The action of the neurotoxins 5,6-dihydroxytryptamine and p-chlorophenylalanine on the electrical activity parameters of the command neurons during long-term sensitization and learning in the snail]

The influence of 5,6-dihydroxytryptamine (5,6-DHT), which selectively destroyed serotonin terminals, and p-chlorphenylalanine, which inhibited serotonin synthesis, was studied on the long-term sensitization (LTS) in a snail. The membrane mechanisms were analyzed by measuring electrical characteristics of command neurons of defensive behavior LPa3, RPa3, LPa2, and RPa2. Snails injected with saline served as an active control. It was shown that the injected drugs inhibited the LTS in certain concentrations. A significant increase was observed in the membrane potential and the threshold of the action potential generation in the command neurons after 5,6-DHT injection in the doses of 20 and 30 mg/kg (in comparison with the active control). Sensitization of snails injected with saline solution led to the LTS and decrease in the membrane and threshold potentials of the command neurons. After the LTS, changes in membrane and threshold potentials in snails injected with 5,6-DHT were negligible in comparison with those injected with 5,6-DHT but without the LTS. Neither the LTS nor subsequent learning resulted in a further decrease in membrane and threshold potentials. Thus, the neurotoxin injection led to an increase in excitability of command neurons and their depolarization, and the LDS did not elicit further excitability increase. Since the shifts of the threshold and membrane potentials were the same, it was concluded that the increase in membrane excitability was induced by the depolarizing shift of the membrane potential.     

Comparative investigation of influence of injections of chlorpromazine and 5,6-dihydroxytryptamine on locomotion, defensive reactions of snall helix lucorum and excitability of command neurons in long-term sensitization

Comparative analysis of the action of chlorpromazine (CPZ) and neurotoxin 5,6-dihydroxytryptamine (5,6-DHT) on defensive reactions and locomotion of grape snail and elaboration of long-term sensitization (LTS), was carried out. Long-term (chronic) injection of chlorpromazine led to significant increasing of a pneumostome closing time and to changing of motor behaviour towards decrease of the velocity of the locomotion. Daily injections of 5,6-DHT in small doses within a week were accompanied by the gradual decrease of the velocity of snails locomotion, which was kept for a week. Similar effect was observed in injection of neurotoxin (30 mgs/kg). Injections of CPZ prevents elaboration of LTS, as well as injections of 5,6-DHT. After the action of CPZ, LTS, LTS followed by CPZ, and also during elaboration of LTS after injection of CPZ, the velocity of locomotion directly depended on the length of leg. During elaboration of LTS after injection of 5,6-DHT, such dependency is not retained. Electrophysiological study revealed that chronic injections of CPZ led to depolarizing shift of membrane potential and decrease of the threshold of action potential generation in command neurons as after injection of neurotoxin 5,6-DHT. Therefore, the action of neuroleptic drug CPZ on the defensive behaviour, locomotion of grape snail and electrical characteristics of identifying neurons is comparable with the action of toxic analogue of serotonin.     

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.     

Neuronal mechanisms contributing to long-term sensitization in Aplysia

1.) Cellular processes that contribute to the acquisition and expression of long-term sensitization have been examined in Aplysia. The tail-siphon withdrawal reflex was studied because the neural circuit for this reflex has been well characterized. Furthermore, the sensory neurons of this neural circuit exhibit cellular changes that accompany short-term sensitization. 2.) Repeated application of noxious stimuli to the animal produces a long-lasting enhancement of reflex withdrawal of the siphon when the animal is tested with a weak stimulus to the tail. These findings confirm the existence of long-term sensitization in Aplysia, first described by Pinkser et al. (1973). 3.) Biophysical correlates of long-term sensitization were examined in the first central relay of the tail-siphon reflex circuit, the sensory neurons that innervate the animal's tail. The net outward membrane currents of these cells reduced after 24 hours as a consequence of long-term sensitization training. 4.) The intracellular signal for the induction of these changes in membrane currents was examined by intracellular injection of cAMP into individual sensory neurons. This procedure mimics at least some of the effects of sensitization training at the single-cell level. cAMP induced a long-term reduction of membrane K+ currents 24 hours after the cells were injected with cAMP. The membrane currents reduced by cAMP were similar to those reduced by long-term sensitization training. 5.) Preliminary experiments indicate that neurotransmitters and agents that induce an evaluation of cAMP in the sensory neurons also alter the incorporation of labeled amino acids into specific proteins in the sensory neurons.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrophysiological study of effects of chronic injection of caffeine on defensive reflex conditioning in grape snail

