C. Janse

Peptidomics of a single identified neuron reveals diversity of multiple neuropeptides with convergent actions on cellular excitability

In contrast to classical transmitters, the detailed structures and cellular and synaptic actions of neuropeptides are less well described. Peptide mass profiling of single identified neurons of the mollusc Lymnaea stagnalis indicated the presence of 17 abundant neuropeptides in the cardiorespiratory neuron, visceral dorsal 1 (VD1), and a subset of 14 peptides in its electrically coupled counterpart, right parietal dorsal 2. Altogether, based on this and previous work, we showed that the high number of peptides arises from the expression and processing of four distinct peptide precursor proteins, including a novel one. Second, we established a variety of posttranslational modifications of the generated peptides, including phosphorylation, disulphide linkage, glycosylation, hydroxylation, N-terminal pyroglutamylation, and C-terminal amidation. Specific synapses between VD1 and its muscle targets were formed, and their synaptic physiology was investigated. Whole-cell voltage-clamp analysis of dissociated heart muscle cells revealed, as tested for a selection of representative family members and their modifications, that the peptides of VD1 exhibit convergent activation of a high-voltage-activated Ca current. Moreover, the differentially glycosylated and hydroxylated α2 peptides were more potent than the unmodified α2 peptide in enhancing these currents. Together, this study is the first to demonstrate that single neurons exhibit such a complex pattern of peptide gene expression, precursor processing, and differential peptide modifications along with a remarkable degree of convergence of neuromodulatory actions. This study thus underscores the importance of a detailed mass spectrometric analysis of neuronal peptide content and peptide modifications related to neuromodulatory function.

Age-related changes in junctional and non-junctional conductances in two electrically coupled peptidergic neurons of the mollusc Lymnaea stagnalis.

Age-related changes in electrotonic coupling ratio of two identified neurons in Lymnaea stagnalis were studied together with the underlying changes in the steady-state conductance properties of the network. Two phases were distinguished in the development of coupling ratio across lifespan. During the first phase (age of 3-13 months), coupling ratio decreased from decreased from 60% to 30%. The second phase (age 13-20 months) was characterized by an increase in coupling ratio. Values of up to 60% were reached again in the oldest animals. Voltage clamp measurements showed that the biphasic trend of the age-related changes in coupling ratio is paralleled by changes in conductance properties of the junction between VD1 and RPD2. During the first phase junctional conductance decreased, whereas during the second phase junctional conductance increased. In addition to the decrease in junctional conductance, a growth-related increase in non-junctional conductance of VD1 and RPD2 contributed to the decrease in coupling ratio observed during the first phase. Thus our results indicate that in Lymnaea junctional connections between neurons may undergo considerable and discontinuous changes after sexual maturation. In addition to these changes in steady-state electrical properties, indications were obtained that age-related changes of kinetically slower conductance(s) may occur in the non-junctional membrane of VD1 and RPD2.

Multiple Synaptic Connections of a Single Neuron Change Differentially with Age

The efficacy of chemical synaptic connections of a single identified interneuron with different types of follower neurons was studied throughout the adult life of the pond snail Lymnaea stagnalis. Simultaneous intracellular recordings were made from the interneuron RPeD1 and its follower neurons in isolated CNS preparations from animals of different age groups (3-18 months of age). The presence of postsynaptic responses to RPeD1 action potentials was tested. With increasing age, the number of A-group neurons that was found with a response to evoked RPeD1 action potentials decreased, yet the number of HIJK-group neurons responding to RPeD1 input increased. The number of G-group neurons and the number of individual neurons VD2/3 and VD4 with RPeD1 input did not differ significantly between age groups. However, there was variability in the presence of responses in these individual neurons. Thus, synaptic connections of the single interneuron RPeD1 change differentially throughout the adult life of L. stagnalis. Within the A-group we found indications that changes in RPeD1 input apply to the entire A-group. In the A-group neurons changes in several electrical properties could not account for the observed age-related changes in the number of neurons responding to RPeD1 action potentials.

