Responses of cerebral GABA-containing CBM neuron to taste stimulation with seaweed extracts in Aplysia kurodai

Aplysia kurodai distributed along Japan feeds well on Ulva pertusa but rejects Gelidium amansii with distinctive patterned movements of the jaws and radula. On the ventral side of the cerebral M cluster, four cell bodies of higher order neurons that send axons to the buccal ganglia are distributed (CBM neurons). We have previously shown that the dopaminergic CBM1 modulates basic feeding circuits in the buccal ganglia for rejection by firing at higher frequency after application of the aversive taste of seaweed such as Gelidium amansii. In the present experiments immunohistochemical techniques showed that the CBM3 exhibited gamma-aminobutyric acid (GABA)-like immunoreactivity. The CBM3 may be equivalent to the CBI-3 involved in changing the motor programs from rejection to ingestion in Aplysia californica. The responses of the CBM3 to taste stimulation of the lips with seaweed extracts were investigated by the use of calcium imaging. The calcium-sensitive dye, Calcium Green-1, was iontophoretically introduced into a cell body of the CBM3 using a microelectrode. Application of Ulva pertusa or Gelidium amansii extract induced different changes in fluorescence in the CBM3 cell body, indicating that taste of Ulva pertusa initially induced longer-lasting continuous spike responses at slightly higher frequency compared with that of Gelidium amansii. Considering a role of the CBM3 in the pattern selection, these results suggest that elongation of the initial firing response may be a major factor for the CBM3 to switch the buccal motor programs from rejection to ingestion after application of different tastes of seaweeds in Aplysia kurodai.     

Responses of cerebral GABA‐containing CBM neuron to taste stimulation with seaweed extracts in Aplysia kurodai

Aplysia kurodai distributed along Japan feeds well on Ulva pertusa but rejects Gelidium amansii with distinctive patterned movements of the jaws and radula. On the ventral side of the cerebral M cluster, four cell bodies of higher order neurons that send axons to the buccal ganglia are distributed (CBM neurons). We have previously shown that the dopaminergic CBM 1 modulates basic feeding circuits in the buccal ganglia for rejection by firing at higher frequency after application of the aversive taste of seaweed such as Gelidium amansii. In the present experiments immunohistochemical techniques showed that the CBM 3 exhibited γ‐aminobutyric acid (GABA)‐like immunoreactivity. The CBM 3 may be equivalent to the CBI‐3 involved in changing the motor programs from rejection to ingestion in Aplysia californica. The responses of the CBM 3 to taste stimulation of the lips with seaweed extracts were investigated by the use of calcium imaging. The calcium‐sensitive dye, Calcium Green‐1, was iontophoretically introduced into a cell body of the CBM 3 using a microelectrode. Application of Ulva pertusa or Gelidium amansii extract induced different changes in fluorescence in the CBM 3 cell body, indicating that taste of Ulva pertusa initially induced longer‐lasting continuous spike responses at slightly higher frequency compared with that of Gelidium amansii. Considering a role of the CBM 3 in the pattern selection, these results suggest that elongation of the initial firing response may be a major factor for the CBM 3 to switch the buccal motor programs from rejection to ingestion after application of different tastes of seaweeds in Aplysia kurodai. © 2005 Wiley Periodicals, Inc. J Neurobiol, 2005     

Patterned jaw movements and the motor neuron activity during rejection of seaweed in Aplysia kurodai

Japanese Aplysia kurodai feeds well on Ulva. In the present experiments we collected several species of seaweed at a location with many animals and initially explored the preference behavior for them. The animals rejected Grateloupia, Pachydictyon, Gelidium and Laurencia with rhythmic jaw and radula movements (active rejection). The animals sometimes bit off a piece of them (biting-off response). Recording activity of muscles contributing to jaw-opening and jaw-closing in freely moving animals showed that the onset of the jaw-closing activity, which always started later than the jaw-opening activity during each cycle of ingestion of Ulva, was advanced toward that of the jaw-opening activity during each cycle of the active rejection. Semi-intact experiments also showed that application of Pachydictyon or Gelidium extract to the lip region advanced the firing onset of the jaw-closing motor neurons at the radula-retraction phase. Video analysis showed that during the Ulva response the jaws opened at the radula-protraction phase and remained half-open at the earlier radula-retraction phase, while the jaws opened similarly at the radula-protraction phase but immediately closed at the radula-retraction phase during the Pachydictyon or Gelidium response. Accepted: 8 October 1997     

