP1 inhibition is dependent on the local release of BCP from bag cell neurites in the pleural ganglion. These results open new possibilities for how bag cell afterdischarges may be initiated and broaden the distribution of their effects.Abbreviations ASW artificial sea water; -BCP -bag cell peptide - ELH egg-laying-hormone - IR immunorective - PB phosphate buffer - PVC pleurovisceral connective     

Cover Caption

The carnivorous spread‐winged damselflies (Lestidae) closely interact with plants only during their egg laying activity. The female chooses the long and narrow monocot plants, like Scirpus or Juncus, to insert its eggs into their tissues. Linear patterns of the egg‐clutches are species‐specific and behaviorally stereotyped characteristics among the lestids studied so far. Most likely, these peculiarities evolved in dragonflies when simultaneously or soon after appearance of monocot plant families in Cretaceous or Paleogene. (see pages 893‐902). Photo provided by Natalia Matushkina     

Effect of synthetic bag cell and atrial gland peptides on identified nerve cells in Aplysia

We have examined the effects of peptides on the neuroendocrine bag cells, the R2 neuron and the left upper quadrant (LUQ) neurons of the abdominal ganglion of Aplysia californica. Peptides include those extracted from the atrial gland, a reproductive organ; those released by an afterdischarge of the bag cells; and 2 synthetic peptides: the amidated 9-amino acid C-terminal portion of atrial gland peptides A/B/ERH (B_26–34), and the 8-amino acid alpha-bag cell peptide (α-BCP_1–8). Peptides were applied by superfusion, arterial perfusion, pressure ejection from micropipettes, or by inducing a bag cell afterdischarge. Both α-BCP_1–8 and B_26–34 are able to produce a bag cell afterdischarge when applied to the abdominal ganglion but are not as effectively able to trigger the bag cells when applied selectively to the ganglia of the head ring. Peptides released by the bag cells inhibit R2 and LUQ neurons; whereas atrial gland extract mildly excites LUQ neurons and powerfully excites R2. The inhibitory effect of the LUQ cells and R2 following an afterdischarge of the bag cells in mimicked by α-BCP_1–8. The excitatory effect of the atrial gland extract cannot be duplicated with B_26–34. Rather, instead of having an excitatory effect on R2 and LUQ cells, B_26–34 seems to mimick α-BCP_1–8 and inhibit these neurons. Both peptides produce a membrane conductance increase in R2 and LUQ cells.     

Association of single nucleotide polymorphism in melatonin receptor 1A gene with egg production traits in Yangzhou geese

In the present study the melatonin receptor 1A gene (MTNR1A) was proposed to be a candidate gene for egg production in Yangzhou geese. A total of 210 goose blood samples were collected to investigate the association of the MTNR1A gene with the number of eggs produced. Using a direct sequencing method, a single nucleotide polymorphism (SNP; g.177G>C) was detected in the 5? regulatory region of the MTNR1A gene (Genbank ss1985399687). Two alleles (G and C) and three genotypes were identified. Association analysis results showed that the g.177G>C SNP significantly affected the level of egg production within a 34‐week egg‐laying period (P < 0.05). Furthermore, the geese with the GG genotype produced significantly more eggs compared to the geese with the CC genotype. Quantitative real‐time PCR analysis showed that the MTNR1A gene was highly expressed in small intestine, granulosa cell and ovary compared to other examined tissues. In addition, the mRNA expression level of MTNR1A in ovary indicated that significantly higher expression levels were recorded for geese with the GG genotype compared to those with the CC genotype. Moreover, a luciferase reporter assay showed that the CC genotype had significantly lower promoter activity than did GG. These results suggest that the identified SNP in the MTNR1A gene may influence the number of eggs produced and mRNA expression levels in Yangzhou geese and could be considered as a useful molecular marker in goose selection and improvement, especially for egg production.     

Egg Deposition Behavior in the Haplodiploid Sawfly Athalia rosae ruficornis Jakovlev (Hymenoptera: Symphyta: Tenthredinidae)

The egg deposition behavior of the turnip sawfly, Athalia rosae (Hymenoptera: Symphyta), is described. Both unmated and mated females lay eggs individually inside of fresh young leaves of cruciferous plants. During an oviposition event, females exhibit a distinct pause in abdominal contractions just before the actual egg deposition act. Unmated females show a longer pause (11.31 s on average) than mated females (4.38 s on overall average). By employing an eye color mutation, the sex of the eggs laid by females was ascertained. Females mated once lay mostly fertilized (diploid female) eggs initially but begin to lay a considerable number of unfertilized (haploid male) eggs later in life. The laying of an unfertilized egg is associated with a longer pause (6.98 s on average) than the laying of a fertilized egg (3.76 s on average). These results are in contrast to previous reports on apocritan Hymenoptera, where the presence of a pause or a longer pause during oviposition was associated with the deposition of fertilized eggs rather than unfertilized eggs. The possibility that mated Athalia rosae females control fertilization and its implications for sex allocation strategies are discussed.     

1D simulation of blood flow characteristics in the circle of Willis using THINkS

One-dimensional (1D) simulation of the complete vascular network, so called THINkS (Total Human Intravascular Network Simulation) is developed to investigate changes of blood flow characteristics caused by the variation of CoW. THINkS contains 158 major veins, 85 major arteries, and 77 venous and 43 arterial junctions. THINkS is validated with available in vivo blood flow waveform data. The overall trends of flow rates in variations of the CoW, such as the missing anterior cerebral artery (missing-A1) or missing posterior cerebral artery (missing-P1), are confirmed by in vivo experimental data. It is demonstrated that the CoW has the ability to shunt blood flow to different areas in the brain. Flow rates in efferent arteries remain unaffected under the variation of CoW, while the flow rates in afferent vessels can be subject to substantial changes. The redistribution of blood flow can cause particular vessels to undergo extra flow rate and hemodynamic stresses.     

A proposed role for prostaglandins in the modulation of the relaxation response to urotensin I in isolated rat arteries

Urotensin I (UI) elicits dose-dependent relaxation responses in isolated helical strips of rat tail and mesenteric arteries contracted by 10⁻⁵M norepinephrine (NE). The rat mesenteric artery demonstrated a 40 fold lower threshold sensitivity to UI (0.25 mU/M1 versus maximal relaxation at 0.25 mU/m1). Complete relaxation of the rat tail artery with UI could not be achieved, even at doses exceeding 10 mU/m1. Pretreatment of the arterial strips with cyclooxygenase inhibitors had no effect on the contractile response to NE in the tail artery, but reduced NE responsiveness in the mesenteric artery. Significant enhancement of UI relaxation responses in both types of arterial strips was achieved by pre-treatment with the cyclooxygenase inhibiters, suggesting a modulatory role for prostaglandins (PGs) in the expression of the UI relaxation response in NE contracted arterial strips. The major enzymatically formed PG (as assessed by [1-¹⁴C] PGH₂ metabolism in broken cell preparations) in both the rat tail and mesenteric arteries was 6-keto PGF_1α, the stable hydrolysis product of PGI₂. Using a specific RIA to quantify 6-keto PGF_1α release, it was found that UI elicited nearly a two-fold increase in the release of this PG compared to the NE control in both rat tail and mesenteric arteries. These data suggest that PGI₂ may modulate the relaxation response to UI either by direct physiological opposition (PGI₂ elicited contractile response in NE contracted tail and mesenteric arteries at doses exceeding 10−8M) and/or by some as yet undefined mechanism (eg. effects on Ca²⁺, cAMP).