Excitability and branching of neuroendocrine cells during reproductive senescence

In the mollusc Lymnaea stagnalis, neuroendocrine caudodorsal cells (CDCs) were studied physiologically and morphologically from egg layers (EL) (aged 154-400), and animals 4 weeks (CEL-4) (342-455 days), and 8 weeks (CEL-8) (477-660 days) after production of their last egg mass. After recording chemical transmission, electrical coupling and stimulation induced afterdischarges (ADs), CDCs then were filled with Lucifer Yellow. Based on the axonal branching revealed by Lucifer Yellow, CDCs were classified as extensively, moderately, or minimally branched. In EL-CDCs, induction of AD activity, which normally (9) precedes egg-laying, only was initiated in the resting state. CEL-4 CDCs exhibited ADs whereas CEL-8 CDCs did not. CEL-8 CDCs exhibited significantly reduced chemical and electrical transmission, and CEL-4 CDCs did not differ from resting state EL-CDCs. CDC branching was significantly reduced with both increasing age and declining egg-laying. Minimally branched CDCs most frequently failed to exhibit an AD and exhibited reduced electrical coupling. We conclude that both physiology and morphology of CDCs are related to age and reproductive state.

Age-related decrease in electrical coupling of two identified neurons in the mollusc Lymnaea stagnalis

Electrophysiological characteristics of two identified giant electrotonically coupled neurosecretory cells in the central nervous system of the mollusc Lymnaea stagnalis were studied in mature animals of different age. The coupling coefficient of the neurons decreased considerably with age. The possibility that the decrease is due to an increase in the junctional resistance between the cells is discussed.

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.

Intracellularly recorded responses to tilt and efferent input of statocyst sensory cells in the pulmonate snail Lymnaea stagnalis

Abstract 1. 1. The morphology of the statocyst sensory cells in Lymnaea was investigated by backfiling the statocyst nerve with HRP. Two types of cells were found: small and large. 2. 2. Responses of statocyst sensory cells were recorded intracellularly upon tilts of different amplitudes. The stimulus response plots showed that there were two types of cells which differed in sensitivity. There was a linear relationship between stimulus and firing rate, stimulus and amplitude of the receptor potential and between receptor potential and firing rate. 3. 3. The statocyst sensory cells receive efferent input from mechanoreceptors in the skin from the eyes and from oxygen-sensitive detectors in the mantle-lung area. This input is probably of synaptic origin. 4. 4. Aminergic drugs applied to the preparation produced hyperpolarizations in the statocyst sensory cells resembling the responses upon exposure of the mantle-lung area to air with low oxygen content. Indications were obtained that noradrenergic β-receptors are involved in the aminergic responses. 5. 5. In the presence of noradrenalin, the responses of the statocyst sensory cells upon tilt decreased.

Serotonergic modulation of junctional conductance in an identified pair of neurons in the mollusc Lymnaea stagnalis

Serotonin (5-HT) is shown to modulate electrotonic coupling between two giant peptidergic neurons in the CNS of Lymnaea stagnalis. The primary effect of 5-HT appears to be a rapid and reversible decrease in gap junctional conductance.

The role of pacemaker properties and synaptic input in generation and modulation of spiking activity in a pair of electrically coupled peptidergic neurons

The origin of patterned electrical activity in two electronically coupled peptidergic neurons, VD1 and RPD2, in the CNS of Lymnaea stagnalis was investigated. VD1 proved to have intrinsic beating pacemaker properties. Hybrid current/voltage clamp experiments demonstrated that in the intact CNS generation of spike activity in the coupled cell system is dominated by VD1. Modulation of spiking activity of VD1/RPD2 appears to originate mainly from chemical synaptic input. The electrical coupling of VD1 and RPD2 proved essential for spike synchronization between the cells.

cAMP and excitability in neuroendocrine cells during reproductive senescence

Excitability changes during reproductive senescence were investigated in the neurosecretory caudodorsal cells (CDCs) that control egg laying in the mollusc Lymnaea stagnalis. CDCs in the isolated central nervous system (CNS) were exposed to different discharge inducing treatments. Senescent CDCs (of animals 8 weeks after laying their last egg mass) and inhibited (I-) state CDCs (of egg-laying (EL) animals) were used. We showed that senescent and I-state CDCs closely resemble each other electrophysiologically. Electrical stimulation did not induce an afterdischarge in either type of CDC but exposure to release products of CDCs from EL animals or to saline with high potassium concentration did induce discharge activity. Also, 8-chlorophenylthio (8-CPT)-cAMP (10(-5) M) induced discharge activity. Exposure to the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) (10(-3) M) or to the adenylate cyclase activator Forskolin (10(-4) M), restored afterdischarge induction by electrical stimulation. Application of IBMX (10(-3) M) and Forskolin (10(-4) M) together induced discharges in the absence of electrical stimulation. Our results suggest that in senescent CDCs changes in the intracellular cAMP pathway may underlie afterdischarge failure.