In eukaryotes, CREB‐binding protein (CBP), a coactivator of CREB, functions both as a platform fo... more In eukaryotes, CREB‐binding protein (CBP), a coactivator of CREB, functions both as a platform for recruiting other components of the transcriptional machinery and as a histone acetyltransferase (HAT) that alters chromatin structure. We previously showed that the transcriptional activity of cAMP‐responsive element binding protein (CREB) plays a crucial role in neuronal plasticity in the pond snail Lymnaea stagnalis. However, there is no information on the molecular structure and HAT activity of CBP in the Lymnaea central nervous system (CNS), hindering an investigation of its postulated role in long‐term memory (LTM). Here, we characterize the Lymnaea CBP (LymCBP) gene and identify a conserved domain of LymCBP as a functional HAT. Like CBPs of other species, LymCBP possesses functional domains, such as the KIX domain, which is essential for interaction with CREB and was shown to regulate LTM. In‐situ hybridization showed that the staining patterns of LymCBP mRNA in CNS are very similar to those of Lymnaea CREB1. A particularly strong LymCBP mRNA signal was observed in the cerebral giant cell (CGC), an identified extrinsic modulatory interneuron of the feeding circuit, the key to both appetitive and aversive LTM for taste. Biochemical experiments using the recombinant protein of the LymCBP HAT domain showed that its enzymatic activity was blocked by classical HAT inhibitors. Preincubation of the CNS with such inhibitors blocked cAMP‐induced synaptic facilitation between the CGC and an identified follower motoneuron of the feeding system. Taken together, our findings suggest a role for the HAT activity of LymCBP in synaptic plasticity in the feeding circuitry.
Mechano- and chemoafferent responsiveness as well as outputs of identified cerebral neurones were... more 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.
Localization and distribution of cerebral neurones sending axons into the three pairs of Helix po... more Localization and distribution of cerebral neurones sending axons into the three pairs of Helix pomatia lip nerves were investigated by the method of retrograde axonal NiCl2 transport. Using electrophysiological technics (extracellular recordings) the dependence of lip nerve's activity on inputs of other lip nerves was studied after application of various types of stimuli to the lip of semi-intact preparations. All lip nerves have neuronal representation in each lobe of the cerebral ganglia but in different proportions. Labelled neurones were located mainly on the ventral surface of the cerebral ganglia, most of them projecting to the medial, the least to the inner lip nerve. Lip nerves differ from each other according to the proportions of neurones of various size. They share in the axons of large (55-70 microns) and medium sized (30-40 microns) neurones in the order inner greater than outer greater than medial and medial greater than outer greater than inner lip nerve, respectively. Most neurones projecting to different nerves are located in discrete groups. According to the electrophysiological results the medial lip nerve has the most prominent afferent, while the inner one has the strongest efferent activity. Both the afferent and efferent activities of the outer lip nerve proved to be the least significant compared to the other lip nerves. Contralateral cerebral connections play an important role in the sensory information processing. The sensory input of a given nerve usually activates the contralateral member of another pair of lip nerves. Mechano- and chemo-afferent pathways have almost the same properties but there are some differences in latencies and other parameters.
The pond snail Lymnaea provides highly valuable experimental models for top-down analyses of asso... more The pond snail Lymnaea provides highly valuable experimental models for top-down analyses of associative learning and memory. Using classical and operant conditioning paradigms, molecular mechanisms of consolidation, reconsolidation, extinction, and forgetting of associative memory have been investigated. Long-term memory (LTM) forms after multi-trial aversive conditioning but, unusually, also after single-trial reward conditioning (‘flash-bulb memory’). Molecular mechanisms of LTM involve highly conserved signaling pathways (nitric oxide/cyclic guanosine monophosphate/protein kinase G, cyclic adenosine monophosphate/protein kinase A, and mitogen-activated protein kinase), transcriptional regulation of gene expression by cAMP response element binding protein and CCAAT/enhancer binding protein, and new protein synthesis. Importantly, a number of molecular processes involved in LTM have been traced from the behavioral level to single identified neurons.
We have photoinactivated identified feeding interneurons known as N1 and N2 neurons. These are pa... more We have photoinactivated identified feeding interneurons known as N1 and N2 neurons. These are pattern-generating neurons that are active in the protraction of the radula and rasping phases, respectively, of the feeding cycle of the pond snail. The N1 or N2 feeding interneurons in the buccal ganglia were filled with the fluorescent dye 5(6)-carboxyfluorescein (5-CF) from the cut end of the nerve that contains their axon. Filling the cerebrobuccal connective (N = 151) stained just one N1 cell in the contralateral buccal ganglion. Filling the postbuccal nerve stained neurons symmetrically in both buccal ganglia (N = 75): only one labeled cell in each ganglion is an N2 interneuron. The feeding rhythm was evoked by depolarizing a modulatory neuron, the SO, located in the buccal ganglia. The axonally filled N1 interneuron was irradiated at its axon in the buccal commissure with blue laser light (intensity of 0.5 MW.m-2). Irradiation of just one N1 completely blocked the feeding rhythm (seven preparations). In seven further preparations, N1 ablation slowed the SO-driven feeding rhythm and weakened the N1 input to the feeding neurons. Irradiation of the cell bodies of both the filled left and right N2 interneurons killed the cells but did not produce any consistent change in the feeding rate (15 preparations). The feeding interneurons and motoneurons still showed the characteristic N2 phase synaptic inputs, so more, as yet unidentified, N2 neurons must be located in other parts of the buccal ganglia. We conclude that the participation of the identified N1 interneurons is essential for the normal feeding pattern while other, still to be identified N2 neurons must be present and must contribute to the feeding rhythm. We suggest that the extra redundancy of the N2 network may be related to the greater necessity of sensory feedback control during rasping than during protraction of the radula.
