Changes in molluscan neurosecretory cells during reproductive cessation: cause or effect?

The pond snail Lymnaea stagnalis has a maximum life span of about 22 months. At the age of about 250 days animals start to decrease egg laying activity and at about 500 days most animals ceased egg laying activity. At the age of cessation of egg laying the neurosecretory caudodorsal cells (CDCs) which control egg laying in Lymnaea exhibit reduced branching patterns. At this stage the cells still exhibit their physiological properties. CDCs still contain biologically active peptides and in the isolated CNS they still exhibit an afterdischarge upon electrical stimulation. Probably in the intact animal cessation of egg laying occurs because the CDCs are not activated anymore by natural egg laying inducing stimuli. In very old animals CDCs exhibit signs of degeneration indicating that cell death occur. After an extended period of no egg laying of Lymnaea physiological changes occur in the CDCs. CDCs from animals after an extended period of no egg laying failed to exhibit an afterdischarge. In such CDCs chemical and electrical coupling among the CDCs are reduced. Morphologically reduced CDCs predominantly fail to exhibit an afterdischarge. However, there are minimally branched CDCs that still could give an afterdischarge. Probably morphological reduction is not the only factor that defines afterdischarge failure. At present we suggest the following sequence of changes. 1. Morphological reduction of CDC branching patterns. 2. Cessation of egg laying. 3. Physiological changes in the CDCs resulting in afterdischarge failure. 4. Further morphological and physiological deterioration of CDCs.     

Pacemaking mechanism of the afterdischarge of the ovulation hormone-producing caudo-dorsal cells in the gastropod mollusc Lymnaea stagnalis

The ovulation hormone-producing caudo-dorsal cells (CDC) of Lymnaea stagnalis have three states of excitability (active, inhibited, and resting), which are related to the egg-laying cycle. Active state CDC produce a firing pattern of prolonged spiking activity (1 spike/2 s), which in the animal occurs shortly before egg laying. In preparations it is evoked as an afterdischarge upon repetitive stimulation of CDC. The afterdischarge is not synaptically driven, but rests on a pacemaking mechanism. CDC are silent in the inhibited and resting states, which follow egg laying. In these states the membrane potential is mainly dependent on [K⁺]₀. In the active state the ratio of the K⁺, Na⁺, and Ca²⁺ permeabilities has changed considerably, probably resulting from an increased permeability to Na⁺ and Ca²⁺. The firing rate in the afterdischarge is dependent on the membrane potential, which is confirmed experimentally by varying [K⁺]₀.[Na⁺]₀ and [Ca²⁺]₀ directly influence the firing rate. Firing stops in Na⁺-free saline, but is enhanced by Ca²⁺-free or high-Mg²⁺ saline. TTX does not affect firing. Relatively high concentrations of Co²⁺ and La³⁺ (2 × 10^?3M) strongly inhibit CDC. Regular firing can be changed into bursting by various means, such as high K⁺ or addition of 1 mM Ba²⁺. Bursting normally occurs at the beginning of the afterdischarge. Postburst hyperpolarizations are reduced in Ca²⁺-free saline and by low Co²⁺ (10^?4-5 10^?4M). Active CDC are driven by a pacemaking mechanism constituted by a voltage-dependent Na⁺/Ca²⁺ channel and a Ca²⁺-dependent K⁺ channel, thus resembling that of bursting pacemakers. The pacemaking mechanism is inactive in the resting and inhibited state.     

Na+ - and Ca2+ -dependent components in action potentials of the ovulation hormone producing caudo-dorsal cells in Lymnaea stagnalis (Gastropoda)

