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.     

Cell and molecular analysis of long-term sensitization in Aplysia

We have found that one cellular locus for the storage of the memory underlying short-term sensitization of the gill and siphon withdrawal reflex in Aplysia is the set of monosynaptic connections between the siphon sensory cells and the gill and siphon motor neurons. These connections also participate in the storage of memory underlying long-term sensitization. In animals that have undergone long-term sensitization, the amplitudes of the monosynaptic connections are significantly larger (2.2x) than the ones in control animals. To study the mechanisms of onset and retention of long-term synaptic facilitation that underly long-term sensitization and the role of protein synthesis in long-term memory, we have developed two types of reduced preparations: the intact reflex isolated from the remainder of the animal, and a dissociated cell culture system in which the monosynaptic component (sensory neurons and motor neurons) of the neuronal circuit mediating the withdrawal reflex is reconstituted. We found that protein synthesis inhibitors, such as anisomycin or emetine, and RNA synthesis inhibitors, such as actinomycin D or alpha-amanitin, blocked long-term facilitation without interfering with short-term facilitation. These results suggest that the acquisition of long-term memory may require the expression of genes and the synthesis of proteins not needed for short-term memory.     

Water-soluble and membrane proteins of the central nervous system of the edible snail at different stages of long-term sensitization

An increase in the amount of water-soluble proteins with the mobility of 0.56 in polyacrylamide gel was observed in the snail central nervous system at the early stages of long-term sensitization of the defensive reflex. Such an increase seems to be associated with the enhanced motor activity and a rise in the excitation level at this stage. 2 hours, 1 day and 3 days after long-term sensitization a dramatic decrease in the amount of water-soluble proteins with the mobility of 0.54, 0.42, 0.40 and membrane protein with the molecular weight of 24000 kD was observed. Such a decrease appears to reflect the stage nature of long-term sensitization and to be linked with shifts in the functional characteristics of some cellular elements of snail central nervous system.     

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     

Effect of thymalin on protein synthesis in the brain and conditioned-reflex activity of the progeny of neurosensitized female rats

Thymalin administration to two-week-old offspring of neurosensitized female rats prevented the development of protein synthesis disturbances in the central nervous system and the retention of conditioned reflex of passive avoidance. Thymalin injection to the offspring of intact female rats improved conditioned reflex retention and did not affect brain protein synthesis. A possible mechanism of thymalin effect in conditions of congenital neuroimmunopathology is discussed.     

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)     

Imitation of neurophysiological effects of long-term sensitization with the action of the blockers of protein synthesis in snailsHelix lucorum

The experiments on command neurons of defensive behavior in snailsHelix lucorum have shown that the synaptic components of responses to the testing stimuli are facilitated within 65 to 75 min after the onset of the application of the protein synthesis inhibitors cycloheximide or anisomycin (30 µM during 30 min) to the central nervous system. At the same time, the parameters of the electrical properties of neuronal membrane (membrane potential level and excitability) were not affected by the blockers. The facilitatory effects were not observed when the blockers were applied for a longer period or when their concentration was raised to 300 µM. The blockade of the responses to different sensory stimuli appeared selective. The facilitation of the responses to tactile stimulation of the head, to weak quinine solution, and to carrot juice lasted for about an hour, 2–3 h and about 1.5 h, respectively. The blockers facilitated only the responses to tactile stimulation of the head, but did not change the responses to stimulation of the middle part of the foot or the mantle bolster. Some characteristics of the blocking effects are similar to those of long-term sensitization. The blocking effects observed are suggested to be due to activation of synthesis of short-living proteins functionally related, in particular, to selective regulation of synaptic transmission.Neirofiziologiya/Neurophysiology, Vol. 26, No. 1. pp. 61–67, January–February, 1994.     