It was found that chronic injection of caffeine to grape snail increases a velocity of elaboration of conditioned defensive reflex. It was shown that after daily injection of caffeine immediately after procedure of learning the conditioned defensive reflex elaborated faster than daily injection before procedure of learning. It has been shown, that chronic injection of caffeine both in naive as well as learned snails led to depolarizing shift of membrane potential and to decrease of threshold potential of command neurons of the defensive behaviour of grape snails. It was also found that addition of caffeine in bath solution led to decrease of threshold of generation of action potential of command neurons both in intact and learned snails. The resting membrane potential of command neurons was not changed.     

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.     

Dynamic defensive and feeding reactions to the acquisition of sensitization in edible snails]

In snails changes of defensive and alimentary behaviour and of reactions of command and modulator neurones of these kinds of behaviour were studied during elaboration of sensitization. After a single action of 50% chinine solution on mollusc's head, a short-term (during 50-70 min) and long-term (hours or days) facilitation was found of animals defensive reactions and responses of command neurones of defensive behaviour to tactile and chemical sensory stimuli. Alimentary behaviour of the snails and reactions of modulator neurones of alimentary behaviour to carrot juice presentation were inhibited in sensitized animals. Dynamics differences of defensive responses to tactile and chemical stimuli in the same sensitized animals were observed. Short-term responses facilitation under sensitization correlated chiefly with depolarization of the membrane potential of defensive behaviour neurones and with an increase of plasmatic membrane excitability. Prolonged facilitation of responses was due mainly to a change of effectiveness of synaptic transmission. The described model of sensitization elaboration can be a base for studying of molecular-cellular mechanisms, underlying the learning.     

Effect of antibodies against S100 proteins on neural plasticity in sensitized and naive snails

The influence of antibodies against total S100 protein fraction (AB-S100) and S100b protein (AB-S100b) on the activity of LP11 and RP11 neurons were studied in naive snails and during the nociceptive sensitization. Application of AB-S100 or AB-S100b (0.1 mg/ml) initiated membrane depolarization, increase in its excitability, and depression of neural responses to sensory stimulation in nonsensitized snails. The sensitization produced facilitation of neural transmission and increase in membrane excitability. Exposure to AB-S100 or AB-S100b (0.1 mg/ml) during sensitization substantially reduced its effects on neural transmission and membrane excitability. The difference between the extent of synaptic facilitation in neurons of sensitized snails and neurons of snails sensitized under conditions of AB-S100 or AB-S100b application was comparable with synaptic depression in neurons of naive snails produced by the isolated application of AB-S100 or AB-S100b. Application of AB-S100 of AB-S100b in the dose of 0.01 mg/ml did not change the parameters of neural activity. The obtained evidence suggest that S100 proteins (in particular, S100b) in L-RP11 neurons are involved in the mechanisms of membrane excitability, regulation of membrane potential and synaptic transmission in naive snails and in the mechanisms of membrane plasticity in the neurons during development of nociceptive sensitization.     

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.     

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).     

