Temperature dependence of monoamine-induced pulmonary respiration in the mollusc <Emphasis Type="Italic">Lymnaea stagnalis</Emphasis>

Pulmonary respiration (spontaneous and mediated by intracavitary administration of monoamines) has been studied in molluscs at different ambient temperatures (5, 15, and 25°C). Monoamines (dopamine, serotonin, and adrenaline) were established not to broaden the temperature diapason realization of the respiratory behavior. Microelectrode studies of the spontaneous electrical activity of the Lymnaea stagnalis respiratory network neurons (RPeD1, VD4, and Vi-cluster cells) revealed that both spontaneous and monoamine-induced respiration programs had been terminated under hypothermia conditions. The indicated effects are suggested to be due to the temperature dependence of the chemical, predominantly peptidergic, transmission of signal between neurons of the central respiratory rhythm generator in Lymnaea.     

Respiratory behavior in the pond snail Lymnaea stagnalis

Summary This study describes the neural basis of respiratory behavior in a pulmonate mollusc, Lymnaea stagnalis. We describe and identify muscles of the respiratory orifice (pneumostome) and mantle cavity as well as relevant motor neurons innervating these muscles. All of these identified motor neurons are active during spontaneously occurring respiratory behavior and a sporadically occurring synaptic input, termed Input 3, controls the activities of these motor neurons. This spontaneous input can also be recorded from isolated brain preparations, suggesting that the respiratory motor program is generated centrally. However, evidence is also presented that in semi-intact preparations the role of peripheral feedback is important for the initiation and termination of respiratory behavior in Lymnaea.     

Effect of acute temperature change on lung respiration of the mollusc Lymnaea stagnalis

• 1.Temperature-dependent effects on respiratory behaviour as well as the corresponding temperature-dependent activities of identified neurons within the respiratory network of the pulmonate snail Lymnaea stagnalis were investigated.
• 2.Lymnaea lung ventilation terminated at low temperatures (under 10 °C) while temperature elevation increased ventilation rates. The respiratory central pattern generator (CPG) functioning was relatively quiescent at temperatures under 12.5±0.44 °C.
• 3.Identified CPG neurons (RPeD1, VD4, VD1/RPaD2) and the respiratory network motor neurons (Vi- and RPa-cells) were found to exhibit varied temperature-dependent electrophysiological parameters (action potential frequency and amplitude, resting potential value) between cell types.
• 4.The observed alterations in the electrical activity of the Lymnaea respiratory network neurons underlie the marked changes of respiratory behaviour observed in the intact animal during temperature changes.

     

Mytilus inhibitory peptides (MIP) in the central and peripheral nervous system of the pulmonate gastropods, Lymnaea stagnalis and Helix pomatia: distribution and physiological actions

The distribution and neuroanatomy of Mytilus inhibitory peptides (MIP)-containing neurons in the central nervous system and their innervation pattern in the peripheral nervous system of the pulmonate snail species, Lymnaea stagnalis and Helix pomatia, have been investigated immunocytochemically, by applying an antibody raised to GSPMFVamide. A significant number of immunoreactive neurons occurs in the central nervous system of both species (Lymnaea: ca 600-700, Helix: ca 400-500), but their distribution is different. In Lymnaea, labeled neurons are found in all central ganglia where a number of large and giant neurons, previously identified physiologically, reveal MIP immunoreactivity. In Helix, most of the immunolabeled neurons are small (12-30 microm) and concentrated in the buccal and cerebral ganglia; the parietal ganglia are free of labeled cells. In both species, the ganglionic neuropils, peripheral nerves, connectives, and commissures are richly supplied with immunolabeled fibers. The MIP-immunoreactive innervation pattern in the heart, intestine, buccal mass and radula, and foot is similar in both species, with labeled axonal bundles and terminal-like arborizations (buccal mass, foot) or a network of varicose fibers (heart, intestine). Intrinsic neurons are not present in these tissues. The application of GSPYFVamide inhibits the spontaneous contractions of the esophageal longitudinal musculature in Helix, indicating the bioactivity of the peptide. An outside-out patch-clamp technique has demonstrated that GSPYFVamide opens the K+ channels in central nerve cells of Helix. Injection of GSPYFVamide into the body cavity inhibits the feeding of starved Helix. A wide modulatory role of MIP at central and peripheral levels is suggested in Lymnaea and Helix, including the participation in intercellular signalling processes and remote neurohormonal-like control effects.     

