Biomedical subjects

W Winlow

Publications and source records attributed to W Winlow.

At least 19 recordsLinked to original sources

PubMed2000

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.

Animals↗

PubMed1999

Effect of volatile anaesthetics on the electrical activity and the coupling coefficient of weakly electrically coupled neurones.

1. The application of the volatile anaesthetics, halothane and isoflurane (1% v/v and 2% v/v), to the CNS of Lymnaea reduced the firing frequency of the small weakly coupled pedal A cluster (PeA) neurones, which eventually become quiescent. There was no change in their resting membrane potential. 2. Met-enkephalin significantly increased the coupling coefficient between PeA neurones. 3. The volatile anaesthetics decreased the coupling coefficient even in the presence of met-enkephalin. 4. These effects were dose dependent and the effects of halothane were more rapid than those of isoflurane, reflecting their different anaesthetic potencies.

Anesthetics, Inhalation↗

PubMed1998

Modulation of reconstructed peptidergic synapses and electrical synapses by general anaesthetics.

1. The actions of clinically relevant concentrations of general anaesthetics on reconstructed peptidergic synapses and electrical synapses in the intact brain of the mollusc Lymnaea stagnalis (L.) are described. 2. At identified, reconstructed, FMRFamidergic synapses, chemical synaptic transmission is completely blocked in 2% halothane. 3. Inhibitory postsynaptic responses to directly applied FMRFamide are maintained in 2% halothane and are enhanced in 1% halothane, unlike excitatory responses which are abolished at this concentration. 4. Met-enkephalin normally produces inhibitory responses on postsynaptic PeA neurones, but these are non-reversibly abolished by halothane, whose presence induces novel, dose-dependent, enkephalinergic depolarising responses. 5. The biophysical effects of volatile anaesthetics and sodium pentobarbital on neuronal membranes have been described and they are shown to have opposite dose-dependent effects on input resistance, input conductance and time constant of the electrically coupled neurones VD1 and RPD2. 6. Volatile anaesthetics decouple the neurones VD1 and RPD2 in a dose dependent manner, whilst sodium pentobarbital either enhances coupling or has no effect, depending on the concentration used.

Anesthetics, General↗

PubMed1998

Serotonergic innervation of the foot of the pond snail Lymnaea stagnalis (L.).

The aminergic innervation of the foot of Lymnaea stagnalis was investigated using electron microscopy, immunocytochemistry, and HPLC. The foot was found to contain large amounts of serotonin and dopamine, though at lower concentrations than are found in nervous tissue. Serotonin containing tissue was concentrated in the ventral surface of the foot, under ciliated areas of the epidermis where it occurred in varicosities, with fine tracts joining these varicosities. Varicosities also occurred in deeper tissues, probably adjacent to mucus cells. Positive fluorescence for serotonin in axons was found in nerves innervating the foot, but few neuronal cell bodies containing serotonin were detected, indicating that most of the innervation was coming from the central ganglia. Axon varicosities were found using TEM on ciliated cells, mucus cells, and muscle cells as well as interaxonal junctions (possibly non-synaptic) within nerves. The neuronal varicosities contacting the ciliated cells and mucus cells contained mostly dense-cored vesicles of between 60 and 100 nm in diameter. Smaller, lucent vesicles also occurred in these terminals. The origin and significance of this innervation is discussed. It is suggested that both serotonin and dopamine may play a large role in controlling ciliary gliding by the foot.

Animals↗

PubMed1997

Low concentrations of caffeine raise intracellular calcium concentration only in the presence of extracellular calcium in cultured molluscan neurons.

1. The effects of low concentrations of caffeine (100 and 300 microM) on the intracellular calcium concentration [Ca2+]i in four cultured, identified neurons of the pond snail Lymnaea stagnalis (L) were investigated. 2. Intracellular CA2+ levels in these neurons were measured with the cell-permeable Ca2+ indicator Fura-2/AM, both in the presence and absence of extracellular Ca2 (o-Ca2+/EGTA). 3. In the presence of Ca2+ in the external medium, caffeine was found to induce a substantial elevation in the free [Ca2+]i in all cell types. 4. In some cases, the rise in [Ca2+]i was found to be both time- and concentration-dependent. 5. Low doses of caffeine did not produce any appreciable rise in [Ca2+]i in the absence of Ca2+ in the external medium, but calcium was still available from stores, as clinical concentrations of halothane rose [Ca2+]i in the absence of extracellular calcium. 6. These results indicate that the actions of caffeine, when applied at low concentrations, are dependent on extracellular calcium.