Effect of intracerebroventricular injection of 6-hydroxydopamine and 5,6-dyhydroxytryptamine on the phasic secretion of luteinizing hormone (author's transl)]

In ovariectomized rats carrying an estradiol sylastic implant the effect of the intraventricular administration of the serotonin neurotoxin 5,6-dihydroxytryptamine and of the norepinephrine neurotoxin 6-hydroxydopamine on the circadian secretion of the luteinizing hormone (LH) was studied. The decrease of 5-HT after injection of 5,6-dihydroxytryptamine reduced the a.m. and p.m. values and suppressed the phasic rhythm of LH. The diminution of noradrenaline after administration of 6-hydroxydopamine increased the a.m. and did not alter the p.m. values, resulting in a partial abolition of the rhythm.     

Electrophysiological study of 5,6-dihydroxytryptamine effect on defensive reflex conditioning in the snail

The role of serotonin in expression of membrane properties of identified neurons was studied during defensive reflex conditioning using the neurotoxic analogue of serotonin 5,6-dihydroxytryptamine (5,6-DHT). The defensive reflex conditioning in snails was destroyed on the second day after second injection of 5,6-DHT. Through the 1st weeks after second injection of 5,6-DHT the snails were learned but worse than snails after injection of saline solution. This result shows the recovery of snail's learning ability within 2 weeks after the second injection of 5,6-DHT. It was found that injection of 5,6-DHT prevented the decrease of membrane and threshold potentials of command neurons during defensive reflex conditioning as compared with the snails injected with 5,6-DHT without learning.     

The duration of the storage of the electrical characteristics of command neurons in the acquisition of long-term sensitization in the snail

The retention of the long-term sensitization (LTS) of defensive reflex and dynamics of change in electric characteristics (membrane potential (Vm) and action potential generation threshold (Vt)) of command neurons of defensive reflex was studied in a snail during behavioral tests. The membrane mechanisms were analyzed by measuring electrical characteristics of the LPa3, RPa3, LPa2, and RPa2 command neurons on the 1st, 4th, 7th, 10th, and 14th days after the LTS formation and 1 month later. The membrane potential and threshold potential in sensitized snails (-54.1 +/- 2.0 and 24.5 +/- 1.4 microV, respectively) were significantly (p < 0.001) decreased in comparison with the control animals (-60.9 +/- 0.8 and 19.9 +/- 0.6 microV respectively). These changes retained within 14 days after the LTS formation. The results suggest the long-term retention of the increased excitability of command neurons. A month after the LTS formation, the duration of the defensive reflex returned to the initial level and the electric characteristics of command neurons did not significantly differ from the control (-61.1 +/- 2.0 and 19.3 +/- 1.4 microV, respectively).     

Nature of Inhibition of Mitochondrial Respiratory Complex I by 6-Hydroxydopamine

Abstract: The catecholaminergic neurotoxin 6-hydroxydopamine causes parkinsonian symptoms in animals and it has been proposed that reactive oxygen species and oxidative stress, enhanced by iron, may play a key role in its toxicity. The present results demonstrate that 6-hydroxydopamine reversibly inhibits complex I (NADH dehydrogenase) of brain mitochondrial respiratory chain in isolated mitochondria. 6-Hydroxydopamine itself, rather than its oxidative products, was responsible for the inhibition. Iron(III) did not enhance inhibition but decreased it by stimulating the nonenzyme oxidation of 6-hydroxydopamine. Inhibition was potentiated to some extent by calcium ion. Desferrioxamine protected complex I activity against the inhibition, but it was not due to its chelator or antioxidative properties. Desferrioxamine was also shown to activate NADH dehydrogenase in the absence of 6-hydroxydopamine. Activation of mitochondrial respiration by desferrioxamine may contribute to the enhanced neuron survival in the presence of desferrioxamine in some neurodegenerative conditions.     