Modulation in firing pattern and oscillation in nerve cells of Lymnaea during network reconstruction

The modulation and reconstruction of the cardio-respiratory neural circuit of Lymnaea stagnalis L. was compared to that of Helix ponatia L. where the input variation and signal molecules were found to have primary importance in network reorganization. From the cardio-respiratory circuit only neurons connected by afferent or efferent pathways to the peripheral chemosensory organ, the osphradium, were used. It was shown that, the general principles of the network reorganization is similar in the two species. The firing pattern of the neurons altered in Lymnaea depending on the input activation or presence of signal molecules in the vicinity of the neurons. The responses of the neurons to the same sensory information, originating from osphradium varied depending on their firing patterns. On central neurones the generation of phasic pattern and/or oscillation was an indicator of network disintegration leading to insensibility to the osphradial sensory inputs. Co-application of signal molecules (5HT, DA, GABA with opioid peptides) to the neurons caused a phasic firing pattern and/or oscillation leading to disintegration of one network and activation of another one. The effect of mu-opioid peptides on GABA-induced and voltage activated ion currents were shown to be the cellular target in reconstruction of neural networks in Lymnaea. The neural network reconstruction in vertebrate brain evoked by signal molecules can be compared to that observed in the identified network of Lymnaea stagnalis making this latter a useful model in further studies, too.     

Influence of HgCl2 on the osphradial multisensory system of Lymnaea stagnalis L

The osphradial multisensory system of Lymnaea stagnalis L. (Pulmonata, Basommatophora) was used to demonstrate the modulation of chemosensory information both at periphery and central nervous system (CNS) following heavy metal treatments. A semi-intact preparation including osphradium, CNS and the right inner parietal nerve (r.i.p.n.) connecting them was used to record both extracellular activity of nerve and intracellular activity of central neurons receiving information from osphradium. The ion currents of osphradium were recorded using patch-clamp method. The changes in nerve and neuronal activity were expressed by averaging of firing frequency and interspike intervals. The chemosensory function of osphradium was shown by application of L-aspartate, urea, saccharose and stagnant water to its surface. The central neurons reacting to the stimulation ofosphradium were located to visceral, right parietal, pedal and cerebral ganglia of Lymnaea. Both the acute and chronic treatments with HgCl2 damaged the sensory function of osphradium traced on the flow of information from periphery to central neurons. At the same time, mercury chloride modified the synaptic connections of respiratory pattern generators as well as the Ca- and K-dependent ion currents of osphradial neurons. The results proved the multisensory role of osphradium sensing the alterations in the environment and its usefulness in monitoring the effects of pollutants at various level of regulation from chemosensory epithelium to CNS.     

A sodium leak current regulates pacemaker activity of adult central pattern generator neurons in Lymnaea stagnalis

The resting membrane potential of the pacemaker neurons is one of the essential mechanisms underlying rhythm generation. In this study, we described the biophysical properties of an uncharacterized channel (U-type channel) and investigated the role of the channel in the rhythmic activity of a respiratory pacemaker neuron and the respiratory behaviour in adult freshwater snail Lymnaea stagnalis. Our results show that the channel conducts an inward leak current carried by Na(+) (I(Leak-Na)). The I(Leak-Na) contributed to the resting membrane potential and was required for maintaining rhythmic action potential bursting activity of the identified pacemaker RPeD1 neurons. Partial knockdown of the U-type channel suppressed the aerial respiratory behaviour of the adult snail in vivo. These findings identified the Na(+) leak conductance via the U-type channel, likely a NALCN-like channel, as one of the fundamental mechanisms regulating rhythm activity of pacemaker neurons and respiratory behaviour in adult animals.     

