Electrophysiological and neurochemical study of long-term sensitization in the snail

The long-term sensitization of avoidance reflex was produced in snail Helix pomatia, which led to the remarkable increase in the pneumostome closures period. The formation of long-term sensitization is also accompanied by increase in excitability of command neurons of this reflex. One of the possible mechanisms of this phenomenon is the depolarization of these cells. The quantitative redistribution of water-soluble proteins with relative mobility 0.54 0.42 0.40 was also observed in the identified neurons, both included in the avoidance reflex (command neurons) and non-included (bursting neurons, nerve cells of pool D). The protein with the relative mobility of 0.75 was unique for the nerve cells of neurosecretory pool D in sensitized snails, and was never found in control animals.     

Water-soluble proteins of the subesophageal ganglionic complex in the edible snail in the early stages of learning

During the early stages of conditioned defensive reflex formation, a dramatic increase in the acidic protein concentration can be seen in the snail CNS (the mobility 0.58 in polyacrylamide gel) with reference to water-soluble proteins of the animals' ganglions under study. Such an increase occurs at the time when the majority of neurons are involved in the learning process. The rise of the protein content is also seen upon the snail arousal, 2-6 h after being awake. It is suggested that the high level of the protein correlates with the appearance of the orientation reflexes. At the cellular level this phenomenon appears to be linked with increased excitability of a great number of neurons.     

Effect of selective protein kinase C inhibitor on synaptic plasticity of command neurons of defensive behaviour during sensitization in snails

Polymyxin B (proteinkinase C inhibitor) effects on nociceptive sensitization of semiintact preparation were investigated in LP11 and RP11 snail neurons. It was found that application of sensitizing stimuli to control snail head initiated neural membrane depolarization, increase its excitability as well as depression of neural responses evoked by sensory stimulation during short-term stage. Polymyxin B application suppressed neural responses evoked by sensitizing (nociceptive) stimuli. At the same time changes in neural membrane excitability as well as neural responses evoked by tactile stimulation of snail foot or chemical stimulation of snail head were similar with ones in control snails. Polymyxin Bdid does not change the depression of neural responses evoked by tactile stimulation of snail head during short-term stages of sensitization but significantly suppressed facilitation of neural responses evoked by tactile stimulation of snail head during long-term stage of sensitization. It was suggested that proteinkinase C is involved in regulation of nociceptive mechanisms as well as in plasticity selective induction mechanisms in command neuron synaptic inputs activated by tactile stimulation of snail head.     

Two types of plasticity in the tentacle withdrawal reflex of Helix aspersa are dissociated by tissue location and response measure

The tentacle withdrawal reflex of the terrestrial snail Helix aspersa was studied in vitro. The reflex is evoked by mechanical stimulation of the nose. Lesion experiments showed that 45% to 75% of the response amplitude is attributable to peripheral pathways alone. The central contribution increases with increasing stimulus intensity.Repeated stimulation produced pure habituation at low stimulus strengths, and habituation mixed with intrinsic sensitization (warm-up effect) at high stimulus strengths. The simultaneous occurrence of habituation and sensitization is consistent with the dual process theory of plasticity. Additional results differentiate the two processes. Habituation can occur without the CNS, whereas intrinsic sensitization requires the CNS. Also, the two processes are differentially effective in their influences on response amplitude and duration: habituation is more effective in determining response amplitude, while sensitization is more effective in determining response duration.Although the establishment of sensitization requires the CNS, 81% of the memory for intrinsic sensitization was localized to the periphery, by lesion experiments. Extrinsic sensitization, caused by stimulation of the medial lip nerve, had similar behavioural effects and a similar memory locus. Both types of sensitization appear to be caused by neuromuscular facilitation mediated by a central pathway.Abbreviations CNS central nervous system - PNS peripheral nervous system - S-R stimulus-response - TRM tentacle retractor muscle     

Inhibitor of protein synthesis blocks long-term behavioral sensitization in the isolated gill-withdrawal reflex of Aplysia

To study the effects of protein synthesis inhibition on long-term sensitization of the gill- and siphon-withdrawal reflex of Aplysia, we have developed an isolated reflex preparation in which we could expose the inhibitor to only that part of the central nervous system involved in mediating the reflex and not to the other parts of the animal's central nervous system, thus minimizing the possible systemic side effects. We have found that long-term sensitization can be obtained in the isolated gill reflex, and that this long-term process, but not the short-term process, is blocked selectively by anisomycin, a reversible inhibitor of protein synthesis. Moreover, to obtain this blockade of long-term sensitization, this drug need only be applied during the training procedure.     