脊髓蛋白激酶Cα和γ亚型在吗啡依赖和戒断反应中的不同作用

在大鼠吗啡依赖和戒断模型上,采用行为学、免疫组织化学和Western blot方法观察鞘内应用蛋白激酶C(protien kinase C,PKC)抑制剂chelerythrine chloride(CHE)对吗啡依赖大鼠纳洛酮催促成断反应、脊髓Fos蛋白表达和脊髓神经元胞膜和胞浆PKCα、γ表达的影响,以探讨不同亚型PKC在吗啡依赖和戒断反应中的作用。结果表明,鞘内注射CHE能明显减轻吗啡成断症状的评分和吗啡戒断引起的痛觉异常,抑制吗啡成断期间脊髓Fos蛋白表达的增加;吗啡依赖可引起脊髓神经元PKCα和γ表达的上调和转位：吗啡戒断期间存在明显的且可被鞘内注射CHE抑制的PKCα转位,但未观察到明显的PKCγ转位。上述结果表明,脊髓PKC表达上调和转何可能参与吗啡依赖的形成和戒断反应的表达,且PKCα和γ亚型在吗啡依赖和戒断反应中的作用存在差异。     

The effect of neurochemical damage of dopaminergic terminals in early ontogenesis on the behavior of adult rats

6-Hydroxydopamine (75 mkg), producing selective degeneration of dopaminergic neurons in the brain, was injected intraamniotically to every rat fetus on 13th or 17th day of mother pregnancy. The other experiment was performed, when 6-hydroxydopamine administered i.p. to newborn rats on 4th or 10th day of life. All rats were growing, and several dopamine-dependent behaviors were investigated in adult animals: open field, rotation behavior, anxiety in elevated plus maze, conditioned placed preference, differentiation of novel and known alleys of Y-maze, aggressive behavior in intruder-resident test, selfs-stimulation of lateral hypothalamus. Prenatal administration of 6-hydroxydopamine initiated rotations and stereotypy, decreased anxiety in elevated maze, reduced reinforcing properties of amphetamine in place preference test, disturbed differentiation of novel and known alley in Y-maze, high aggression and decreased self-stimulation in less degree that postnatal injection of neurotoxin. Therefore, the early postnatal period is more sensitive to neurotoxin action than prenatal period of development. This phenomenon is connected with critical periods of development of dopaminergic system in ontogeny.     

Comparative Effects of Acute or Chronic Administration of Levodopa to 6-OHDA-lesioned Rats on the Expression and Phosphorylation of <Emphasis Type="Italic">N</Emphasis>-methyl-<Emphasis Type="SmallCaps">d</Emphasis>-aspartate Receptor NR1 Subunits in the Striatum

N-methyl-d-aspartate receptor (NMDA) has been increasingly implicated in the formation and maintenance of various forms of behavioral and synaptic plasticity. Recent evidence has linked striatal NMDA function to the adverse effects of long-term dopaminergic treatment in Parkinson’s disease. The subcellular distribution and phosphorylation of NMDA subunit, NR1, reflects NMDA receptor activity. To elucidate molecular mechanisms that underlie the persisting alterations in motor response occurring with levodopa treatment of parkinsonian patients, we evaluated the effects of unilateral nigrostriatal depletion with 6-hydroxydopamine and subsequent levodopa treatment on motor responses and NR1 alterations. Three weeks of levodopa administration to rats shortened the rotational duration and increased the peak turning responses, which lasted after withdrawal of chronic levodopa treatment. We found a significant reduction in the abundance of both phosphorylated NR1 on serine residues 890 and 896 (pNR1S890 and pNR1S896) and NR1 in the cell plasma membrane of lesioned striatum. Chronic treatment of lesioned rats with levodopa markedly upregulated pNR1S890, pNR1S896, and pNR1S897 in lesioned striatum with a concomitant normalization of the plasma membrane NR1 abundance. The magnitude of increased pNR1S890, pNR1S896, and pNR1S897 is dependent on the number of levodopa injections and is paralleled by a sensitization of the rotational response. Our data indicate that glutamate signaling is triggered during the levodopa administration. Activated NMDA receptor NR1-mediated mechanisms are involved in the persistent expression of the motor response alterations that appear during chronic levodopa therapy of parkinsonian rats and continue after treatment withdrawal. M. Kong and M. Ba are contributed equally to this work.