Animals↗

PubMed1996

Halothane affects both inhibitory and excitatory synaptic transmission at a single identified molluscan synapse, in vivo and in vitro.

In the isolated CNS of Lymnaea, a peptidergic neuron termed VD4 makes monosynaptic connections with identified pedal A cluster neurons. In this study, the pedal A (PeA) neurons were further divided into two subgroups depending upon whether they received an inhibitory or excitatory input from VD4. PeA cells inhibited by VD4 were designated PeA(I), whereas those excited by VD4 were termed PeA(E). Both inhibitory and excitatory effects of VD4 stimulation on the PeA(I) and PeA(E) cells, respectively, were mimicked by exogenous FMRFamide in culture (in vitro), implicating this or a related peptide as the transmitter utilized at the VD4-to-PeA synapses. We tested the ability of the general anesthetic, halothane, to affect either the inhibitory or the excitatory peptidergic synapses between VD4 and the PeA neurons, both in the isolated CNS (in vivo) and at the in vitro reconstructed synapses. In the presence of 1% halothane, the excitatory synaptic potential between VD4 and the PeA(E) cells was either depressed or completely abolished, whereas the inhibitory synaptic potential between VD4 and the PeA(I) cells was unaffected in the presence of 1% halothane. The inhibitory potential between VD4 and the PeA(I) cells was, however, blocked in 2% halothane. In order to determine halothane' 5 site of action, exogenous FMRFamide was applied to both PeA(E) and PeA(I) cells in the presence of 1 or 2% halothane. In 1% halothane, the excitatory responses produced by FMRFamide were substantially reduced or abolished, whereas the inhibitory responses to FMRFamide were maintained and enhanced in duration in 1% halothane. In 2% halothane, the inhibitory responses to exogenous FMRFamide remained unchanged. It, therefore, appears that halothane exerts effects at both the pre- and postsynaptic level of the synapse, although presynaptic transmitter release is probably not substantially affected until a concentration of 2% halothane is reached. Our data provide the first evidence that clinically relevant concentrations of halothane (1-2%) affect both excitatory and inhibitory peptidergic synaptic transmission between identified neurons in the nervous system. Furthermore, excitatory transmission is abolished at lower anesthetic concentrations than inhibitory transmission.

Animals↗

PubMed1995

Optical monitoring of movements in small animals and in semi-intact preparations.

A system has been developed for monitoring motor patterns both in small animals and in semi-intact preparations. Using a video recording system, the optical image of the selected object is projected onto a TV monitor to which is attached a pair of photosensitive resistors or diodes. The signals from the photosensor are amplified, filtered prior to being recorded, or stored using commercially available data capture systems. Use of the paired photosensitive elements allows us to avoid changes in background illumination, using a differential amplification function, during different patterns of activity of monitored organs or animals and also allows us to record activity from several different organs or systems simultaneously. The simplicity of the system enables us to perform simultaneous monitoring of both effector movements and neuronal activity via multi-channel microelectrode recordings. We have tested this system using semi-intact preparations of the freshwater pulmonate snail, Lymnaea stagnalis. Examples of different types of recordings are presented.

Animals↗

PubMed1995

5-HT receptors on identified Lymnaea neurones in culture: pharmacological characterization of 5-HT3 receptors.

1. The selective agonist, 1-(m-chlorophenyl)-biguanide (m-CPBG) and antagonist, 3-tropanyl-3,5-dichlorobenzoate (MDL 72222) were used to characterize the 5-HT3 receptors in cultured identified neurones; the serotonin-containing cerebral giant cells (CGCs) and some follower neurones in the buccal ganglia of Lymnaea stagnalis. 2. 5-HT and its agonists were pressure ejected, while the 5-HT antagonists were bath applied. 3. Although m-CPBG evoked mostly depolarizing responses, hyperpolarizing responses were sometimes evoked. 4. At 10(-4) M, m-CPBG failed to mimic the responses of 5-HT, but at a concentration higher, 10(-3) M, pressure-ejected m-CPBG mimicked most 5-HT responses. 5. The 5-HT2 antagonist ketanserin failed to block the m-CPBG-evoked responses, whilst partially blocking the 5-HT responses. 6. These results suggest the presence of 5-HT3 receptors similar to those found in mammalian neurones, and that multiple subtypes of these receptors may be present in Lymnaea neurones.