Sources of activator calcium ions in the contraction of smooth muscles in Aplysia kurodai

The physiological and pharmacological properties of contraction and the ultrastructure of buccal mass retractor muscle (I4) and gill-pinnule closure muscle (GPCM) in Aplysia kurodai were studied to learn more about the sources of activator Ca2+ in molluscan smooth muscle. Acetylcholine (ACh) and high K+-induced contractions were reduced by lowering the external Ca2+ concentration, and eliminated by the removal of extracellular Ca2+. Nifedipine appreciably reduced ACh- and high K+-induced contractions, while amiloride decreased only ACh-induced contractions and had no significant effect on high K+-induced contractions. When nifedipine and amiloride were applied together, either type of contraction was still appreciable. Serotonin (5-HT) could potentiate subsequent ACh- and high K+-induced contractions in I4; potentiated tension was significantly reduced by nifedipine and amiloride, whereas 5-HT inhibited ACh-and high K+-induced contractions in GPCM. The potentiating effects of 5-HT may be mediated by the activation of the Ca2+-channel to increase the influx from extracellular Ca2+. Caffeine caused contractions in Ca2+-free solution in both muscles. Electron microscopy revealed sarcolemmal vesicles underneath the plasma membrane in both muscle fibers. Electron microscopical cytochemistry demonstrated that pyroantimonate precipitates were localized in the sarcolemmal vesicles and in the inner surface of plasma membranes in the resting fibers. Present results indicate that the contractions of I4 and GPCM fibers are caused not only by Ca2+-influx but also by Ca2+ release from the intracellular storage sites, such as the sarcolemmal vesicles and the inner surface of plasma membranes.     

A vasotocin-like peptide in Aplysia kurodai ganglia: HPLC and RIA evidence for its identity with Lys-conopressin G.

The presence of a vasopressin (VP)- or vasotocin (VT)-like peptide in the central nervous system of the gastropod mollusc Aplysia has been indicated previously. In the case of Aplysia californica, HPLC and RIA evidence suggested the peptide was VT-like but not identical with the nonmammalian vertebrate peptide [Arg8]VT (AVT). In the present study, anterior ganglia extracts from the related species Aplysia kurodai were analyzed by HPLC followed by RIA. Further analysis of the major AVT-IR peak showed it to be indistinguishable, in three distinct solvent systems, from the sea snail venom peptide Lys-conopressin G, but to be different from the vertebrate peptides [Arg8]VP (AVP), [Lys8]VP (LVP), AVT, oxytocin (OT), mesotocin, isotocin, aspargtocin, glumitocin, and valitocin, from the sea snail venom peptide Arg-conopressin S, and from the peptides [Lys8]VT and [Gln8]OT. In addition, the carboxymethylated (CM) A. kurodai peptide had the same HPLC retention time as CM-Lys-conopressin G. The HPLC/RIA results suggest that (i) based on the properties of the solvent systems used, the A. kurodai peptide has two basic amino acids (like the conopressins but unlike the vertebrate peptides), and (ii) there is a high probability that the A. kurodai peptide is identical with Lys-conopressin G.     

