Biomedical subjects

K Lukowiak

Publications and source records attributed to K Lukowiak.

At least 37 records · Page 2Linked to original sources

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↗

PubMed1996

Hypothalamic and extrahypothalamic sauvagine-like immunoreactivity in the bullfrog (Rana catesbeiana) central nervous system.

In the present study, immunocytochemistry and radioimmunoassay were used to investigate the presence of sauvagine in both hypothalamic and extrahypothalamic areas of the central nervous system (CNS) of the bullfrog (Rana catesbeiana) using a specific antiserum raised against synthetic non-conjugated sauvagine (SVG), a frog (Phyllomedusa sauvagei) skin peptide of the corticotropin-releasing factor (CRF) family. Sauvagine-immunoreactive (SVG-ir) bipolar neurons were found in the nucleus of the fasciculus longitudinalis medialis located in the rostral mesencephalic tegmentum. In the tectal mesencephalon, beaded SVG-ir fibres were present in the optic tectum, and in the torus semicircularis. Abundant SVG-ir varicose fibres were seen in the granulosa layer of the cerebellum, the nucleus isthmi, and the obex of the spinal cord. SVG-ir fibres were also seen by the alar plate of the rombencephalon. In the diencephalon, the antiserum stained parvocellular neurons of the preoptic nucleus (PON) which extended their dendrites into the cerebro-spinal fluid (CSF) of the third ventricle and projected their ependymofugal fibres to the zona externa (ZE) of the median eminence. Immunopositive fibres were also present in the medial forebrain bundle at the chiasmatic field, the posterior thalamus, the pretectal gray, and the ventrocaudal hypothalamus. In the telencephalon (forebrain), SVG-ir fibres were seen in the medial septum, the lateral septum, and the amygdala. The SVG immunoreactivity could not be detected after using the SVG antiserum previously immunoabsorbed with synthetic SVG (0.1 microM), but immunoblock of the antiserum with sucker (Catostomus commersoni) urotensin I (sUI), sole (Hippoglossoides elassodon) urotensin I, sucker CRF, rat/human CRF, or ovine CRF (0.1-10 microM) did not eliminate visualization of the immunoreactivity. In radioimmunoassay, the SVG antiserum did not crossreact with sUI, or the SVG fragments SVG1-16, SVG16-27, and SVG26-34, but it recognized the C-terminal fragment SVG35-40. Crossreaction with mammalian ovine CRF and rat/human CRF was negligible. Both hypothalamic and mesencephalic extracts gave parallel displacement curves to SVG. The results suggest the presence in the bullfrog brain of a SVG-like neuropeptide, i.e., a peptide of the CRF family, that either is SVG or shares high homology with the C-terminus of that peptide. The function of this neuropeptide in amphibians is not known at this time, but based on its anatomical distribution to the ZE it could affect the release of adrenocorticotropin (ACTH) or other substances from the amphibian pars distalis. Involvement of the SVG-like peptide in behavioural (forebrain), visual (thalamus-tegmentum mesencephali-pretectal gray-optic tectum), motor coordination (cerebellum), and autonomic (spinal) functions, as well as an undefined interaction with the CSF in the bullfrog, seems likely.

Amphibian Proteins↗

PubMed1996

Rhythmic activities of isolated and clustered pacemaker neurons and photoreceptors of Aplysia retina in culture.

Each eye of Aplysia contains a circadian clock that produces a robust rhythm of optic nerve impulse activity. To isolate the pacemaker neurons and photoreceptors of the eye and determine their participation in the circadian clock and its generation of rhythmic autoactivity, the retina was dissociated and its cells were placed in primary cell culture. The isolated neurons and photoreceptors survived and vigorously extended neurites tipped with growth cones. Many of the photoreceptors previously described from histological sections of the intact retina were identified in culture, including the large R-type photoreceptor, which gave robust photoresponses, and the smaller tufted, whorled, and flared photoreceptors. The pacemaker neurons responsible for the rhythmic impulse activity generated by the eye were identified by their distinctive monopolar morphology and recordings were made of their activity. Isolated pacemaker neurons produced spontaneous action potentials in darkness, and pacemaker neurons attached to fragments of retina or in an isolated cluster interacted to produce robust spontaneous activity. This study establishes that isolated retinal pacemaker neurons retain their innate autoactivity and ability to produce action potentials in culture and that clusters of coupled pacemaker neurons are capable of generating robust autoactivity comparable to pacemaker neuron rhythmic activity recorded in the intact retina, which was previously shown to correspond to 1:1 with the optic nerve compound action potential activity.