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。     

The role of rapid, local, postsynaptic protein synthesis in learning-related synaptic facilitation in aplysia

The discovery that dendrites of neurons in the mammalian brain possess the capacity for protein synthesis stimulated interest in the potential role of local, postsynaptic protein synthesis in learning-related synaptic plasticity. But it remains unclear how local, postsynaptic protein synthesis actually mediates learning and memory in mammals. Accordingly, we examined whether learning in an invertebrate, the marine snail Aplysia, involves local, postsynaptic protein synthesis. Previously, we showed that the dishabituation and sensitization of the defensive withdrawal reflex in Aplysia require elevated postsynaptic Ca(2+), postsynaptic exocytosis, and functional upregulation of postsynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors. Here, we tested whether the synaptic facilitation that underlies dishabituation and sensitization in Aplysia requires local, postsynaptic protein synthesis. We found that the facilitatory transmitter, serotonin (5-HT), enhanced the response of the motor neuron to glutamate, the sensory neuron transmitter, and this enhancement depended on rapid protein synthesis. By using individual motor neurites surgically isolated from their cell bodies, we showed that the 5-HT-dependent protein synthesis occurred locally. Finally, by blocking postsynaptic protein synthesis, we disrupted the facilitation of the sensorimotor synapse. By demonstrating its critical role in a synaptic change that underlies learning and memory in a major model invertebrate system, our study suggests that local, postsynaptic protein synthesis is of fundamental importance to the cell biology of learning.     

Differential effects of serotonin depletion on sensitization and dishabituation in the leech, Hirudo medicinalis.

The goal of these experiments was to test the hypothesis that serotonin (5-HT) is involved in facilitation of the shortening reflex in the leech Hirudo medicinalis. For this reason, we have used the toxin 5-hydroxytryptamine (5,7-DHT) to deplete serotonin from the nervous systems of intact leeches and have assessed the effect on early facilitation, dishabituation, and sensitization of the touch-elicited shortening reflex using behavioral procedures previously developed in our lab (Boulis and Sahley, 1988). We find that 5,7-DHT lesions completely attenuate early facilitation and sensitization but only reduce dishabituation of the touch-elicited shortening reflex. Histological analyses of the ganglia from these leeches using glyoxilic acid staining procedures revealed an absence of staining in the Retzius cell of experimental leeches. All other serotonin-containing neurons showed glyoxilic acid staining comparable to that observed in the control leeches.     

Differential effects of serotonin depletion on sensitization and dishabituation in the leech,Hirudo medicinalis

The goal of these experiments was to test the hypothesis that serotonin (5-HT) is involved in facilitation of the shortening reflex in the leech Hirudo medicinalis. For this reason, we have used the toxin 5-hydroxytryptamine (5,7-DHT) to deplete serotonin from the nervous systems of intact leeches and have assessed the effect on early facilitation, dishabituation, and sensitization of the touch-elicited shortening reflex using behavioral procedures previously developed in our lab (Boulis and Sahley, 1988). We find that 5,7-DHT lesions completely attenuate early facilitation and sensitization but only reduce dishabituation of the touch-elicited shortening reflex. Histological analyses of the ganglia from these leeches using glyoxilic acid staining procedures revealed an absence of staining in the Retzius cell of experimental leeches. All other serotonin-containing neurons showed glyoxilic acid staining comparable to that observed in the control leeches.     

Encoding of the cough reflex in anesthetized guinea pigs

We have previously described the physiological and morphological properties of the cough receptors and their sites of termination in the airways and centrally in the nucleus tractus solitarius (nTS). In the present study, we have addressed the hypothesis that the primary central synapses of the cough receptors subserve an essential role in the encoding of cough. We found that cough requires sustained, high-frequency (≥8-Hz) afferent nerve activation. We also found evidence for processes that both facilitate (summation, sensitization) and inhibit the initiation of cough. Sensitization of cough occurs with repetitive subthreshold activation of the cough receptors or by coincident activation of C-fibers and/or nTS neurokinin receptor activation. Desensitization of cough evoked by repetitive and/or continuous afferent nerve activation has a rapid onset (<60 s) and does not differentiate between tussive stimuli, suggesting a central nervous system-dependent process. The cough reflex can also be actively inhibited upon activation of other airway afferent nerve subtypes, including slowly adapting receptors and pulmonary C-fibers. The sensitization and desensitization of cough are likely attributable to the prominent, primary, and unique role of N-methyl-d-aspartate receptor-dependent signaling at the central synapses of the cough receptors. These attributes may have direct relevance to the presentation of cough in disease and for the effectiveness of antitussive therapies.     