Action potentials in the afterdischarge of the ovulation hormone producing caudo–dorsal cells (CDC) of Lymnaea stagnalis are strikingly different from electrically evoked spikes in the silent resting and inhibited states of these cells. Spikes evoked in the silent states consist of one fast peak (80–100 mV; 10–15 ms). The overshoot is Na⁺ - and Ca²⁺ -dependent. Spikes are blocked in Na⁺ -free saline and by TTX. Repolarization is retarded by TEA. Co²⁺ increases the overshoot. Active state action potentials (60–80 mV) last up to 125 ms, due to activation of a slow component following the TTX-sensitive spike. The slow component is Na⁺ - and Ca²⁺ -dependent. In normal saline it is blocked by Co²⁺ and La³⁺. In Ca²⁺ -free saline the remaining part of the slow component is blocked by La³⁺ only. The slow component is voltage-dependent in a graded fashion. Activation is bound to the active state in which the CDC are depolarized by 20 mV. TEA and Ca²⁺ -free saline greatly increase spike duration in the active state. This suggests that, in addition to the classical TEA-sensitive channel, a Ca²⁺ -dependent K⁺ channel is involved in repolarization of active state action potentials. The underlying membrane properties and the functional significance are discussed in relation to the pacemaking mechanism of the CDC.     

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.     

Membrane mechanism of neuroendocrine caudo-dorsal cell inhibition by the ring neuron in the pond snail Lymnaea stagnalis

Ovulation in the pond snail Lymnaea stagnalis is controlled by the neuroendocrine caudo-dorsal cells (CDCs) in the cerebral ganglia, which release an ovulation hormone during a period of impulse activity. Firing of the single RN in the right cerebral ganglion hyperpolarizes the CDCs. This hyperpolarization is caused by the opening of potassium channels in the axons that connect both the CDC clusters. By this action, that presumably is mediated by axonal branches of the RN in the intercerebral commissure closely associated with these CDC axons, the RN decouples both the CDC clusters. Although the RN has negative feedback on the CDC, it does not control afterdischarge characteristics. The authors suggest that the RN, next to the egg-laying behavior, is involved in another behavior, not compatible with ovulation. Male reproductive activity is presented as a possible candidate for such behavior.     

Hyperosmotic media inhibit voltage-dependent calcium influx and peptide release in Aplysia neurons

Summary The bag cell neurons of Aplysia provide a model system in which to investigate the effects of hyperosmolality on the electrical and secretory properties of neurons. Brief stimulation of these neurons triggers an afterdischarge of action potentials that lasts approximately 20–30 min, during which time they release several neuroactive peptides. We have found that pre-incubation of intact clusters of bag cell neurons in hyperosmotic media prior to stimulation prevents the initiation of afterdischarges. Furthermore, an increase in osmolality of the external medium during an ongoing afterdischarge causes its premature termination. Hyperosmotic media attenuate the release of peptide evoked by both electrically stimulated afterdischarges and potassium-induced depolarization. The ability of high potassium to depolarize the bag cell neurons is, however, not impaired. Exposure of isolated bag cell neurons to hyperosmotic media also inhibits the amplitude of action potentials evoked by depolarizing current injection and attenuates the voltage-dependent calcium current. In isolated bag cell neurons loaded with the calcium indicator dye, fura-2, hyperosmotic media reduced the rise in intracellular calcium levels that normally occurs in response to depolarization. Our results suggest that the effects of hyperosmotic media on peptide secretion in bag cell neurons can largely be attributed to their effects on calcium entry.This work was supported by NIH Grant NS-18492 to L.K. Kaczmarek.     

Intracellular modulation of membrane channels by cyclic AMP-mediated protein phosphorylation in peptidergic neurons of Aplysia

The peptidergic bag cell neurons of the opisthobranch mollusc Aplysia control egg laying and its correlated behavior by release of the neuroactive peptide, egg-laying hormone, during the extended electrical discharge termed afterdischarge. This paper examines the evidence for the involvement of cyclic AMP (cAMP) and protein phosphorylation in the mediation of this electrical afterdischarge. It is concluded that an important component in the mechanism of afterdischarge is the suppression of a potassium channel, mediated by cAMP-dependent protein kinase-induced protein phosphorylation. The exact identity of the potassium channel remains to be worked out.     