Inhibitor of protein synthesis blocks longterm behavioral sensitization in the isolated gill-withdrawal reflex of Aplysia

To study the effect of protein synthesis inhibition on long-term sensitization of the gill- and siphon-withdrawal reflex of Aplysia, we have developed an isolated reflex preparation in which we could expose the inhibitor to only that part of the central nervous system involved in mediating the reflex and not to the other parts of the animal's central nervous system, thus minimizing the possible systemic side effects. We have found that long-term sensitization can be obtained in the isolated gill reflex, and that this long-term process, but not the short-term process, is blocked selectively by anisomycin, a reversible inhibitor of protein synthesis. Moreover, to obtain this blockade of long-term sensitization, this drug need only be applied during the training procedure.     

The dynamics of the defensive and feeding conditioned reactions of the edible snail during long-term sensitization]

During the long-term sensitization in a snail there occurs an enhancement of a defensive reaction and a significant decrease of food intake. Defensive conditioning is facilitated and feeding conditioning deteriorated. The results give evidence that a formation of the state of the long-term sensitization is accompanied by a generation of a dominant defense focus.     

Immunocytochemical localization of insulin-related peptide(s) in the central nervous system of the snailHelix aspersa müller: Involvement in growth control

1. The presence of insulin-like substances has been demonstrated by immunocytochemistry in the central nervous system of the snail Helix aspersa. 2. The immunopositivity has been observed especially in the large perikarya of the mesocerebral green cells [the cerebral green cells (CeGC) stained in green by the alcian blue:alcian yellow technique]. 3. The removal of either the mesocerebrum or the CeGC stops the growth of the snail and induces the increase of the glycogen content in the mantle edge. 4. Our results show the existence of insulin-like material in the neurosecretory cells. Previous data having demonstrated the presence of specific binding sites to insulin in the cephalic ganglia of Helix aspersa, one may suggest that insulin could play a neuromodulatory or a neurotransmittory role in the central nervous system and might control the growth.     

Selective involvement of opioids in mechanisms of synapse-specific plasticity in Helix lucorum snail during sensitization acquisition

Effects of met-enkephalin (opioid peptide) and naloxone (opioid antagonist) on nociceptive sensitization were studied in L-RP11 Helix neurons. In control snails sensitizing stimulation produced reversible membrane depolarization and depression of neural responses evoked by sensory stimuli during the short-term stage of sensitization and facilitation of these responses at the long-term stage. Met-enkephalin (10 but not 0.1 microM) suppressed the neural responses evoked by nociceptive stimuli. Sensitizing stimulation during metenkephalin application prevented the facilitation of neural responses evoked by tactile stimulation of snail head, whereas facilitation of neural responses evoked by chemical stimulation of head or tactile stimulation of foot were similar to that in control sensitized snails. Sensitizing stimulation during met-enkephalin and/or naloxone application prevented the facilitation of neural responses evoked by chemical stimulation of snail head, whereas responses evoked by tactile stimulation of snail head or foot were facilitated (as in neurons of control sensitized snails). Opioids are suggested to be involved in regulation of nociceptive mechanisms and selective induction of long-term plasticity in L-RP11 neural inputs activated by tactile of chemical stimulation of snail head.     

Critical role of intracellular calcium in plasticity mechanisms of defense behavior command neurons LPl1 and PPl1 of Helix lucorum during nociceptive sensitization

The role of intracellular calcium in changes in excitability and responses of defense behavior command neurons LP11 and PP11 of Helix lucorum to sensory stimulation was investigated in semi-intact preparation of a snail during nociceptive sensitization. It was found that application of sensitizing stimuli onto the snail's head initiated membrane depolarization, increase in its excitability as well as depression of neural responses evoked by sensory stimuli in short-term period of sensitization and significant facilitation of neural responses in long-term period of sensitization. To elucidate the contribution of LP11 and PP11 neurons in plasticity rearrangements involved in the mechanisms of sensitization, we applied sensitizing stimuli during strong hyperpolarization of the neurons or after intracellular injection of calcium chelators. Application of sensitizing stimuli during hyperpolarization of the neurons suppressed the increase in membrane excitability and depressed the neural responses evoked by chemical stimulation of snail's head i.m. short- and long-term periods of sensitization. At the same time, synaptic facilitation of neural responses evoked by tactile stimulation of snail's head and foot was observed, which was similar to synaptic facilitation in the control sensitized snail. Intracellular injection of EGTA or BARTA (calcium chelators) before sensitization suppressed synaptic facilitation in neural responses evoked by sensory stimulation. Under these conditions, the increase in excitability was more pronounced then in the control snail neurons. The experimental results suggest the changes in neural responses evoked by sensory stimulation in sensitized snails involve postsynaptic calcium-dependent mechanisms of plasticity in LP11 and PP11 neurons.     

Behavioral changes during the acquisition of long-term sensitization of the defensive reflex in the edible snail

Reaction of long-term sensitization (LTS) of defensive reflex was elaborated in the snail. It was accompanied by a considerable increase of the time of closing of pneumostome. The results show that LTS is preserved for more than two week and may be a model of long-term memory.     