Philosophical Transactions of the Royal Society B, May 29, 1992
The N1 neurons are a population of interneurons active during the protraction phase of the feedin... more The N1 neurons are a population of interneurons active during the protraction phase of the feeding rhythm . All the N1 neurons are coupled by electrical synapses which persist in a high Mg/low Ca saline which blocks chemical synapses. Individual N1 spikes produce discrete electrotonic postsynaptic potentials (PSPS) in other N1 cells, but the coupling is not strong enough to ensure 1:1 firing. Bursts of N1 spikes generate com pound PSPS in the feeding motoneurons. The sign (excitation or inhibition) of the N1 input corresponds with the synaptic barrage recorded during the protraction phase. Discrete PSPS are only resolved in a Hi-Di saline. Their variation in latency and number can be explained by variation in electrotonic propagation within the electrically coupled network of N1 cells. The excitatory postsynaptic potentials (EPSPS) in the 1 cell are reduced by 0.5 mM antagonists hexamethonium (HMT), atropine (ATR), curare (d-TC) and by methylxylocholine (MeXCh), all of which block the excitatory cholinergic receptor (Elliott et al. ( Phil. Trans. R. Soc. Lond. 336, 157-166 (Preceding paper.) (1992)). The 1 cell EPSPS were transiently blocked by phenyltrimethylammonium (PTMA), which is both an agonist and antagonist at the 1 cell excitatory acetylcholine (ACh) receptor (Elliott et al. 1992). The inhibitory postsynaptic potential (IPSP) in the 3 cell is blocked by bath applications of MeXCh and PTM A , which both abolish the response of the 3 cell to ACh (Elliott et al. 1992). It is concluded that the population of N1 cells are multiaction, premotor cholinergic interneurons.
In eukaryotes, CREB‐binding protein (CBP), a coactivator of CREB, functions both as a platform fo... more In eukaryotes, CREB‐binding protein (CBP), a coactivator of CREB, functions both as a platform for recruiting other components of the transcriptional machinery and as a histone acetyltransferase (HAT) that alters chromatin structure. We previously showed that the transcriptional activity of cAMP‐responsive element binding protein (CREB) plays a crucial role in neuronal plasticity in the pond snail Lymnaea stagnalis. However, there is no information on the molecular structure and HAT activity of CBP in the Lymnaea central nervous system (CNS), hindering an investigation of its postulated role in long‐term memory (LTM). Here, we characterize the Lymnaea CBP (LymCBP) gene and identify a conserved domain of LymCBP as a functional HAT. Like CBPs of other species, LymCBP possesses functional domains, such as the KIX domain, which is essential for interaction with CREB and was shown to regulate LTM. In‐situ hybridization showed that the staining patterns of LymCBP mRNA in CNS are very similar to those of Lymnaea CREB1. A particularly strong LymCBP mRNA signal was observed in the cerebral giant cell (CGC), an identified extrinsic modulatory interneuron of the feeding circuit, the key to both appetitive and aversive LTM for taste. Biochemical experiments using the recombinant protein of the LymCBP HAT domain showed that its enzymatic activity was blocked by classical HAT inhibitors. Preincubation of the CNS with such inhibitors blocked cAMP‐induced synaptic facilitation between the CGC and an identified follower motoneuron of the feeding system. Taken together, our findings suggest a role for the HAT activity of LymCBP in synaptic plasticity in the feeding circuitry.
Mechano- and chemoafferent responsiveness as well as outputs of identified cerebral neurones were... more 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.
Localization and distribution of cerebral neurones sending axons into the three pairs of Helix po... more Localization and distribution of cerebral neurones sending axons into the three pairs of Helix pomatia lip nerves were investigated by the method of retrograde axonal NiCl2 transport. Using electrophysiological technics (extracellular recordings) the dependence of lip nerve's activity on inputs of other lip nerves was studied after application of various types of stimuli to the lip of semi-intact preparations. All lip nerves have neuronal representation in each lobe of the cerebral ganglia but in different proportions. Labelled neurones were located mainly on the ventral surface of the cerebral ganglia, most of them projecting to the medial, the least to the inner lip nerve. Lip nerves differ from each other according to the proportions of neurones of various size. They share in the axons of large (55-70 microns) and medium sized (30-40 microns) neurones in the order inner greater than outer greater than medial and medial greater than outer greater than inner lip nerve, respectively. Most neurones projecting to different nerves are located in discrete groups. According to the electrophysiological results the medial lip nerve has the most prominent afferent, while the inner one has the strongest efferent activity. Both the afferent and efferent activities of the outer lip nerve proved to be the least significant compared to the other lip nerves. Contralateral cerebral connections play an important role in the sensory information processing. The sensory input of a given nerve usually activates the contralateral member of another pair of lip nerves. Mechano- and chemo-afferent pathways have almost the same properties but there are some differences in latencies and other parameters.