Survival of egg-laying controlling neuroendocrine cells during reproductive senescence of a mollusc

During brain aging neuronal degradation occurs. In some neurons this may result in degeneration and cell death, still other neurons may survive and maintain their basic properties. The present study deals with survival of the egg-laying controlling neuroendocrine caudodorsal cells (CDCs) during reproductive senescence of the pond snail Lymnaea stagnalis. In senescent animals CDCs exhibited reduced branching patterns but still maintained their electrophysiological characteristics. In the isolated CNS the cells could still respond with an afterdischarge upon electrical stimulation. After an extended period of no egg laying of Lymnaea CDCs failed to exhibit an afterdischarge. In senescent CDCs that failed an afterdischarge, discharge activity could be restored by exposure to peptides released by CDCs from reproductive animals. Moreover, raising the intracellular cAMP level could induce discharge activity in CDCs with afterdischarge failure. Discharge activity also occurred during depolarization of senescent CDCs by exposure of the cells to saline with a high potassium concentration. These results indicate that in senescent CDCs the pacemaking mechanism of the afterdischarge is still intact but that the initial activation fails. Chemical (auto)transmission of CDCs in such animals was indeed reduced as indicated by the small amplitude of the depolarizing afterpotential (DAP) induced by electrical stimulation. Interestingly, CDCs of senescent animals contained a relative large amount of a particular small peptide. The artificially synthesized peptide appeared to suppress DAP induction in CDCs. Possibly, release of the peptide contributes to the prevention of afterdischarge induction in senescent CDCs. The results so far indicate that in senescent Lymnaea neurons electrophysiological functions persist even after long periods of inactivity and severe morphological reduction.     

Measurement of fasciculations as motor nerve ending discharges in the rat: a dose related effect of neostigmine

The muscle fasciculations caused by neostigmine and similar agents are the result of a primary drug action on motor nerve endings. Asynchronous, repetitive firing of action potentials are evoked at motor nerve endings which are then transmitted to muscle. A dose-response relationship between neostigmine dose and the rate of/or total neural activity has been established in the rat. This fasciculatory response to neostigmine can serve as an index of motor nerve ending excitability and may be useful in assessing the effects of certain pathological states or drug actions at the neuromuscular junction.     

Use of aminopterin in selecting electrically active neuroblastoma cells

A marked increase in electrical excitability and process formation occurs in the N-18 clone of mouse neuroblastoma as these cells go from the logarithmic phase of growth to the stationary state in confluent cultures. Even more excitable cells can be selected by growth in culture medium containing 10⁻⁵ M aminopterin which kills about 90% of the cells. Clone 1A-103 does not develop significant processes or exhibit marked electrical excitability under any of the culture conditions studied. Thus, our results show that one or more of the steps required for generation of the action potential is sensitive to regulation in cultured cells. Methods are presented for obtaining populations of either electrically passive cells or electrically excitable cells which can easily be maintained for several weeks. Clones differ markedly in their capacity to extend processes and their ability to generate action potentials.     

Spontaneous action potentials produced by Na and Cl channels in maturing Rana pipiens oocytes

The electrical excitability of maturing Rana pipiens oocytes was studied using intracellular recording and voltage-clamp techniques. Naturally ovulated oocytes, removed from the body cavity within a few hours after ovulation, possess voltage-sensitive Na and Cl channels that can produce action potentials (ap's). Young oocytes (sometime during metaphase I to first polar body stage) can generate trains of spontaneous action potentials: no chemical treatment or current injection is required. This is the first report of spontaneous repetitive firing in an egg cell membrane. As the oocyte matures, the duration of each ap increases because the outward Cl- current decreases. Middle-aged oocytes (about first polar body stage to metaphase II) have continuously positive membrane potentials (Vm's). Mature, activatable (metaphase II) oocytes have negative Vm's when impaled but can produce a long-lived ap when depolarizing current is injected. The ap's differ fundamentally from ap's in other excitable cells, including eggs: the Na+ current develops slowly and does not inactivate; most of the outward current is carried by Cl-, not by K+; the Cl channel is lost or is rendered insensitive to voltage as the oocyte matures.     