Activation of cerebral neurons by static nerve inputs in Aplysia californica

A number of cerebral B-neurons of Aplysia californica were activated by tactile stimulation of the statocysts and by electrical stimulation of the static nerve. Types of responses recorded included antidromic spike, monosynaptic EPSP with or without spike and polysynaptic EPSPs. Some of the B-neurons were inhibited by trains of electrical stimulation of the static nerve. Hyperpolarization was mostly preceded by a short increase of spiking, usually during stimulation. Some A-neurons also responded with inhibition. The statocyst nerve contained axons carrying information not only from the statocysts to the cerebral ganglion but also from the cerebral ganglion to the statocyst. The latter pathway could be activated by tactile stimulation of the tentacles. Activation of either static nerves resulted in the increase of activity of the other static nerve through the cerebral ganglion, suggesting interaction between the two statocysts.     

Organization of neurons responding to photic stimulation in the Clare-Bishop area in cats

Neuronal organization in the Clare-Bishop cortical association area was studied by consecutive vertical penetration of an electrode and analysis of unit responses to photic stimulation during each penetration. Activity of one or two neurons was recorded during 131 penetrations, and activity of over 3 neurons responding to photic stimulation (visually driven) during 55 penetrations. In 8 of the 55 penetrations all neurons discovered in each had identical characteristics; this type of organization corresponded most of all to the columnar organization of the cortical neurons. In 24 penetrations the neurons were arranged in groups: two or three neurons of one type intermingled with neurons of other types. In 18 penetrations considerable overlapping of the receptive fields of neurons in the same column was observed. A chaotic distribution of neurons with different characteristics was found in 5 penetrations. It is suggested that the organization of neurons in the Clare-Bishop area in columns as functional units of cortical structure is not the principal type of their organization.L. A. Orbeli Institute of Physiology, Academy of Sciences of the Armenian SSR, Erevan. Translated from Neirofiziologiya, Vol. 11, No. 4, pp. 297–302, July–August, 1979.     

Activation of a calcium entry pathway by sodium pyrithione in the bag cell neurons of Aplysia

The ability of sodium pyrithione (NaP), an agent that produces delayed neuropathy in some species, to alter neuronal physiology was accessed using ratiometric imaging of cytosolic free Ca(2+) concentration ([Ca(2+)](i)) in fura PE-filled cultured Aplysia bag cell neurons. Bath-application of NaP evoked a [Ca(2+)](i) elevation in both somata and neurites with an EC(50) of approximately 300 nM and a Hill coefficient of approximately 1. The response required the presence of external Ca(2+), had an onset of 3-5 min, and generally reached a maximum within 30 min. 2-Methyl-sulfonylpyridine, a metabolite and close structural analog of NaP, did not elevate [Ca(2+)](i). Under whole-cell current-clamp recording, NaP produced a approximately 14 mV depolarization of resting membrane potential that was dependent on external Ca(2+). These data suggested that NaP stimulates Ca(2+) entry across the plasma membrane. To minimize the possibility that a change in cytosolic pH was the basis for NaP-induced Ca(2+) entry, bag cell neuron intracellular pH was estimated with the dye 2',7'-bis(carboxyethyl-5(6)-carboxy-fluorescein acetoxy methylester. Exposure of the neurons to NaP did not alter intracellular pH. The slow onset and sustained nature of the NaP response suggested that a cation exchange mechanism coupled either directly or indirectly to Ca(2+) entry could underlie the phenomenon. However, neither ouabain, a Na(+)/K(+) ATPase inhibitor, nor removal of extracellular Na(+), which eliminates Na(+)/Ca(2+) exchanger activity, altered the NaP-induced [Ca(2+)](i) elevation. Finally, the possibility that NaP gates a Ca(2+)-permeable ion channel in the plasma membrane was examined. NaP did not appear to activate two major forms of bag cell neuron Ca(2+)-permeable ion channels, as Ca(2+) entry was unaffected by inhibition of voltage-gated Ca(2+) channels using nifedipine or by inhibition of a voltage-dependent, nonselective cation channel using a high concentration of tetrodotoxin. In contrast, two potential store-operated Ca(2+) entry current inhibitors, SKF-96365 and Ni(2+), attenuated NaP-induced Ca(2+) entry. We conclude that NaP activates a slow, persistent Ca(2+) influx in Aplysia bag cell neurons.     