Animals↗

PubMed1995

Halothane-induced synaptic depression at both in vivo and in vitro reconstructed synapses between identified Lymnaea neurons.

1. In the present study we tested the ability of the general anesthetic, halothane, to affect synaptic transmission at in vivo and in vitro reconstructed peptidergic synapses between identified neurons of Lymnaea stagnalis. 2. An identified respiratory interneuron, visceral dorsal 4 (VD4), innervates a number of postsynaptic cells in the central ring ganglia of Lymnaea. Because VD4 has previously been shown to exhibit immunoreactivity for FMRFamide-related peptides, it was hypothesized that these peptides may be utilized by VD4 during synaptic transmission. In the intact, isolated CNS of Lymnaea, we have identified novel connections between VD4 and the pedal A (PeA) cells. We demonstrate that VD4 makes inhibitory connections with the PeA neurons, in particular PeA4, and that these synaptic responses are mimicked by exogenous application of FMRFamide. 3. The synaptic transmission between VD4 and the PeA cells in an intact, isolated CNS preparation was completely blocked in 2%, but not 1% halothanc. Interestingly, the postsynaptic responses (PeA) to exogenous FMRFamide were maintained in the presence of both 1 and 2% halothane. 4. To determine the specificity of the observed responses and to determine the precise synaptic site of anesthetic action, we reconstructed the VD4/PeA synapses in vitro. After isolation from their respective ganglia, both cell types extended processes and established neuritic contact. We demonstrated that not only did the presynaptic neuron reestablish the appropriate inhibitory synapses with the PeA neurons, but that the PeA cells also maintained their responsiveness to exogenous FMRFamide. 5. Superfusion of the in vitro synaptically connected VD4 and PeA cells with 2% halothane completely abolished the synaptic transmission between these cells. However, even higher concentrations of 4% halothane failed to block the responsiveness of the PeA neurons to exogenous FMRFamide. Moreover, both 1 and 2% halothane enhanced the duration of the postsynaptic response to exogenously applied FMRFamide. These data suggest that the halothane-induced depression of synaptic transmission most likely occurred at the presynaptic level. 6. This study provides the first direct evidence that peptidergic transmission in the nervous system may also be susceptible to the actions of general anesthetics. In addition, we utilized a novel approach of in vitro reconstructed synapses for studying the effects of general anesthetics on monosynaptic transmission in the absence of other synaptic influences.

Anesthetics, Inhalation↗

PubMed1995

Effects of hydrogen peroxide and nitric oxide (NO) on neuronal discharges and intracellular calcium concentration in the molluscan CNS.

In the isolated brain of the freshwater pulmonate snail, Lymnaea stagnalis, application of H2O2 (0.003-0.0003%) activated specific discharge patterns both in neurones of the respiratory and feeding networks, as well as in other identified central neurones and these observed effects were different from the effects of NO or NO-donors. At least part of these effects could be mediated by changes in intracellular calcium concentration, [Ca2+]i, because both the NO-donor, sodium nitroprusside (10(-6)-10(-4) M) and H2O2 (0.003-0.0003%) increased [Ca2+]i in a concentration-dependent manner in isolated neurones in culture. The level of increased [Ca2+]i depended on the drug tested and was characteristic of the identified neurone investigated. We suggest that both NO and H2O2 may be functionally different components of the same neuronal signalling system associated with NO synthase or related enzyme(s).

Animals↗

PubMed1995

The use of NO-sensitive microelectrodes for direct detection of nitric oxide (NO) production in molluscs.