Vital investigation of the genome activity and neuronal synaptic plasticity in snail in the course of learning

Vital investigation of genome activity and its role in mechanisms of long-term synaptic plasticity formation were studied in LP11 neuron (command neurone of defence behaviour) during sensitisation (simple form of learning) in semi-intact preparation of snail Helix lucorum. Genome activity was investigate by means of specific fluorescent dye SYTO 16 and image analysis system. It was found that application of sensitizing stimulation (10% quinine solution) onto the snail head initiate two-phase changes in neural responses evoked by tactile or chemical stimulation. Depression of neural responses was obtained during short-term phase (during 1 hour after sensitization) and facilitation of neural responses--during long-term phases (over 1 hour). At the same time (15-20 min after learning), a significant increase in SYTO 16 fluorescent level was found in nucleus site LP11 neurone. Initial SYTO 16 fluorescence level was registered in 4-5 hours after sensitization. If the sensitization was produced during actinomycin D application (inhibitor of RNA synthesis, 20 microM) then facilitation of neural responses evoked by sensory stimulation were suppressed during long-term phase of sensitization and was the same as in control sensitized snails during short-term phase of learning. Increase in SYTO 16 fluorescence level in nucleus region site of LP11 neurone was completely prevented in sensitization during actinomycin D application. If actinomycin D was applied 30 min after sensitization (1 hour after first sensitizing stimulation) then synaptic facilitation and fluorescent dynamics was the same as in control sensitized snails. Our experimental data showed that nociceptive sensitization development was followed by quick (15-20 min) DNA activation and long-term synaptic facilitation (1 hour after sensitization), while induction of the processes was suppressed by inhibitor of RNA synthesis during short time interval (during 1 hour of learning).     

NMDA受体在痛觉过敏中的作用

N-甲基-D-天冬氨酸受体(NMDA受体)是中枢神经系统中兴奋性递质谷氨酸受体的一种类型,属于离子型受体。它涉及了体内许多复杂的生理和病理过程,包括wind-up、中枢敏化、长时程增强、外周敏化和内脏疼痛、细胞坏死和凋亡,除此以外,还参与了痛觉过敏的产生和维持。对NMDA受体在痛觉过敏中作用的探讨为研发新一代的镇痛药提供了广阔的思路和前景。     

Diurnal variations in physiological activities in the garden snail,Cryptozona ligulata

Summary Acetylcholinesterase (AChE) activity in the central nervous system and foot muscle in the garden snail,Cryptozona ligulata, was maximum at 20.00 h and minimum at 08.00 h during the 24 h period of the day. The cyclic variation in acetylcholine (ACh) was out of phase with that of AChE. In the body fluid, ACh content showed a rhythm with maximum at 00.00 h and minimum at 12.00 h, with AChE activity being in phase with it.The rhythm of spontaneous electrical activity of the nervous system was in phase with that of AChE activity in tissues. Perfusion with body fluid collected from snails at 20.00 h elevated the spontaneous electrical activity, while body fluid collected at 08.00 h inhibited the activity. Perfusion with extract prepared from the central nervous tissue isolated at 08.00 h elevated the electrical activity, while the extract prepared from nervous tissue isolated at 20.00 h inhibited the activity. Perfusion with 10^–4 M acetylcholine chloride solution elevated the electrical activity.It is suggested that the synthesis and release of ACh occur in a regular diel cycle in tissues. These changes, among others, may be responsible for the observed diurnal rhythmicity in electrical activity in the snail.     

Partial deficiencies in the myelin proteins of developing mice heterozygous for the shiverer mutation

The central nervous system of the shiverer mouse is known to be severely deficient in myelin. Animals heterozygous for this autosomal-recessive mutation were crossed, and the myelin proteins were examined in the brains and spinal cords of shiverers and unaffected littermates among the offspring. In the brains and spinal cords of nine of the 14 unaffected littermates examined, the quantities of the myelin basic and proteolipid proteins were lower than normal. Furthermore, in the brains of heterozygotes 33 to ～ 150 days old, the myelin basic and proteolipid proteins were reduced in amount, compared to wild-type controls; the myelin basic protein was also present in subnormal amounts in the spinal cords from heterozygous animals at the ages of 17 to 150 days. More severe reductions in the quantities of the myelin proteins were observed in central nervous system tissue from homozygous shiverer mice, and the quantity of the myelin proteolipid protein in the central nervous system of the shiverer mouse, expressed as a ratio to the control value at each age, underwent a developmental decline. In heterozygotes, as well as shiverers, the peripheral nerves were also deficient in the P₁ and P_r proteins, which are the same as the basic proteins in rodent central nervous system myelin. The findings regarding heterozygotes suggest that the defective primary gene product in the shiverer mouse could be the myelin basic protein itself or a protein required for a rate-limiting step in the processing of the myelin basic protein.     