Respiratory behavior in the pond snail Lynmaea stagnalis

Summary Previously (Syed et al. 1991) we described the ventilatory behavior of the pond snail Lymnaea stagnalis and identified motor neurons that innervate various muscles involved in this behavior. In the present study we describe an interneuronal network that controls ventilatory behavior in Lymnaea. An identified interneuron, termed the input 3 interneuron (Ip.3.I), was found to be involved in the opening movement of the pneumostome (expiration), whereas another identified interneuron known as visceral dorsal 4 (V.D.4) caused its closure (inspiration). These cells have reciprocal inhibitory connections with each other, which accounts for their opposing effects on common follower motor neurons. In isolated brain preparations a third identified interneuron, right pedal dorsal 1 (R.Pe.D.1) initiated the respiratory cycle by the excitation of Ip.3.I. Whereas Ip.3.I in turn excited R.Pe.D.1, the connections between R.Pe.D.1 and V.D.4 were mutually inhibitory. Both Ip.3.I and V.D.4 were active during spontaneously occurring respiratory behavior as recorded from semi-intact preparations, and selective hyperpolarization of V.D.4 during such spontaneous activity disrupted the respiratory behavior. Regarding peripheral feedback, the mechanical stimulation of the pneumostome during its opening movements not only caused closure but also inhibited Ip.3.I in the middle of its discharge. Ip.3.I and V.D.4 were also found to be multifunctional, inhibiting both locomotor and whole body withdrawal neural networks. We conclude from these results that the rhythmic patterned activity underlying respiratory behavior in Lymnaea is generated centrally, and that the network described here therefore comprises a central pattern generator.     

The effect of leu-enkephalin on membrane potential and activity of the rat respiratory center neurons in vitro

In frontal brainstem slices of Wistar rats, the whole-cell patch-clamp recordings showed the effect of opioid peptide leu-enkephalin (10 nM-1 microM) on membrane potential and spontaneous activity pattern of neurons in two divisions of the respiratory center, ventro-lateral area of the solitary tract nucleus, and the pre-B?tzinger complex. Leu-enkephalin induced a membrane hyperpolarization of the respiratory centre neurons and reduction of the spike activity level in spontaneously active units. After administration of leu-enkephalin, a decrease in frequency of bursts was found in bursting cells of the pro-B?tzinger complex; in two cases, there was a transition of bursting activity to tonic one. The data suggest that the mechanism of the central respiratory activity of leu-enkephalin is based on its direct action at the level of membrane of the respiratory centre neurons.     

Effects of temperature on respiration defensive behavior and locomotion of fresh water snail Lymnaea stagnalis

Temperature dependence of lung respiration, defensive behavior and locomotion of Lymnaea stagnalis snail was studied. At the temperature in the range of 4-6 degrees C the rates of locomotion and respiration were reduced (as compared to control temperature of 14-16 degrees C), whereas defensive reactions were much more intense. Vice versa, the temperature rise to 24-26 degrees C activates respiration and locomotion but inhibits defensive behavior. It is suggested that the observed changes in Lymnaea behavior result from temperature-dependent reactions of neurons underlying these activities.     

Dependence of Electric Activity of Motoneurons and Locomotor Behavior of Lymnaea stagnalis on Environmental Temperature

Experiments on serotoninergic motoneurons of the pedal A-cluster innervating the foot ciliated epithelium of Lymnaea stagnalis L. have shown that changes of the environmental temperature lead to a change of several electrophysiological parameters of these neurons. The firing rate of action potential (AP) progressively increases with elevation of the temperature up to the level of about 30°C, after that an abrupt drop of the rate occurs. The membrane potential and AP amplitude decrease as the temperature rises. The revealed changes may be explained by an increase of the membrane permeability, specifically, to sodium ions. The characteristic activity pattern of these neurons disappears in the temperature range of 34–36°C. The peculiarities observed in the neuronal activity at changes of temperature correlate with a change of characteristics of locomotor behavior of L. stagnalis.     

Synaptic and intrinsic activation of GABAergic neurons in the cardiorespiratory brainstem network