The pond snail Lymnaea provides highly valuable experimental models for top-down analyses of asso... more The pond snail Lymnaea provides highly valuable experimental models for top-down analyses of associative learning and memory. Using classical and operant conditioning paradigms, molecular mechanisms of consolidation, reconsolidation, extinction, and forgetting of associative memory have been investigated. Long-term memory (LTM) forms after multi-trial aversive conditioning but, unusually, also after single-trial reward conditioning (‘flash-bulb memory’). Molecular mechanisms of LTM involve highly conserved signaling pathways (nitric oxide/cyclic guanosine monophosphate/protein kinase G, cyclic adenosine monophosphate/protein kinase A, and mitogen-activated protein kinase), transcriptional regulation of gene expression by cAMP response element binding protein and CCAAT/enhancer binding protein, and new protein synthesis. Importantly, a number of molecular processes involved in LTM have been traced from the behavioral level to single identified neurons.
We have photoinactivated identified feeding interneurons known as N1 and N2 neurons. These are pa... more We have photoinactivated identified feeding interneurons known as N1 and N2 neurons. These are pattern-generating neurons that are active in the protraction of the radula and rasping phases, respectively, of the feeding cycle of the pond snail. The N1 or N2 feeding interneurons in the buccal ganglia were filled with the fluorescent dye 5(6)-carboxyfluorescein (5-CF) from the cut end of the nerve that contains their axon. Filling the cerebrobuccal connective (N = 151) stained just one N1 cell in the contralateral buccal ganglion. Filling the postbuccal nerve stained neurons symmetrically in both buccal ganglia (N = 75): only one labeled cell in each ganglion is an N2 interneuron. The feeding rhythm was evoked by depolarizing a modulatory neuron, the SO, located in the buccal ganglia. The axonally filled N1 interneuron was irradiated at its axon in the buccal commissure with blue laser light (intensity of 0.5 MW.m-2). Irradiation of just one N1 completely blocked the feeding rhythm (seven preparations). In seven further preparations, N1 ablation slowed the SO-driven feeding rhythm and weakened the N1 input to the feeding neurons. Irradiation of the cell bodies of both the filled left and right N2 interneurons killed the cells but did not produce any consistent change in the feeding rate (15 preparations). The feeding interneurons and motoneurons still showed the characteristic N2 phase synaptic inputs, so more, as yet unidentified, N2 neurons must be located in other parts of the buccal ganglia. We conclude that the participation of the identified N1 interneurons is essential for the normal feeding pattern while other, still to be identified N2 neurons must be present and must contribute to the feeding rhythm. We suggest that the extra redundancy of the N2 network may be related to the greater necessity of sensory feedback control during rasping than during protraction of the radula.
Philosophical Transactions of the Royal Society B, May 29, 1992
The N1 neurons are a population of interneurons active during the protraction phase of the feedin... more The N1 neurons are a population of interneurons active during the protraction phase of the feeding rhythm . All the N1 neurons are coupled by electrical synapses which persist in a high Mg/low Ca saline which blocks chemical synapses. Individual N1 spikes produce discrete electrotonic postsynaptic potentials (PSPS) in other N1 cells, but the coupling is not strong enough to ensure 1:1 firing. Bursts of N1 spikes generate com pound PSPS in the feeding motoneurons. The sign (excitation or inhibition) of the N1 input corresponds with the synaptic barrage recorded during the protraction phase. Discrete PSPS are only resolved in a Hi-Di saline. Their variation in latency and number can be explained by variation in electrotonic propagation within the electrically coupled network of N1 cells. The excitatory postsynaptic potentials (EPSPS) in the 1 cell are reduced by 0.5 mM antagonists hexamethonium (HMT), atropine (ATR), curare (d-TC) and by methylxylocholine (MeXCh), all of which block the excitatory cholinergic receptor (Elliott et al. ( Phil. Trans. R. Soc. Lond. 336, 157-166 (Preceding paper.) (1992)). The 1 cell EPSPS were transiently blocked by phenyltrimethylammonium (PTMA), which is both an agonist and antagonist at the 1 cell excitatory acetylcholine (ACh) receptor (Elliott et al. 1992). The inhibitory postsynaptic potential (IPSP) in the 3 cell is blocked by bath applications of MeXCh and PTM A , which both abolish the response of the 3 cell to ACh (Elliott et al. 1992). It is concluded that the population of N1 cells are multiaction, premotor cholinergic interneurons.
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