Action Potentials↗

PubMed1996

Dopamine regulation of neurite outgrowth from identified Lymnaea neurons in culture.

1. An identified dopaminergic interneuron (RPeD1) of the snail Lymnaea stagnalis, makes specific synaptic connections with a number of target (VI and VJ) but not non-target (VF and RPB) neurons in vivo. When cultured in vitro with both target and non-target cells, RPeD1 re-establishes synapses with target cells only. 2. To test whether exogenous dopamine exerts effects on the neurite outgrowth of both target and non-target neurons respectively, these cells were cultured in conditioned media (CM) in the presence of dopamine (10(-5) M). The growth of the non-target cells was severely restricted and retarded in the presence of dopamine. These data suggest that dopamine may regulate neurite outgrowth of non-target cells in culture. 3. The growth regulatory effects of dopamine on the non-target cells were blocked in the presence of a dopamine receptor antagonist (R(+) SCH-23390, 10(-4) M). These results indicate that dopamine-induced growth regulation of the non-target cells is mediated via dopamine receptors on these cells. 4. In the absence of conditioned media, dopamine was not sufficient to exert growth promoting effects on either target or non-target cells. 5. Taken together, our data show that dopamine differentially regulates growth of identified Lymnaea neurons in culture. Dopamine alone, however, is not sufficient to initiate and support neurite outgrowth from these cells. Rather, it functions to suppress the neurite outgrowth of the non-target cells, initiated by the conditioned media.

Animals↗

PubMed1996

Presence of sauvagine-like epitopes in the interrenal gland of the bullfrog Rana catesbeiana.

Immunocytochemistry was used to investigate the presence of corticotropin-releasing factor-like peptides in the interrenal (adrenal) glands of the bullfrog Rana catesbeiana by using specific antisera raised against synthetic nonconjugated rat/human corticotropin-releasing factor, urotensin I, and sauvagine. From these three antisera, covering a broad range of corticotropin-releasing factor-like immunoreactivities, only the sauvagine antiserum gave positive immunoreactivity. Sauvagine immunoreactivity was found in cortical cells grouped into cords in the renal zone of the interrenal gland. The central and subcapsular cords were less stained. Tyrosine hydroxylase-positive chromaffin cells were not sauvagine-immunoreactive. The immunoreactivity was abolished, in all cases, by previous immunoabsorption of the sauvagine antiserum with synthetic sauvagine (0.1 microM), but it was not eliminated by sucker (Catostomus commersoni) urotensin I, sole (Hippoglossoides elassodon) urotensin I, sucker corticotropin-releasing factor, rat/human corticotropin-releasing factor, or ovine corticotropin-releasing factor (0.1-10 microM). In a sauvagine radioimmunoassay, interrenal extracts displaced 125I-sauvagine from antiserum only partially, and not in parallel with the sauvagine standard curve. The results suggest that the sauvagine immunoreactivity in the R. catesbeiana interrenal gland may represent a novel sauvagine-like peptide.

Amphibian Proteins↗

PubMed1996

Glutamate as a putative neurotransmitter in the mollusc, Lymnaea stagnalis.

Bath-applied glutamate (10-1000 microM) produced excitatory and inhibitory responses on numerous identified neurons of the mollusc Lymnaea stagnalis. Using both in situ and in vitro preparations, glutamate or glutamate agonists produced a depolarization in identified neurons right pedal dorsal 1 and right pedal dorsal 2 and 3. However, attempts to block glutamate-evoked responses with glutamate antagonists were unsuccessful. We examined a potential glutamatergic neuron, visceral dorsal 4. Exogenous application of the peptides (GDPFLRFamide and SDPFLRFamide) could mimic the inhibitory, but not the excitatory effects of visceral dorsal 4 on its postsynaptic cells, implying the presence of a second transmitter. We tested the possibility that glutamate is this second neurotransmitter by using excitatory synapses between visceral dorsal 4 and postsynaptic cells right pedal dorsal 2 and 3, right pedal dorsal 1, visceral F group and right parietal B group neurons. Of all the putative neurotransmitters tested, only glutamate had consistent excitatory effects on these postsynaptic cells. Also, the amplitude of the right pedal dorsal 2 and 3 excitatory postsynaptic potentials was reduced in the presence of N-methyl-D-aspartate and other glutamate agonists, suggesting desensitization of the endogenous transmitter receptor. In conclusion, some identified Lymnaea neurons respond to glutamate via a receptor with novel pharmacological properties. Furthermore, a Lymnaea interneuron may employ glutamate as a transmitter at excitatory synapses.