The endogenous production of nitric oxide (NO) from the CNS and a peripheral sensory structure (osphradium) of the pulmonate molluscs, Lymnaea stagnalis and Limax sp. as well as from the rat aorta was studied using two different types of NO-sensitive microelectrodes. Both NO-sensitive electrodes gave complementary, but comparable results. From our data it was possible to compile a hierarchy of tissues with respect to estimated NO production: the rat aorta (300-600 nM) > Lymnaea osphradium (100-300 nM) > Lymnaea buccal ganglia (30-100 nM) > Limax protocerebrum (10-50 nM). In the preparations tested the administration of L-arginine (10 nM) caused an increased level of the recorded signals. This effect was suppressed by NG-Nitro-L-arginine (10 nM), an inhibitor of NOS. It may be concluded that NO can be detected directly from the CNS and peripheral tissues of Lymnaea, and rat aorta, despite the limitations of the techniques used. The putative level of NO production in the osphradium is higher than that in areas of the mammalian CNS and can be compared with release from the aorta. The NO release from the buccal ganglia and the protocerebrum was comparable with that of the rat cerebellum. Such high levels of NO production lend themselves to further analysis of the biological role of this molecule in molluscs.

Animals↗

PubMed1995

Multiple cellular and subcellular actions of general anaesthetics on cultured molluscan neurones.

The pond snail Lymnaea stagnalis has been used as a model system to study the cellular and subcellular actions of general anaesthetics. Here we describe the actions of general anaesthetics mainly on cultured, identified neurones, maintained in isolation to prevent the actions of synaptic inputs upon them. Using the whole-cell patchclamp technique, we have found that application of clinical concentrations of inhalational anaesthetics (halothane and isoflurane) and barbiturates depresses whole-cell calcium currents and potassium currents in a concentration-dependent manner. After loading cultured neurones with the ratiofluorescent dye fura-2AM, we find that halothane raises intracellular calcium concentration in a concentration-dependent manner, both in the presence and absence of extracellular calcium. Thus anaesthetics have multiple cellular and subcellular actions, some of which we have described, but most of which are yet to be discovered.

Anesthetics, General↗

PubMed1994

Nitric oxide synthase-immunoreactive cells in the CNS and periphery of Lymnaea.

The presence and distribution of nitric oxide synthase (NOS) in the CNS and peripheral organs (buccal muscles, oesophagus, salivary glands, foot, mantle and pneumostome) of the pulmonate mollusc, Lymnaea stagnalis were studied using an antiserum developed against rat cerebellar NOS. NOS-immunopositive neurones in Lymnaea were localized predominantly in the buccal ganglia as well as in distinct areas of the cerebral and suboesophageal ganglia. NOS-immunoreactive terminals were also found on the somata of some central neurones. In the periphery, NOS-immunostaining was detected only in a few neurones in the pneumostome area and in the osphradial ganglion. In addition, approximately 100 NOS-immunopositive cells have been found in the salivary glands. Our data supports other recent reports indicating that NO may be a signal molecule in the CNS of molluscs.

Amino Acid Oxidoreductases↗

PubMed1994

5-HT receptors on identified Lymnaea neurones in culture: pharmacological characterization of 5-HT2 receptors.

1. Pressure ejection techniques were used to investigate the identify of receptors mediating 5-HT (5-Hydroxytryptamine) effects on the serotonin-containing cerebral giant cells (CGCs) of the cerebral ganglia and some of their follower motorneurones from the buccal ganglia of Lymnaea stagnalis in culture. 2. The vertebrate 5-HT2 receptor agonist alpha-methylserotonin maleate (10(-4) M), inhibited most of the neurones inhibited by 5-HT (10(-3) M). Others were excited by both agonists. In cells where 5-HT failed to evoke any effects, the 5-HT2 agonist also lacked an effect. 3. Bath application of the 5-HT2 receptor antagonists ketanserin and methysergide (10(-4) M), not only blocked spike generation, but also reduced both the excitatory and inhibitory responses to both 5-HT and alpha-methylserotonin maleate, while the 5-HT3 antagonist MDL 72222 (10(-4) M) failed to block alpha-methylserotonin maleate effects. 4. At 10(-3) M, alpha-methylserotonin maleate increased the amplitudes of the hyperpolarizing responses in a dose-dependent manner. These responses were blocked by ketanserin (10(-4) M). 5. The above results suggest that 5-HT2 receptors are involved in the responses of the CGCs and the buccal motorneurones to 5-HT in Lymnaea stagnalis. The pharmacological characterization of these receptors indicates that the compounds that interact with the 5-HT2 receptors in mammals also interact with the 5-HT2 receptors in molluscs.