Nitric oxide as a modulator of central respiratory rhythm in the isolated brainstem of the bullfrog (Rana catesbeiana)

Nitric oxide (NO) is a unique interneuronal neurotransmitter and/or neuromodulator that is involved in a variety of physiological functions within the central nervous system (CNS). In neural tissue, NO is generated from an oxygen-dependent, constitutive NO synthase (NOS) by glutamatergic stimulation of N-methyl-D-aspartate (NMDA) receptors. Recent studies indicate that NO has excitatory effects on breathing within the CNS and mediates a central component of the hypoxic ventilatory reflex in mammals. Because NMDA receptors are important in central respiratory rhythmogenesis, we hypothesized that NO would have significant effects on the central pattern generator (CPG) for breathing in the brainstem. To test this hypothesis, the effects of NO on respiratory-related neural activity were investigated using an in vitro brainstem preparation from North American bullfrogs (Rana catesbeiana). Extracellular recordings of respiratory-related burst activity were made from cranial nerves V, X and XII before and during superfusion of the brainstem with NO-generating compounds, or inhibitors of NO synthesis. Addition of the NO donor, sodium nitroprusside (SNP; 0.1-1.0 mM), or the amino acid precursor for NO synthesis, L-arginine (L-Arg; 0.01-1.0 mM), caused significant increases in respiratory-related burst frequency. Inhibition of NOS with N omega-nitro-L-arginine (L-NA; 5-10 mM), a non-selective NOS inhibitor, caused a significant reduction in burst frequency or reversibly abolished neural activity. Brainstem perfusion with the specific neuronal NOS (nNOS) inhibitor, 7-nitro indazole (7-NI), produced significant, dose-dependent reversible reductions in burst frequency at concentrations of 0.1, 0.5 and 1.0 mM. These results suggest that production of NO, probably via nNOS, provides an excitatory input to the respiratory CPG in the amphibian brainstem. Our results suggest that NO may be a necessary inter- or intracellular messenger for neurotransmission and/or neuromodulation of central respiratory drive to motor effectors in the bullfrog.     

Brief exposure to sensory cues elicits stimulus-nonspecific general sensitization in an insect

The effect of repeated exposure to sensory stimuli, with or without reward is well known to induce stimulus-specific modifications of behaviour, described as different forms of learning. In recent studies we showed that a brief single pre-exposure to the female-produced sex pheromone or even a predator sound can increase the behavioural and central nervous responses to this pheromone in males of the noctuid moth Spodoptera littoralis. To investigate if this increase in sensitivity might be restricted to the pheromone system or is a form of general sensitization, we studied here if a brief pre-exposure to stimuli of different modalities can reciprocally change behavioural and physiological responses to olfactory and gustatory stimuli. Olfactory and gustatory pre-exposure and subsequent behavioural tests were carried out to reveal possible intra- and cross-modal effects. Attraction to pheromone, monitored with a locomotion compensator, increased after exposure to olfactory and gustatory stimuli. Behavioural responses to sucrose, investigated using the proboscis extension reflex, increased equally after pre-exposure to olfactory and gustatory cues. Pheromone-specific neurons in the brain and antennal gustatory neurons did, however, not change their sensitivity after sucrose exposure. The observed intra- and reciprocal cross-modal effects of pre-exposure may represent a new form of stimulus-nonspecific general sensitization originating from modifications at higher sensory processing levels.     

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.     

Receptors and transmission in the brain-gut axis: potential for novel therapies. V. Fast and slow extrinsic modulation of dorsal vagal complex circuits

Vago-vagal reflex circuits in the medulla are responsible for the smooth coordination of the digestive processes carried out from the oral cavity to the transverse colon. In this themes article, we concentrate mostly on electrophysiological studies concerning the extrinsic modulation of these vago-vagal reflex circuits, with a particular emphasis on two types of modulation, i.e., by "fast" classic neurotransmitters and by "slow" neuromodulators. These examples review two of the most potent modulatory processes at work within the dorsal vagal complex, which have dramatic effects on gastrointestinal function. The reader should be mindful of the fact that many more different inputs from other central nervous system (CNS) loci or circulating humoral factors add to this complex mix of modulatory inputs. It is likely that similar long-term modulations of synaptic transmission occur with other neurotransmitters and may represent an important mechanism for the integration and regulation of neuronal behavior. Of course, this fact strongly militates against the success of any single drug or approach in the treatment of motility disorders having a CNS component.     