Animals↗

PubMed1996

Identification of a putative mechanosensory neuron in Lymnaea: characterization of its synaptic and functional connections with the whole-body withdrawal interneuron.

1. In this study, we identified a putative mechanosensory neuron in the freshwater pond snail Lymnaea stagnalis. This sensory neuron, termed right parietal dorsal 3 (RPD3), mediates part of the whole-body withdrawal behavior via the activation of a withdrawal interneuron. 2. RPD3 is located in the central ring ganglia, where its soma is situated on the dorsal surface of the right parietal ganglion. Intracellular injection of the dye Lucifer yellow revealed that RPD3 has both central and peripheral axonal projections. 3. In isolated-CNS preparations, RPD3 was quiescent. In semi-intact preparations, however, a gentle/moderate mechanical touch (by a pair of blunt forceps) to the mantle cavity or columellar musculature elicited action potentials in RPD3 in the absence of prepotential activity. Furthermore, mechanical stimulus-induced action potentials in RPD3 persisted in the presence of zero Ca2+/ high Mg2+ and high Ca2+/high Mg2+ salines. Together, these data suggest that RPD3 is most likely to be a primary sensory neuron. 4. In both isolated-CNS and semi-intact preparations, intracellular depolarization of RPD3 excited the whole-body withdrawal interneuron right pedal dorsal 11 (RPeD11). This synaptic connection persisted in the presence of high Ca2+ and high Mg2+ saline, suggesting that it is likely to be monosynaptic. Moreover, when stimulated electrically, the interneuron RPeD11 induced an hyperpolarizing response in RPD3. The possibility of this connection being monosynaptic was not tested, however, in the present study. Together, these data demonstrate that RPD3 excites RPeD11, which in turn may inhibit RPD3 activity. 5. In the semi-intact preparation, a mechanical touch to the mantle edge excited RPD3, which in turn generated action potentials in RPeD11. Zero Ca2+ saline blocked this synaptic connection between RPD3 and RPeD11, suggesting that it is chemical. 6. To demonstrate that RPD3 was sufficient to induce the withdrawal response and that the withdrawal behavior was mediated indirectly via RPeD11, we made simultaneous intracellular recordings from these two neurons while monitoring muscle contractions via a tension transducer. Intracellular depolarization of RPD3 elicited action potentials in RPeD11, followed by the contraction of the columellar muscle. Similar stimulation of RPD3 failed to excite a simultaneously hyperpolarized RPeD11 and as a result, no contraction of the columellar muscle occurred. Direct intracellular depolarization of RPeD11, however, induced the contraction of the columellar muscle. These data suggest that RPD3-induced withdrawal behavior is mediated in part via RPeD11.

Animals↗

PubMed1996

Operant conditioning of aerial respiratory behaviour in Lymnaea stagnalis

In this study, we operantly conditioned the aerial respiratory behaviour of the freshwater snail Lymnaea stagnalis. Aerial respiration in Lymnaea stagnalis is accomplished by the spontaneous opening and closing of its respiratory orifice, the pneumostome, at the water surface. Weak tactile stimulation of the pneumostome area, when the pneumostome is open, evoked only the pneumostome closure response, which is one aspect of the escape-withdrawal reflex. Pneumostome stimulation resulted in its closure and the termination of aerial respiratory activity. A contingent tactile stimulation paradigm was used to operantly condition the animals. Stimulation of the pneumostome whenever the animal attempted to breathe resulted in significantly fewer attempts to open the pneumostome as training progressed. The latency of the first breath (subsequent to stimulation), the number of breaths and the total breathing time were measured before and after each training period. Significant, quantifiable changes in these behavioural parameters were observed only in the operant conditioning group animals. Control animals receiving tactile stimulation to their pneumostome not contingent upon pneumostome opening movements (yoked controls) or those that were physically prevented from surfacing to breathe (hypoxic controls), did not exhibit significant changes in these behavioural parameters. Our data provide the first direct evidence for operant conditioning of respiration in any animal.