Electrical properties of motoneurons in the spinal cord of rat embryos

Electrical properties of immature motoneurons were studied in vitro using isolated segments of spinal cords of rat embryos aged 14-21 days of gestation. Stable resting potentials and evoked synaptic potentials were recorded for more than 9 hr, indicating that motoneurons remain viable for many hours. Motoneurons are electrically excitable at 14 days of gestation and from the onset of excitability the action potentials are Na+-dependent but slow rising long-duration Ca2+-dependent action potentials can be evoked if K+ conductance is reduced. Thus, during embryonic development the regenerative potential inward current is Na+-and Ca2+-dependent. During motoneurons' differentiation there are some changes in their electrical properties: resting membrane potential increases, input resistance decreases, input capacitance increases, threshold for action potential decreases, and maximum rate of rise of action potential increases. Afferent motoneuron contacts are formed at 16-18 days of gestation when excitatory synaptic potentials can first be evoked in response to dorsal root stimulation. The changes in input capacitance and threshold for action potential occur at the onset of functional afferent motoneuron contacts, but it is not known whether these changes are autonomous or are influenced by the newly formed sensory inputs.     

Selective effect of the antibody to protein SMP-69 on activity of the defence behavior command neurons in grape snail

Effects of antibody against serotonin-modulated protein SMP-69 on defence behavior command neurons L-RP11 were studied in semi-intact preparation of snail Helix lucorum. An increase in membrane excitability as well as selective facilitation of neural responses evoked with chemical sensory stimulation of the snail head (0.25-0.5% quinine solution) were determined 1-1.5 hours after antibody application to the neurons. The antibody did not change neural responses evoked with tactile stimulation of the snail head. These effects were similar to those found in L-RP11 neurons after serotonin or cAMP applications as well as after nociceptive sensitization of the snail. It was suggested that protein homologically related the SMP-69 in mammalians was involved in mechanisms of excitability as well as long-term specific plasticity regulation of L-RP11 neurons synaptic inputs from the head chemoreceptors in snail Helix lucorum.     

Generation of action potentials in a mathematical model of corticotrophs.

Corticotropin-releasing hormone (CRH) is an important regulator of adrenocorticotropin (ACTH) secretion from pituitary corticotroph cells. The intracellular signaling system that underlies this process involves modulation of voltage-sensitive Ca2+ channel activity, which leads to the generation of Ca2+ action potentials and influx of Ca2+. However, the mechanisms by which Ca2+ channel activity is modulated in corticotrophs are not currently known. We investigated this process in a Hodgkin-Huxley-type mathematical model of corticotroph plasma membrane electrical responses. We found that an increase in the L-type Ca2+ current was sufficient to generate action potentials from a previously resting state of the model. The increase in the L-type current could be elicited by either a shift in the voltage dependence of the current toward more negative potentials, or by an increase in the conductance of the current. Although either of these mechanisms is potentially responsible for the generation of action potentials, previous experimental evidence favors the former mechanism, with the magnitude of the shift required being consistent with the experimental findings. The model also shows that the T-type Ca2+ current plays a role in setting the excitability of the plasma membrane, but does not appear to contribute in a dynamic manner to action potential generation. Inhibition of a K+ conductance that is active at rest also affects the excitability of the plasma membrane.     

Repetitive firing triggers clustering of Kv2.1 potassium channels in Aplysia neurons

The Kv2.1 gene encodes a highly conserved delayed rectifier potassium channel that is widely expressed in neurons of the central nervous system. In the bag cell neurons of Aplysia, Kv2.1 channels contribute to the repolarization of action potentials during a prolonged afterdischarge that triggers a series of reproductive behaviors. Partial inactivation of Aplysia Kv2.1 during repetitive firing produces frequency-dependent broadening of action potentials during the afterdischarge. We have now found that, as in mammalian neurons, Kv2.1 channels in bag cell neurons are localized to ring-like clusters in the plasma membrane of the soma and proximal dendrites. Either elevation of cyclic AMP levels or direct electrical stimulation of afterdischarge rapidly enhanced formation of these clusters on the somata of these neurons. In contrast, injection of a 13-amino acid peptide corresponding to a region in the C terminus that is required for clustering of Kv2.1 channels produced disassociation of the clusters, resulting in a more uniform distribution over the somata. Voltage clamp recordings demonstrated that peptide-induced dissociation of the Kv2.1 clusters is associated with an increase in the amplitude of delayed rectifier current and a shift of activation toward more negative potentials. In current clamp recording, injection of the unclustering peptide reduced the width of action potentials and reduced frequency-dependent broadening of action potentials. Our results suggest that rapid redistribution of Kv2.1 channels occurs during physiological changes in neuronal excitability.     