Animals↗

PubMed1993

Effects of met-enkephalin on electrical coupling between identified neurons in the pulmonate snails, Helix and Lymnaea.

1. The effects of met-enkephalin on electrical coupling between molluscan neurons have been investigated using the isolated brains of Helix pomatia and Lymnaea stagnalis. 2. In the presence of both serotonin and met-enkephalin, non-rectifying electrical coupling is strongly facilitated between identified respiratory neurons in Helix, whilst coupling between putative, serotonin-containing, ciliomotoneurons in Lymnaea is facilitated by met-enkephalin alone. 3. Facilitation of coupling by met-enkephalin is weaker in the strongly coupled neurons, VD1/RPaD2 of Lymnaea. 4. These data suggest that met-enkephalin can modulate different groups of electrically coupled cells and may be involved in coordination of motor patterns.

Animals↗

PubMed1993

Nitric oxide activates buccal motor patterns in Lymnaea stagnalis.

The mollusc, Lymnaea stagnalis, has been used as a model to study the mechanisms of nitric oxide (NO)-dependent processes in the CNS. Putative NO-containing neurones in Lymnaea are localized in the buccal ganglia, predominantly in areas where sensory neurones known to regulate feeding are found. The NO-generating substance, S-nitrosocysteine (S-NC, 5 x 10(-5)-10(-3 M) activates feeding movements of the buccal mass and modulates the activity of buccal motoneurones. An inhibitor of NO synthase, NG-methyl-L-arginine (10(-4) M) decreases the frequency of background buccal movements and has opposite effects to S-NC on the buccal motoneurones. We suggest that NO is a messenger in the CNS of Lymnaea and may be involved in coordination of feeding motor patterns.

Animals↗

PubMed1993

Effects of general anaesthetics on cultured Lymnaea neurones.

In order to elucidate the mode of action of general anaesthetics we are using neurones of Lymnaea stagnalis as a model system. Neurones exhibit mainly two types of responses to anaesthetics delivered at clinical concentrations, i.e., either gradually going into quiescence or exhibiting paroxysmal depolarizing shifts (PDS). In order to determine whether these differences are due to intrinsic membrane properties or because of synaptic effects, cultured neurones are being used so that cells can be studied in isolation from any synaptic effects. Cells in culture retain their basic electrophysiological characteristics and behave in a similar manner to the applied anaesthetics as do whole brain preparations. Demonstration of PDS and quiescence in cultured neurones shows that these phenomena are due to membrane effects and not due to synaptic inputs. The effects of anaesthetics observed seem to be consistent with the suggestion that anaesthetics may influence the inward calcium current or other calcium-dependent currents.

Action Potentials↗

PubMed1992

Differential effects of general anaesthetics on identified molluscan neurones in situ and in culture.

1. The only unifying principle of general anaesthesia is that general anaesthetics interact with membrane components and no single cellular mechanism appears to explain their widespread effects in the central nervous system. 2. The gastropod mollusc, Lymnaea stagnalis, provides an excellent model system for studies on general anaesthetics because it has large, uniquely identifiable nerve cells. Several of these cells are interneurones with identified neurotransmitters and monosynaptic connections to other cells. 3. Recent work on Lymnaea neurones suggests that calcium currents are depressed by volatile general anaesthetics applied in the clinical range, whilst evidence from other preparations indicates that there is a rise in intracellular calcium concentration following application of these substances. 4. Identified Lymnaea neurones have different responses to applied anaesthetics, irrespective of the anaesthetic used. Following application of halothane, barbiturates and several other anaesthetic agents, some cells gradually become quiescent after a short period, whilst in others a series of paroxysmal depolarizing shifts occur prior to quiescence. 5. Cultured neurones of Lymnaea, Helisoma and related species retain their characteristic action potential types and neurotransmitter identity. Their responses to anaesthetics are similar to those in the intact brain. They may also form synapses in culture. Thus, they are a useful tool for studying the cellular and subcellular actions of general anaesthetics.

Anesthetics↗