Journal Article↗

PubMed1996

Inhibition of the respiratory pattern-generating neurons by an identified whole-body withdrawal interneuron of Lymnaea stagnalis

Respiration and the whole-body withdrawal are two incompatible behaviors in the freshwater snail Lymnaea stagnalis. Whole-body withdrawal behavior is believed to be higher on the behavioral hierarchy than respiratory behavior. A central pattern generator (CPG) underlies respiratory behavior; whole-body withdrawal is mediated by a network of electrically coupled neurons. In this study, we provide evidence that the behavioral hierarchy between the whole-body withdrawal and the respiratory behaviors is established at the interneuronal level. We demonstrate that an identified whole-body withdrawal interneuron inhibits both muscular and neuronal components of the respiratory behavior in Lymnaea stagnalis. A pair of identified, electrically coupled interneurons, termed left and right pedal dorsal 11 (L/RPeD11), coordinates the whole-body withdrawal behavior in Lymnaea stagnalis. In the present study, RPeD11 inhibited spontaneously occurring respiratory CPG activity in isolated brain preparations. In addition, electrical stimulation of RPeD11 in a semi-intact preparation also inhibited respiratory CPG interneuron RPeD1. The synaptic connections between RPeD11 and the respiratory CPG neurons RPeD1 and visceral dorsal 4 (VD4) persisted in the presence of high-Ca2+/high-Mg2+ saline, suggesting the possibility that they may be monosynaptic. In a semi-intact preparation (lungmantle, pneumostome and central nervous system), electrical stimulation of RPeD11 induced pneumostome and columellar muscle contractions while inhibiting the activity of RPeD1. Moreover, mechanical stimulation of the respiratory orifice, the pneumostome, excited RPeD11, while its effects on the respiratory CPG neuron (RPeD1) were inhibitory. To determine the monosynaptic nature of connections between RPeD11 and the respiratory CPG neurons in the intact nervous system, we constructed these synapses in culture. RPeD11 and individual respiratory interneurons were isolated from their respective ganglia and co-cultured under conditions that support neurite outgrowth. Following neuritic overlap, RPeD11 was found to establish inhibitory synapses with the respiratory interneurons, supporting the hypothesis that these synaptic connections are likely to be monosynaptic in the intact central nervous system.

Journal Article↗

PubMed1995

Angiotensinogen-like epitopes are present in the CNS of Aplysia california and co-localize with urotensin I- and urotensin II-like immunoreactivities in the cerebral ganglia.

Immunohistochemistry was used to demonstrate urotensin I (UI), urotensin II (UII), and angiotensinogen (Ao)-like immunoreactivities (ir) in the CNS of Aplysia californica. The fish UI is a 41 amino acid peptide that has 50% identity with mammalian corticotropin-releasing factor (CRF). Identity also exists between UI and angiotensinogen in a tetrapeptide at the N-terminus. Ao-ir neurones were found in the F cluster of the Aplysia cerebral ganglia. Beaded Ao-ir fibres were seen in the neuropile and commissure of the cerebral, pleural and pedal ganglia. Ao neurosecretory material was also seen in the perineural region of the proximal supralabial nerve. Previously we have demonstrated UI and UII immunoreactivities were present in the CNS of Aplysia. A comparison of adjacent sections of the cerebral ganglia immunostained sequentially for UI, UII and Ao revealed that all three immunoreactivities co-existed in the same cells of the F cluster of the cerebral ganglia. Liquid-phase immunoabsorption of the Ao antiserum revealed that porcine or human angiotensinogen but not UI or UII were able to quench Ao immunostaining. Conversely UI and UII staining were quenched by white sucker (Catatomus commersoni) UI and goby (Gillichtys mirabilis) UII, respectively, but they were not modified by angiotensinogen. These results suggest that UI-, UII-, and Ao-like peptides might co-exist as separate entities in the cerebral ganglia of Aplysia californica where they can act in an integrated and/or independent modulatory way.

