A. W. Pieneman

Multiple mating suppresses fecundity in the hermaphrodite freshwater snail Lymnaea stagnalis: a laboratory study

Abstract The oviposition rate of the pond snail Lymnaea stagnalis was determined under conditions of grouping and isolation. Isolated snails appeared to have a higher oviposition rate than animals kept in groups. The removal of a particular part of the vas deferens eliminates copulation activity completely. This technique was used to demonstrate that the reduction of the oviposition rate as a consequence of grouping is caused by mating. Further experiments revealed that this effect of mating was due to a considerable prolongation of the oviposition interval in female copulants. Since these hermaphrodite observed reduction of the oviposition rates in groups can be explained by the prolongation of the oviposition interval due to mating as a female. The lower oviposition rate is not compensated for by increased clutch size, thus an actual decrease of fecundity occurs. The effect of mating on fecundity is discussed in the context of life history tactics of freshwater snails.

Spontaneous and induced egg laying behavior of the pond snail lymnaea-stagnalis

SummaryInLymnaea egg laying is initiated by the discharge of the neurosecretory caudodorsal cells (CDCs), which release an ovulatory hormone (caudodorsal cell hormone; CDCH) and other peptides. Egg laying is a complex behavioral pattern composed of various postures and action patterns. This paper describes the egg laying behavior of intact and freely behaving animals as it occurs spontaneously after an environmental stimulus (clean water stimulus; CWS), and following injections of highly purified CDCH. CDC discharges were monitored in vivo with chronically implanted electrodes. In these animals the egg laying behavior following spontaneous and electrically elicited CDC discharges was analyzed.1.The egg laying behavior induced by the CWS consisted of three separate phases that occurred in a fixed temporal sequence. Shortly after transferral to clean water, animals became quiescent (resting). This was followed by an active phase (turning) during which behaviors were performed to prepare the substrate for the subsequent oviposition phase. Following oviposition, animals examined the deposited egg mass.2.After spontaneous CDC discharges and CDC discharges elicited by selective electrical stimulation of the CDCs (with a chronically implanted fine wire electrode) the egg laying behavior was the same as that following the CWS. All phases of egg laying behavior followed the CDC discharge.3.In contrast, the egg laying behavior following injections of highly purified CDCH was different to that following the CWS. These animals showed no resting phase. Instead, they continued locomoting and entered directly into the turning phase. The animals that did not lay eggs after injection because they were refractory did not show egg laying behavior.4.The durations of the joint turning and oviposition phases depended strongly on clutch size, suggesting that the ovulated eggs are instrumental in causing this behavior.5.We conclude that whenever egg laying is preceded by a CDC discharge, either occurring spontaneously or induced by CWS or electrical stimulation, the behavior differs from that following injections of CDCH directly into the blood. This demonstrates that the presence of CDCH in the blood is not sufficient to produce the full complement of egg laying behaviors and suggests that CDCH, or one of the other peptides released during a CDC discharge, has important local effects within the CNS.

Brain-derived neurotrophic factor signaling in the HVC is required for testosterone-induced song of female canaries.

Testosterone-induced singing in songbirds is thought to involve testosterone-dependent morphological changes that include angiogenesis and neuronal recruitment into the HVC, a central part of the song control circuit. Previous work showed that testosterone induces the production of vascular endothelial growth factor (VEGF) and its receptor (VEGFR2 tyrosine kinase), which in turn leads to an upregulation of brain-derived neurotrophic factor (BDNF) production in HVC endothelial cells. Here we report for the first time that systemic inhibition of the VEGFR2 tyrosine kinase is sufficient to block testosterone-induced song in adult female canaries, despite sustained androgen exposure and the persistence of the effects of testosterone on HVC morphology. Expression of exogenous BDNF in HVC, induced locally by in situ transfection, reversed the VEGFR2 inhibition-mediated blockade of song development, thereby restoring the behavioral phenotype associated with androgen-induced song. The VEGFR2-inhibited, BDNF-treated females developed elaborate male-like song that included large syllable repertoires and high syllable repetition rates, features known to attract females. Importantly, although functionally competent new neurons were recruited to HVC after testosterone treatment, the time course of neuronal addition appeared to follow BDNF-induced song development. These findings indicate that testosterone-associated VEGFR2 activity is required for androgen-induced song in adult songbirds and that the behavioral effects of VEGFR2 inhibition can be rescued by BDNF within the adult HVC.

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.

Assessing aversive emotional states through the heart in mice: implications for cardiovascular dysregulation in affective disorders

Beat-to-beat fluctuations of heart rate (HR) convey information of the brain state with the cardiac time series reflecting the flow of efferent nerve traffic of the autonomic nervous system. Instantaneous HR was studied in mice during exposure to novelty and the expression of fear conditioned to an auditory cue as affective challenge to characterize baseline dynamics and conditioned adjustments to learned fear. These studies included pharmacological and genetic interventions of brain systems implicated in aversive emotional states, the corticotropin-releasing factor (CRF) system and the serotonin (5-HT)1A receptor. Non-linear analyses of neuroautonomic cardiac control provide for functionally adequate measures of dynamical properties. Both CRF1 and 5-HT1A receptor agonists elicited profound sympatho-vagal antagonism with pathological HR dynamics indicative of central autonomic dysregulation via mechanisms resulting in impaired fear adjustment. Non-linear measures provide for a qualitative assessment of dynamical features with regard to physiological or pathological state, are crucial for the translation of results from mouse to man, and may improve our understanding of brain-heart interactions for autonomic dysregulation in affective disorders.