Projections from the ventral subiculum to supraoptical hypothalamic region neurons not responding to stimulation of the hypophyseal pedunculus

Effects of subiculum stimulation on spike activity of neurons localized in the supraoptical nucleus (SON) and perinuclear region were studied in experiments on rats; special attention was paid to neurons that did not respond to stimulation of the hypophyseal pedunculus. With rare exception, the SON cells did not respond to subiculum stimulation; 47% of neurons in the perinuclear region were activated after subiculum stimulation, whereas in 15% the frequency of spike activity decreased. Some neurons were found in the perinuclear region that responded to subiculum stimulation by antidromic spike generation.Organization of the studied afferent input to neurons of the supraoptical region and probability of interconnections between investigated structures are discussed.Neirofiziologiya/Neurophysiology, Vol. 25, No. 4, pp. 253–257, July–August, 1993.     

Input and output organization of cerebral neurones responding to tactile and taste stimuli applied to the lip of Helix pomatia L

Mechano- and chemoafferent responsiveness as well as outputs of identified cerebral neurones were investigated by electrophysiological methods in Helix pomatia L. the axonal projections of the identified cells were studied by intracellular staining. The studied neurones proved to be unipolar, their main axon branches were found in ipsilateral lip nerves. They could be divided into several groups according to their spontaneous activity, input and output organization and the selectivity of their responses to different tactile and taste stimuli applied to the lip. The activity of most of the neurones could be influenced by both ipsi- and contralateral inputs. They receive afferent input mostly through the medial lip nerves and their efferent information is transferred to the periphery mainly through the pair of inner lip nerves. There were seven neurones among the identified cells which responded selectively to taste stimuli identified in previous behavioural tests as phagostimulants. They can be considered as elements of the cerebral system regulating taste discrimination and feeding.     

Evaluation of cellular mechanisms for modulation of calcium transients using a mathematical model of fura-2 Ca2+ imaging in Aplysia sensory neurons.

A theoretical model of [Ca++]i diffusion, buffering, and extrusion was developed for Aplysia sensory neurons, and integrated with the measured optical transfer function of our fura-2 microscopic recording system, in order to fully simulate fura-2 video or photomultiplier tube measurements of [Ca++]i. This allowed an analysis of the spatial and temporal distortions introduced during each step of fura-2 measurements of [Ca++]i in cells. In addition, the model was used to evaluate the plausibility of several possible mechanisms for modulating [Ca++]i transients evoked by action potentials. The results of the model support prior experimental work (Blumenfeld, Spira, Kandel, and Siegelbaum, 1990. Neuron. 5: 487-499), suggesting that 5-HT and FMRFamide modulate action potential-induced [Ca++]i transients in Aplysia sensory neurons through changes in Ca++ influx, and not through changes in [Ca++]i homeostasis or release from internal stores.     

A model for the stimulation of taste receptor cells by salt.

A taste cell mucosal surface is regarded as a planar region containing bound anionic sites and openings to ionic channels. It is assumed that the bulk aqueous properties of the exterior phase are not continuous with the surface but terminate at a plane near the surface. The region between the (Stern) plane and the membrane is regarded as having a lower dielectric constant than bulk water. This fact admits the possibility of ion pair formation between fixed sites and mobile cations. Mobile ion pairs entering the region may also bind to a fixed anionic site. Thus, it is assumed that mobile cations and ion pairs are potential determining species at the surface. Binding cations neutralizes surface charges, whereas binding mobile ion pairs does not. This competition accounts for the observed anion effect on stimulation of tast receptors by sodium salts. The potential profile is constructed by superimposing the phase boundary potentials with an ionic diffusion potential across the membrane. The model accounts for the anion effect on receptor potential, pH effects, the reversal of polarity when cells are treated with FeCl3, and the so-called "water reponse," depolarization of the taste cell upon dilution of the stimulant solution below a critical lower limit. The proposed model does not require both bound cationic and anionic receptors, and further suggests that limited access to a Stern-like region continuous with membrane channels may generally serve to control transport of ions.     