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.     

迷走神经感觉输入诱发的鲫鱼Mauthner细胞胞内电位变化

实验运用微电极穿刺技术,初步探索了刺激鲫鱼右侧迷走神经在双侧Mauthner(M)细胞胞体诱发的胞内电位变化。结果表明：（1）直接刺激鲫鱼右侧迷走神经,可在同侧或对侧M细胞胞体记录到一种短潜伏期、长持续时间、分级的、复合的突触后电位（postsynaptic potentials,PSPs)。此PSPs表现出明显的强度依从性和频率依赖性。（2）刺激迷走神经诱发的PSPs可使逆向锋电位的幅度降低。（3）肌注士的宁后,PSPs的幅度增高、平均持续时间增加、峰值前移。并且可爆发两个以上的动作电位,上述结果提示：迷走神经到M细胞的通路可能 是由长短不等的神经链群组成的。且此通路中不仅包含有兴奋性成分还包含有抑制性成分,而兴奋和抑制之间的相互关系可能起着调节M细胞兴奋性的作用。     

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.     

Characteristics of apple snail giant neurons capable of generating action potentials in sodium-free solutions

The ability of apple snail giant neurons to generate action potentials in solutions that lack sodium ions is associated with the input resistance of these neurons in such a way that the higher the input resistance is, the more pronounced is this ability. Neurons in which this ability is well expressed usually exhibit low resting potential values and a slow repolarization phase. When calcium ions are replaced with barium ions, the neurons retain their excitability in a sodium-free medium for a longer period of time. Raising the calcium ion concentration to 30 µmole may exert a restorative effect on neurons that have lost their excitability in a solution that originally lacked sodium ions but contained 10 µmole of calcium ions. Increasing the calcium ion concentration to 60 µmole leads to loss of excitability, which under these conditions can be restored by means of depolarizing the neuron with an outward current. The results are discussed from the point of view of the theory of ionic conductivity of the surface membrane of neurons. It is hypothesized that the ability of the surface membrane of neurons to make use of sodium or calcium ions in generation of action potentials depends upon its permeability to potassium ions.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 2, No. 1, pp. 100–106, January–February, 1970.     

Immuno-electron microscopy of formation,degradation and exocytosis of the ovulation neurohormone in Lymnaea stagnalis

Summary The cerebral caudodorsal cells (CDC) of the pulmonate snail Lymnaea stagnalis are involved in the control of egg laying and associated behaviour by releasing various peptides. One of these is the ovulation hormone (CDCH). The cellular dynamics of this peptide have been studied using an antiserum raised to a synthetic portion of CDCH comprising the 20–36 amino acid sequence. With the secondary antibody-immunogold technique, specific immunoreactivity was found in all CDC. Rough endoplasmic reticulum and Golgi apparatus showed very little reactivity as did secretory granules that were in the process of being budded off from the Golgi apparatus. However, secretory granules that were being discharged from the Golgi apparatus, were strongly reactive. Secretory granules within lysosomal structures revealed various degrees of immunoreactivity, indicating their graded breakdown. Large electrondense granules, formed by the Golgi apparatus and thought to be involved in intracellular degradation of secretory material, were only slightly reactive. In the axon terminals secretory granules released their contents into the haemolymph by the process of exocytosis. The exteriorized contents were in most cases clearly immunopositive.The possibility has been discussed that CDCH is cleaved from its polypeptide precursor within secretory granules during granule discharge from the Golgi apparatus; subsequently, the mature secretory granules would be transported towards the neurohaemal axon terminals where they release CDCH into the haemolymph. Superfluous secretory material would be degraded by the lysosomal system including the large electron-dense granules.