Angiotensinogen↗

PubMed1995

Sensorin-A immunocytochemistry reveals putative mechanosensory neurons in Lymnaea CNS.

The pond snail Lymnaea stagnalis is a useful model system for studying the neural basis of behaviour but the mechanosensory inputs that impact on behaviours such as respiration, locomotion, reproduction and feeding are not known. In Aplysia, the peptide sensorin-A appears to be specific to a class of central mechanosensory neurons. We show that in the Lymnaea central nervous system sensorin-A immunocytochemistry reveals a discrete pattern of staining involving well over 100 neurons. Identifiable sensorin positive clusters of neurons are located in the buccal and cerebral ganglia, and a single large neuron is immunopositive in each pedal ganglion. These putative mechanosensory neurons are not in the same locations as previously identified motoneurons, interneurons or neurosecretory cells. As would be expected for a mechanoafferent, sensorin positive fibres were found in nerve tracts innervating the body wall. This study lays the foundation for future electrophysiological and behavioural analysis of these putative mechanosensory neurons.

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↗

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

Modulation of ionic currents by dopamine in an interneurone of the respiratory central pattern generator of Lymnaea stagnalis.

Dopamine elicits alternating bursts of activity in the respiratory interneurones of the snail Lymnaea stagnalis. One of the neurones (VD4) was isolated in culture, and the effects of dopamine on both membrane voltage and current were studied utilising the whole-cell tight-seal recording technique. Dopamine had little effect on resting potentials near -60 mV, nor did it affect spike threshold or input resistance measured near -60 mV. However, it did alter the excitability of the cell, changing the response to current injection from one of repetitive spiking to one of rapid accommodation. Under voltage-clamp, VD4 responded to dopamine (EC50 = 92 nmol l-1) with increased net outward current at all potentials more positive than -60 mV. This was due primarily to an increase in voltage-gated potassium current and a decrease in calcium current. A reduction of Cd(2+)-sensitive outward current, possibly calcium-gated potassium current, was also evident at potentials more positive than +60 mV. The physiological actions of dopamine on these cells in vivo are consistent with the inhibitory mechanisms presented in this study.

Action Potentials↗

PubMed1994

DIFFERENTIAL TRACER COUPLING BETWEEN PAIRS OF IDENTIFIED NEURONES OF THE MOLLUSC LYMNAEA STAGNALIS

Electrical coupling is a common means of cell-to-cell communication in both neuronal and non-neuronal tissues (Lowenstein, 1985). Within the nervous system, many electrically coupled neurones exhibit dye coupling (Bennett, 1973; Stewart, 1978; Glantz and Kirk, 1980; Spencer and Satterlie, 1980; Fraser and Heitler, 1993); however, some electrically coupled cells do not dye-couple (Audesirk et al. 1982; Murphy et al. 1983; Berdan, 1987; Robinson et al. 1993; Veenstra et al. 1993). Electrical coupling and dye coupling, often considered in parallel, are in fact two different parameters that can vary independently (e.g. Audesirk et al. 1982; Perez-Armendariz et al. 1991). The giant identified neurones of pulmonate and opisthobranch molluscs have frequently been used for studies of neuronal communication and its plasticity (Winlow and McCrohan, 1987; Bulloch, 1989). In the present study, we explored the relationship between electrical and tracer coupling in both strongly and weakly coupled pairs of molluscan neurones. Specifically, we examined electrically coupled, identified neurones in a freshwater pond snail, Lymnaea stagnalis L., and tested for tracer coupling with Lucifer Yellow CH and biocytin. The cells examined were the strongly electrically coupled neurones, visceral dorsal 1 (VD1) and right parietal dorsal 2 (RPD2) (Boer et al. 1979; Benjamin and Pilkington, 1986), and the weakly coupled neurones, left buccal 1 (LB1) and right buccal 1 (RB1) (Benjamin and Rose, 1979). The use of these particular neurones made it possible to compare electrical coupling with tracer coupling in the molluscan central nervous system (CNS). All experiments were performed on laboratory-bred Lymnaea stagnalis (Mollusca, Pulmonata), maintained as previously described (Ridgway et al. 1991). The CNS was dissected from mature animals (1618 mm shell length) and pinned to the silicone rubber (RTV 616 GE) base of a recording dish in normal saline (51.3 mmol l-1 NaCl, 1.7 mmol l-1 KCl, 4.1 mmol l-1 CaCl2, 1.5 mmol l-1 MgCl2 and 5 mmol l-1 Hepes, pH 7.9). Following removal of the outer connective tissue sheath, a small Pronase crystal (Sigma, type XIV, P-5147), held by forceps, was carefully applied to specific ganglia; this treatment softened the inner sheath and facilitated microelectrode penetration. The CNS was then rinsed several times at 5 °C in normal saline.