Ultrastructural localization of calcium pyroantimonate in Mauthner neurons of goldfish fry adapted to natural stimulation

The localization of Ca2+ in control and adapted goldfish fry Mauthner cells (M-cells) revealed by sedimentation with potassium pyroantimonate technique was investigated. It has been shown the following. 1. In the control M-cells electron dense precipitates are present in the extracellular space, commonly within the active zone clefts of chemical synapses, throughout the whole apposition of the mixed synapses and in the synaptoplasm of both type afferent boutons. No precipitates were seen in the cytoplasm of M-cells. 2. After long term natural (vestibular) stimulation (LTNS), resulting in a strong functional suppression of M-cells, precipitates disappeared entirely from active zones but remained numerous in the cytoplasm of M-cells. The distribution of precipitates within the cytoplasm was non-uniform, the highest density was observed on the surfaces of intracellular organelles and elements of the cytoskeleton. 3. In fatigued M-cells after LTNS and after a subsequent one day rest the distribution of precipitates was less intensive, while in the whole it resembled that of fatigued M-cells. 4. In adapted M-cells the distribution of precipitates was similar to that observed in control. M-cells after LTNS, but the amount and size of the precipitated grains were noticeably increased. 5. The most numerous precipitates were seen in adapted M-cells after LTNS. They were localized throughout the postsynaptic cytoplasm and in a lesser order in the presynaptic cytoplasm. 6. After one day rehabilitation the intensitivity of cytochemical reaction of Ca2+ ion precipitation restored to the initial stage characteristic of adapted M-cells before LTVS. The results obtained suggest that the total concentration of Ca2+ ions in adapted M-cells and the dynamics of their exchanges between cytosole and intracellular depots, such as the smooth endoplasmic reticulum, may increase to keep a normal or even increased functional activity of M-cells, both before and after the LTNS.     

Responsiveness of neurons in the hamster parabrachial nuclei to taste mixtures

Responses from hamster parabrachial nuclei neurons to stimulation of the anterior tongue with sucrose, NaCl, HCl, quinine hydrochloride, and the six two-component mixtures of these stimuli were recorded. A cell's response to a mixture approached its response to the mixture's more effective component in the majority of cases, but was sometimes greater or smaller than this response. The best predictor of a neuron's response to a mixture, then, was its response to the mixture's more effective component. The single-component stimulus producing the maximum response was determined for each neuron and the response to this stimulus was compared with the responses evoked by the six mixtures. For 30% of the cells, a mixture elicited a response reliably, but only 1.1-2.1 times greater than the response to the best single-component stimulus. Thus, there were no neurons specialized to respond to these mixtures. The across-neuron patterns elicited by mixtures and the responses of best-stimulus classes to mixtures were studied for comparison with psychophysical data on taste mixtures. Mixtures were usually correlated with single-component stimuli in the mixture, but not with stimuli not in the mixture. In fact, five of the six mixtures fell directly between their components in a multidimensional scaling plot. In addition, a mixture was most effective in stimulating only those classes of neurons maximally stimulated by the mixture's components. These results correlate with psychophysical data suggesting that mixtures of taste stimuli evoke the same taste qualities as evoked by the mixture's components.     

Basis for the calcium specificity in the plant response to extracellular stimulation

The Ca2+ cation is fully recognized as an important intracellular second messenger coupling a wide range of extracellular stimuli to characteristic responses in plant cells. Such a pleiotropic effect raises questions regarding the mechanisms by which the signalling pathways, all of then involving an increase in intracellular calcium concentration, can be specific to a given stimulus. Here, we present recent results which shed light into different concepts which may explain the response specificity in signalling processes, such as "the cross-talk between signalling pathways", "the Ca2+ signatures" and "the compartmentation of Ca(2+)-signalling".