Effect of galanin on isolated strips of smooth muscle from the gastrointestinal tract of chickens

The contractile effects of galanin on isolated longitudinal smooth muscle strips of pre-crop esophagus, proventriculus, duodenum, colon, and cecum of chickens were investigated. Application of galanin (5.0-100.0 nM) evoked strong contractions from the colon and cecum (hindgut), but evoked minimal responses from the pre-crop esophagus, proventriculus, and duodenum (foregut). Previous studies have demonstrated that the central administration of galanin stimulates food consumption in rats. Since galanin-like immunoreactivity is present in the chicken brain, we speculate that the central release of galanin may increase food intake and possibly be involved in a hypothalamic-colonic reflex modulating hindgut motility and generating a defecation. Thus, the results of this study demonstrate the presence of galanin receptors in the chicken gut and suggest a possible link with their functional presence in the hindgut to the chicken central nervous system.     

Neuronal mechanisms of learning in an in vitro Aplysia preparation: sites other than the sensory-motor neuron synapse are involved

Classical conditioning of the gill withdrawal reflex can be demonstrated in two different in vitro Aplysia preparations. The data obtained show that as conditioning of the gill withdrawal reflex proceeds there are changes in synaptic efficacy at the central sensory-motor neurone synapse. These changes in synaptic efficacy, however, are not necessary nor are they sufficient for the observed changes in gill reflex behaviour. Changes must be occurring at other loci within the nervous system to mediate the associative learning. We hypothesized, based on data obtained from one type of in vitro preparation, that changes occur in the ability of the motor neurone to elicit a gill withdrawal response as a result of classical conditioning training. In order to test this hypothesis we depolarized an identified gill motor neurone before and after classical conditioning and found that the motor neurone's ability to elicit a gill movement was facilitated following classical conditioning training. In control preparations that received an explicitly unpaired stimulus paradigm (which does not lead to classical conditioning of the reflex) there was a decrease in the efficacy of a gill motor neurone to elicit a gill withdrawal response. There are a number of possible sites within the integrated central (CNS) and peripheral (PNS) nervous systems where changes could occur to bring about the alterations in motor neurone efficacy. Our results suggest that changes in neuronal activity which underlie learning occur at multiple sites within the nervous system and that a complete understanding of the mechanisms of associative learning can only be obtained when all of these sites are taken into account.     

The gill withdrawal reflex is suppressed in sexually active Aplysia

In Aplysia, the central nervous system and peripheral nervous system interact and form an integrated system that mediates adaptive gill withdrawal reflex behaviours evoked by tactile stimulation of the siphon. The central nervous system (CNS) exerts suppressive and facilitatory control over the peripheral nervous system (PNS) in the mediation of these behaviours. We found that the CNS's suppressive control over the PNS was increased significantly in animals engaged in sexual activity as either a male or female. In control animals, the evoked gill withdrawal reflex met a minimal response amplitude criterion, while in sexually active animals the reflex did not meet this criterion. At the neuronal level, the increased CNS suppressive control was manifested as a decrease in excitatory input to the central gill motor neurons.     

Localization and in vitro actions of atrial natriuretic peptide in the cat carotid body.

Previous studies of atrial natriuretic peptide (ANP) have indicated that its release from the heart and from discrete areas of the central nervous system evokes coordinated physiological and behavioral adjustments that mitigate the adverse hypertensive effects of volume overload and/or acute increases in sodium intake. Because the reflex activity initiated by arterial chemoreceptors of the carotid body directly contributes to the integrated regulation of systemic blood pressure, we have investigated the possibility that ANP has a significant role in the chemosensory process as well. Our immunocytochemical studies show that ANP-like immunoreactivity is present in the preneural chemosensitive type I cells in the cat carotid body. Furthermore we found that the biologically active ANP fragment atriopeptin III is a potent inhibitor of carotid sinus nerve activity evoked by hypoxia. Our findings suggest that circulating and/or endogenous ANP may modulate carotid body function as part of a coordinated response to changes in systemic volume and solute balance.