GABAergic pathways in the brainstem play an essential role in respiratory rhythmogenesis and interactions between the respiratory and cardiovascular neuronal control networks. However, little is known about the identity and function of these GABAergic inhibitory neurons and what determines their activity. In this study we have identified a population of GABAergic neurons in the ventrolateral medulla that receive increased excitatory post-synaptic potentials during inspiration, but also have spontaneous firing in the absence of synaptic input. Using transgenic mice that express GFP under the control of the Gad1 (GAD67) gene promoter, we determined that this population of GABAergic neurons is in close apposition to cardioinhibitory parasympathetic cardiac neurons in the nucleus ambiguus (NA). These neurons fire in synchronization with inspiratory activity. Although they receive excitatory glutamatergic synaptic inputs during inspiration, this excitatory neurotransmission was not altered by blocking nicotinic receptors, and many of these GABAergic neurons continue to fire after synaptic blockade. The spontaneous firing in these GABAergic neurons was not altered by the voltage-gated calcium channel blocker cadmium chloride that blocks both neurotransmission to these neurons and voltage-gated Ca(2+) currents, but spontaneous firing was diminished by riluzole, demonstrating a role of persistent sodium channels in the spontaneous firing in these cardiorespiratory GABAergic neurons that possess a pacemaker phenotype. The spontaneously firing GABAergic neurons identified in this study that increase their activity during inspiration would support respiratory rhythm generation if they acted primarily to inhibit post-inspiratory neurons and thereby release inspiration neurons to increase their activity. This population of inspiratory-modulated GABAergic neurons could also play a role in inhibiting neurons that are most active during expiration and provide a framework for respiratory sinus arrhythmia as there is an increase in heart rate during inspiration that occurs via inhibition of premotor parasympathetic cardioinhibitory neurons in the NA during inspiration.     

Central representation of internal and external sensory information in the CNS of Helix pomatia L. and Lymnaea stagnalis L

The central representation of intero- and exteroreceptors located in visceral organs and the osphradium were compared in the CNS of Helix pomatia L. (Gastropoda, Stylommatophora) and Lymnaea stagnalis L. (Gastropoda, Basommatophora), two pulmonate snail species inhabiting a terrestrial and anaquatic environment, respectively. Semi-intact preparations were used comprising the CNS connected by the corresponding nerves either to the cardio-renal, respiratory and genital systems or to the osphradium. Spike discharges of central neurons and the nerves were recorded simultaneously. The central representation of intero- and exteroreceptors was found to be distributed throughout the CNS and involved about 300 neurons. The majority of the neurons received sensory information from all the studied visceral organs and the osphradium. Among the neurons responding to intero- and exteroreceptors a multimodal reaction to tactile, chemical and osmotic stimuli prevailed while in the osphradium specific reactions also were demonstrated. Central neurons receiving sensory information from visceral organs and the osphradium form overlapping and reorganizing neural circuits using the same neurons in the regulation of heart activity, respiration or reproduction producing the appropriate behaviour. In the selection of sensory information the firing pattern appears to be the main determining factor as bursting neurons do not receive sensory information. The central representation of intero- and exteroreceptors and its variability can be a model system for cellular studies of motivational state and self-perception.     

Met-enkephalin modulates rhythmic activity in central neurons of Lymnaea stagnalis

1. The effects of met-enkephalin (10⁻⁶-10⁻⁴m) on electrical activity of identified neurons in the isolated CNS and semi-intact preparations of Lymnaea stagnalis have been investigated.2. Met-enkephalin (in concentrations up to 10⁻⁴M) induced very weak hyperpolarisation or depolarisation (1–4 mV) on the majority of neurons tested here.3. Met-enkephalin inhibited the 5-HT-induced respiratory rhythm during the first few minutes of its action.4. Met-enkephalin later (5–30 min after its administration) induced slow oscillations of the membrane potential in central neurons related to respiratory and locomotory programmes as well as in electrically coupled neurosecretory cells.     

The presence and specificity of crustacean cardioactive peptide (CCAP)-immunoreactivity in gastropod neurons

CCAP-like immunoreactivity was detected in central neurons with small and medium diameters in both Helix and Lymnaea CNS. The intensity of immunoreactivity showed seasonal changes with a maximum intensity during spring. The overwhelming majority of nerve cell bodies exhibiting CCAP immunoreactivity is located in the cerebral and parietal ganglia of both Helix and Lymnaea. The neurons of pleural and buccal ganglia were devoid of CCAP-immunoreactivity. Following preabsorbtion of CCAP antibody in 1:15000 dilution with 10(-3) M CCAP or CCAP-related peptide (Helix -CCAP), immunoreactivity could not be observed in neurons, demonstrating the specificity of the antibody to CCAP-related molecules in both Helix and Lymnaea.     