The role of excitatory amino acid receptors and intracellular messengers in persistent nociception after tissue injury in rats

Increased pain sensitivity (hyperalgesia) and persistent nociception following peripheral tissue injury depends both on an increase in the sensitivity of primary afferent nociceptors at the site of injury (peripheral sensitization), and on an increase in the excitability of neurons in the central nervous system (central sensitization). We will review evidence that central sensitization, and the persistent nociception it leads to, are dependent on an action of glutamate and aspartate at excitatory amino acid (EAA) receptors. Additional evidence will be presented implicating a role of various intracellular second messengers that are coupled to EAA receptors (nitric oxide, arachidonic acid, and protein kinase C) to central sensitization and persistent nociception following tissue injury. Finally, we will examine the evidence for a contribution of molecular events, including noxious stimulus-induced expression of immediate-early genes such as c-fos to persistent nociception.     

The nervous system and the mapping of the neurons in the gastropod Helix lucorum L.]

Summarized literature and experimental author's data are presented concerning the structure of the nervous system and identification of individual neurons in the snail Helix lucorum. Information about especially well-known neurons is given in a table, maps of the ganglia are presented altogether with the results of retrograde staining of different cerebral and suboesophageal nerves. Are given the references concerning morphology of the central nervous system of the snail and identifiable neurons.     

Electrical connection between two bursting neurons inHelix pomatia

In some preparations of the CNS ofHelix pomatia, two neurons with bursting activity may be present in the right parietal ganglion, where usually there is only one bursting neuron RPal. If electrical activity of these neurons is recorded simultaneously, fluctuations of membrane potential are almost completely synchronized. Artificial depolarization and hyperpolarization of the membrane of one neuron caused depolarization or hyperpolarization of the other neuron. During long-term recording of the activity of both neurons synchronous modulation of their bursting activity was observed. Modulating factor (a peptide fraction obtained from the water-soluble part of snail brain homogenate) led to potentiation of the bursting activity of both neurons. It is concluded from the results of these experiments that two bursting RPal neurons, connected electrically with one another, may exist in the snail nervous system. In cases when the parameters of pacemaker activity of these two neurons are closely similar, electrical connection guarantees synchronization of their bursting activity and ensures a common frequency of changes in their membrane potential.     

The neurochemical basis of recurrent inhibition in the reflex arch of the defensive reaction]

Mechanisms of habituation in the network of identified neurones were investigated in isolated preparation of central nervous system in the snail Helix. It has been found that intracellularly induced spike discharge in premotor command neurones decreases synaptic responses to repeated nerve stimulation in all recorded command neurones. Application of the neuropeptide FMRFamide elicits similar changes in the network. Taking into account that the investigated command neurones contain FMRFamide, as was shown immunochemically, it is possible to assume the existence of recurrent inhibition in the network underlying avoidance reactions. This recurrent inhibition causes habituation of the network output in the cases when the repeated stimuli do not evoke sensitization via activation of serotonergic cells.     

A study of protein structure changes during hydration by diffuse X-ray scattering: II. Fourier transform analysis of diffuse X-ray scattering data

Radial distribution functions were deduced by Fourier transform analysis of the angular dependences of diffuse X-ray scattering intensities for the following proteins with different hydration degrees: water-soluble α-protein myoglobin, water-soluble (α + β) protein lysozyme, and transmembrane proteins from the photosynthetic reaction centers of purple bacteria Rhodobacter sphaeroides and Blastochlorii (Rhodopseudomonas) viridis. The results of Fourier transform analysis of X-ray scattering intensities give quantitative characteristics of the mechanism underlying the influence of water on the formation of biological macromolecules. On the one hand, water loosens the network of hydrogen bonds, which results in a considerable conformational mobility in the molecules of lysozyme and myoglobin and the reaction centers. On the other hand, water stabilizes and orders the protein globule. A strict correlation was found between the shift of the “first” maximum of the radial distribution function, loosening of the intraglobular hydrogen bonds, increase in the intramolecular mobility, and appearance of pronounced functional activity in macromolecules. The pattern of behavior of the first maximum in the transmembrane proteins of the reaction center was similar to that observed for the water-soluble proteins. However, the first maximum reached the limiting value of 2.9 Å at a considerably lower hydration degree compared with the water-soluble proteins. A quick transition of the protein complex of the reaction center to its native state is due to the fact that the dehydrated conformation of this complex is very close to the native conformation. Comparison of the radial distribution function for water, water-soluble proteins, and transmembrane proteins suggests a quantitative conclusion that water is the least densely packed and ordered system, the water-soluble proteins are more densely packed than water, and the transmembrane proteins are the most densely packed and ordered system.