GABA(A) receptor activation in the CA1 area of the dorsal hippocampus impairs consolidation of conditioned contextual fear in C57BL/6J mice

Local infusion of the GABA(A) receptor agonist muscimol is used for reversible inactivation of septohippocampal brain structures associated with cognitive functions. However, information on the effective duration, affected processes and site(s) of action of muscimol in the hippocampus is lacking. Therefore, the dose- and time-dependent effects of bilateral dorsohippocampal infusion of muscimol (0.01-2.0 μg/mouse) below the CA1 area were examined on processing of fear memory in male C57BL/6J mice. Infusion of muscimol 15 min-6 h but not 9 h or 24 h before training impaired conditioned context-dependent fear tested 24 h or 48 h after training. Post-training infusion of muscimol also impaired context-dependent fear when applied either 4 h or 6 h after training, although with lower efficacy. Muscimol was ineffective when administered immediately, 1 h or 24 h after training. Infusion of muscimol 15 min before training impaired context-dependent fear 4-6 h after training indicating preserved short-term but impaired long-term memory. Regardless of infusion time and dose, muscimol had no effect on tone-dependent (cued) fear memory. The impairment by the fluorescently-labeled muscimol-bodipy (5.3 μg/mouse) were similar to those of an equimolar dose of muscimol (1 μg/mouse). The distribution profile after local infusion indicated that muscimol-bodipy (5.3 μg/mouse) was confined to the CA1 area of the dorsal hippocampus. These results demonstrated that GABA(A) receptor activation in the CA1 area of the dorsal hippocampus causes a long-term memory impairment of conditioned context-dependent fear mediated by a long-lasting (≥6 h) muscimol action most likely affecting consolidation processes.

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.

Display of individuality in avoidance behavior and risk assessment of inbred mice

Factors determining individuality are still poorly understood. Rodents are excellent model organisms to study individuality, due to a rich behavioral repertoire and the availability of well-characterized isogenic populations. However, most current behavioral assays for rodents have short test duration in novel test environments and require human interference, which introduce coercion, thereby limiting the assessment of naturally occurring individuality. Thus, we developed an automated behavior system to longitudinally monitor conditioned fear for assessing PTSD-like behavior in individual mice. The system consists of a safe home compartment connected to a risk-prone test compartment (TC). Entry and exploration of the TC is solely based on deliberate choice determined by individual fear responsiveness and fear extinction. In this novel ethological assay, C57BL/6J mice show homogeneous responses after shock exposure (innate fear), but striking variation in long-lasting fear responses based on avoidance and risk assessment (learned fear), including automated stretch-attend posture quantification. TC entry (retention) latencies after foot shock differed >24 h and the re-explored TC area differed >50% among inbred mice. Next, we compared two closely related C57BL/6 substrains. Despite substantial individual differences, previously observed higher fear of C57BL/6N vs. C57BL/6J mice was reconfirmed, whereas fear extinction was fast and did not differ. The observed variation in fear expression in isogenic mice suggests individual differences in coping style with PTSD-like avoidance. Investigating the assumed epigenetic mechanisms, with reduced interpretational ambiguity and enhanced translational value in this assay, may help improve understanding of personality type-dependent susceptibility and resilience to neuropsychiatric disorders such as PTSD.

Maximized song learning of juvenile male zebra finches following BDNF expression in the HVC

During song learning, vocal patterns are matched to an auditory memory acquired from a tutor, a process involving sensorimotor feedback. Song sensorimotor learning and song production of birds is controlled by a set of interconnected brain nuclei, the song control system. In male zebra finches, the beginning of the sensorimotor phase of song learning parallels an increase of the brain‐derived neurotrophic factor (BDNF) in just one part of the song control system, the forebrain nucleus HVC. We report here that transient BDNF‐mRNA upregulation in the HVC results in a maximized copying of song syllables. Each treated bird shows motor learning to an extent similar to that of the selected best learners among untreated zebra finches. Because this result was not found following BDNF overexpression in the target areas of HVC within the song system, HVC‐anchored mechanisms are limiting sensorimotor vocal learning.

Central 5‐HT1A receptor‐mediated modulation of heart rate dynamics and its adjustment by conditioned and unconditioned fear in mice

The beat‐by‐beat fluctuation (dynamics) of heart rate (HR) depends on centrally mediated control of the autonomic nervous system (ANS) reflecting the physiological state of an organism. 5‐HT1A receptors are implicated in affective disorders,associated with ANS dysregulation which increases cardiac risk but their role in autonomic HR regulation under physiological conditions is insufficiently characterized.