Seasonal plasticity of synaptic connections between identified neurones in Lymnaea

Here we investigate the synaptic connectivity of the giant dopamine containing neurone (RPeDI) of Lymnaea stagnalis during the winter months, in wild and laboratory bred animals. RPeD1 is one of the three neurones forming the respiratory central pattern generator (CPG) in Lymnaea and initiates ventilation under normal circumstances. Many of the follower cells of RPeD1 are ventilatory motor neurones. The connections of RPeD1 to its follower cells were investigated using standard intracellular recording techniques and dopamine was applied to the follower cells using a puffer pipette. During February and early March, RPeD1 was functionally disconnected from its follower cells, but connections reappeared towards the end of March. Most functionally disconnected cells failed to respond to applied dopamine, consistent with the hypothesis that there is down regulation of dopamine receptors in the follower cells of RPeD1 in the winter months. Behaviourally, Lymnaea that survive the winter, are not active at this time and do not indulge in lung ventilation, but stay quiescent. Thus functional disconnection of neurones from the CPG may be either a cause or a consequence of this change in behaviour.     

Identification and functional expression of a family of nicotinic acetylcholine receptor subunits in the central nervous system of the mollusc Lymnaea stagnalis

We described a family of nicotinic acetylcholine receptor (nAChR) subunits underlying cholinergic transmission in the central nervous system (CNS) of the mollusc Lymnaea stagnalis. By using degenerate PCR cloning, we identified 12 subunits that display a high sequence similarity to nAChR subunits, of which 10 are of the alpha-type, 1 is of the beta-type, and 1 was not classified because of insufficient sequence information. Heterologous expression of identified subunits confirms their capacity to form functional receptors responding to acetylcholine. The alpha-type subunits can be divided into groups that appear to underlie cation-conducting (excitatory) and anion-conducting (inhibitory) channels involved in synaptic cholinergic transmission. The expression of the Lymnaea nAChR subunits, assessed by real time quantitative PCR and in situ hybridization, indicates that it is localized to neurons and widespread in the CNS, with the number and localization of expressing neurons differing considerably between subunit types. At least 10% of the CNS neurons showed detectable nAChR subunit expression. In addition, cholinergic neurons, as indicated by the expression of the vesicular ACh transporter, comprise approximately 10% of the neurons in all ganglia. Together, our data suggested a prominent role for fast cholinergic transmission in the Lymnaea CNS by using a number of neuronal nAChR subtypes comparable with vertebrate species but with a functional complexity that may be much higher.     

Effects of the afferent stimulation on neurons in the medial septal region slices and their modulation by biologically active compounds in hibernating and waking ground squirrels

Responses of the medial septal (MS-DB) neurons to electrical stimulation of the medial forebrain bundle (MFB) and their modulation by some neuropeptides and monoamines were investigated in brain slices taken from two groups of ground squirrels-hibernating (HGS) and waking (WGS). Electrical stimulation evoked mostly inhibitory effects of various duration. Besides, responses by phase reset of the background rhythmic bursts and short-latency single spike responses were observed. The neuropeptides identified in the brain of hibernators differentially and reversibly modulated responses even in those neurons where they did not influence the level and pattern of the background activity. Effects of the peptides were state-dependent. E.g., the peptide TSKYR increased the duration of inhibitory effects in the HGS but shortened them in the WGS, while TSKY which had low efficacy in the HGS, increased the duration of inhibition in the WGS. Dipeptide DY depressed inhibitory components and augmented excitatory components of responses in the HGS but was much less effective in the WGS. Effects of noradrenaline and serotonin had stronger correlation with their influence on spontaneous activity. It is suggested that endogenous substances provide for the increased latent excitability and reactivity of the MS-DB neurons during seasonal hibernation. Thus, the MS-DB may function as a "sentry post" participating in signal detection and urgent arousal during hibernation.     

Nerve growth factor (NGF) induces sprouting of specific neurons of the snail, Lymnaea stagnalis

Nerve growth factor (NGF) was examined for its ability to elicit sprouting by adult molluscan neurons. Motoneurons and interneurons (but not neurosecretory cells) from Lymnaea exhibited a sprouting response to murine 2.5S NGF in defined medium with a half-maximal response at about 150 ng/mL. Furthermore, an NGF antiserum blocked sprouting by all normally responsive neurons. We tested whether an NGF-like molecule is a component of conditioned medium (CM) by attempting to preabsorb its sprout-inducing activity with NGF antiserum. Treatment of CM with immune (but not nonimmune) serum largely blocked the response of motoneurons, but not that of neurosecretory cells, to CM. We conclude that NGF exerts neurotrophic activity on specific adult Lymnaea neurons, and suggest the possibility that an NGF-like molecule may exist in the molluscan nervous system.