Journal Article↗

PubMed1993

Effects on enflurane on gill withdrawal behaviors and the ability of gill motor neurones to elicit gill contractions in aplysia.

We used the Aplysia gill withdrawal reflex model system in order to study how enflurane effected both gill withdrawal adaptive behaviors and the activity of single identified neurones which are involved with the mediation of the gill withdrawal response. We found that a continuous superfusion of enflurane (0.5 and 1.0%) solution over the abdominal ganglion (the CNS) resulted in an increase in the spontaneous gill respiratory movements; an increase in the spontaneous discharges in identified central motor neurones; and a depolarizing shift in the resting membrane potential of these neurones. Enflurane also significantly effected the ability of the gill motor neurones to elicit a gill contraction when the motor neurone was depolarized to produce action potentials by passing depolarizing current into the neurone. Although in most cases the ability of the motor neurone to elicit a gill withdrawal contraction was decreased, that in one third of the cases was increased. Enflurane may exert its actions by effecting the activity of CNS control neurones which exert both facilitatory and suppressive control over the peripheral nervous system in the gill as well as by having direct effects on the motor neurones.

Journal Article↗

PubMed1993

Identification, characterisation and in vitro reconstruction of an interneuronal network of the snail Helisoma trivolvis.

1. We describe three interneurones and their follower cells in the central ganglionic ring of Helisoma trivolvis. 2. The largest neurone on the dorsal surface of the left pedal ganglion is shown to be an interneurone that contains dopamine and makes monosynaptic connections with a large number of follower cells in the visceral and left parietal ganglia. This neurone is designated as left pedal dorsal 1 (LPeD1). 3. Another giant neurone is located on the dorsal surface of the right pedal ganglion. Although the position and morphology of this cell, designated right pedal dorsal 1 (RPeD1), are similar to those of LPeD1, it contains serotonin rather than dopamine. This neurone was found to synapse only on LPeD1, no other follower cells have so far been discovered. The connections between LPeD1 and RPeD1 are mutually inhibitory. 4. A small FMRFamide-immunoreactive neurone, identified here as visceral dorsal 4 (VD4), is located on the dorsal surface of the visceral ganglion. This neurone has a large number of follower cells throughout the central ganglionic ring. Among these follower cells are LPeD1 and RPeD1. The transmitter utilized by VD4 at these synapses is probably FMRFamide. In addition, VD4 receives excitatory inputs from LPeD1 that appear to be chemical and monosynaptic. 5. To test further the monosynaptic and specific nature of the connections within the network, the three interneurones were isolated and cultured in vitro. In these circumstances, the three neurones extended neurites and formed synapses which, with one exception (occasional electrical coupling between LPeD1 and RPeD1), were of similar type to those observed in vivo. 6. The identification and characterization of these three interneurones and their follower cells should greatly facilitate future studies of the Helisoma trivolvis nervous system. Furthermore, the possibility that this three-cell network can be reconstructed in vitro should aid our understanding of the mechanisms underlying synapse formation and neuronal plasticity.

Animals↗

PubMed1993

Neuronal mapping studies on the central nervous system of the pulmonate snail Helisoma trivolvis.

The need for a generalized system for mapping neurons in the central ring ganglia of the freshwater pulmonate snail, Helisoma trivolvis, has recently become apparent. In the present study we have used retrograde staining of nerves to identify neurons projecting to the periphery. This map was combined with histochemical information to produce a map of neuronal clusters and landmark cells. Our mapping strategy was formulated to make direct comparison with maps already established for closely related species, i.e., Lymnaea stagnalis.

Animals↗