Biomedical subjects

Ken Lukowiak

Publications and source records attributed to Ken Lukowiak.

At least 19 recordsLinked to original sources

PubMed2006

Stressful stimuli modulate memory formation in Lymnaea stagnalis.

Stress has been shown to be a strong modulator of learning and memory in animals. We employ operant training of aerial respiratory behaviour in our model system, the pond snail Lymnaea stagnalis, to show that application of an acute consistent physical stressor enhances memory formation. A single 30 min operant conditioning training session, which normally results in intermediate-term memory (ITM) persisting 3h, results in long-term memory (LTM) persisting 24h if immediately preceded or followed by a stressor, for example a 30s exposure to 25 mM KCl. Other physical stressors (0.3% quinine-HCl or quick cooling and warming) similarly enhance memory formation. The memory is context specific and is not seen after the application of too much or too little stress. The memory can be extinguished by exposing snails to the hypoxic training environment and withholding reinforcing stimuli. The LTM that results from 30 min of training and stressor exposure is dependent on de novo protein synthesis and gene transcription in a single neuron, RPeD1. Because the soma of RPeD1 must be present for memory augmentation by the application of a stressor we are well placed for future investigations to directly determine the specific molecular alterations by which stress primes the formation of LTM.

Adaptation, Physiological↗

PubMed2006

Altered gene activity correlated with long-term memory formation of conditioned taste aversion in Lymnaea.

The pond snail Lymnaea stagnalis is capable of learning conditioned taste aversion (CTA) and then consolidating that learning into long-term memory (LTM) that persists for at least 1 month. LTM requires de novo protein synthesis and altered gene activity. Changes in gene activity in Lymnaea that are correlated with, much less causative, memory formation have not yet been identified. As a first step toward rectifying this situation, we constructed a cDNA microarray with mRNAs extracted from the central nervous system (CNS) of Lymnaea. We then, using this microarray assay, identified genes whose activity either increased or decreased following CTA memory consolidation. We also identified genes whose expression levels were altered after inhibition of the cyclic AMP response element-binding protein (CREB) that is hypothesized to be a key transcription factor for CTA memory. We found that the molluscan insulin-related peptide II (MIP II) was up-regulated during CTA-LTM, whereas the gene encoding pedal peptide preprohormone (Pep) was down-regulated by CREB2 RNA interference. We next examined mRNAs of MIP II and Pep using real-time RT-PCR with SYBR Green. The MIP II mRNA level in the CNS of snails exhibiting "good" memory for CTA was confirmed to be significantly higher than that from the CNS of snails exhibiting "poor" memory. In contrast, there was no significant difference in expression levels of the Pep mRNA between "good" and "poor" performers. These data suggest that in Lymnaea MIP II may play a role in the consolidation process that forms LTM following CTA training.

Analysis of Variance↗

PubMed2006

Extinction: [corrected] does it or doesn't it? The requirement of altered gene activity and new protein synthesis.

Many accounts of memory suggest that an initial learning experience initiates a cascade of cellular and molecular events that are required for the consolidation of memory from a labile into a more permanent state. Studies of memory in many species have routinely found that altered gene activity and new protein synthesis are the critical components of this memory consolidation process. During extinction, when organisms learn that previously established relations between stimuli have been severed, new memories are formed and consolidated. However, the nature of the learning that underlies extinction remains unclear and there are many processes that may contribute to the weakening of behavior that occurs during extinction. In this review, we suggest that the molecular mechanisms that underlie extinction may differ depending on the learning process that is engaged by extinction. We review evidence that extinction, like initial learning, requires transcription and translation, as well as evidence that extinction occurs when protein synthesis is inhibited. We suggest that extinction occurs through the interaction of multiple behavioral and molecular mechanisms.

Animals↗

PubMed2006

Modulation of aerial respiratory behaviour in a pond snail.

Aerial respiratory in Lymnaea is driven by a three-neuron CPG whose sufficiency and necessity has been directly demonstrated. While this CPG is 'hard-wired' it displays a tremendous amount of plasticity. That is, it is possible by employing specific training procedures to alter how it functions in a specific hypoxic environment. Thus, it is possible to study directly the causal mechanisms of long-term memory formation, forgetting, and modulation of the memory at a single cell level. Thus, it is possible to use a relatively simple three-neuron CPG to study not only important questions concerning regulation of important homeostatic mechanisms but to also use it to study how learning and non-declarative memory are mediated at a cellular level.

Animals↗

PubMed2006

Canadian Association of Neurosciences Review: learning at a snail's pace.

While learning and memory are related, they are distinct processes each with different forms of expression and underlying molecular mechanisms. An invertebrate model system, Lymnaea stagnalis, is used to study memory formation of a non-declarative memory. We have done so because: (1) We have discovered the neural circuit that mediates an interesting and tractable behaviour; (2) This behaviour can be operantly conditioned and intermediate-term and long-term memory can be demonstrated; and (3) It is possible to demonstrate that a single neuron in the model system is a necessary site of memory formation. This article reviews how Lymnaea has been used in the study of behavioural and molecular mechanisms underlying consolidation, reconsolidation, extinction and forgetting.

Animals↗

PubMed2006

An identified central pattern-generating neuron co-ordinates sensory-motor components of respiratory behavior in Lymnaea.

Defining the attributes of individual central pattern-generating (CPG) neurons underlying various rhythmic behaviors are fundamental to our understanding of how the brain controls motor programs, such as respiration and locomotion. To this end, we have explored a simple invertebrate preparation in which the neuronal basis of respiratory rhythmogenesis can be investigated from the whole animal to a single cell level. An identified dopaminergic neuron, termed right pedal dorsal 1 (RPeD1), is a component of the CPG network which controls hypoxia-driven, aerial respiration in the fresh water snail Lymnaea stagnalis. Using intact, semi-intact and isolated brain preparations, we have discovered that in addition to its role as a respiratory CPG neuron, RPeD1 co-ordinates sensory-motor input from the pneumostome (the respiratory orifice) at the water/air interface to initiate respiratory rhythm generation. An additional, novel role of RPeD1 was also found. Specifically, direct intracellular stimulation of RPeD1 induced pneumostome openings, in the absence of motor neuronal activity. To determine further the role of RPeD1 in the respiratory behavior of intact animals, either its axon was severed or the soma selectively killed. Many components of the respiratory behavior in the intact animals were found to be perturbed following RPeD1 axotomy or 'somatomy' (soma removed). Taken together, the data presented provide a direct demonstration that RPeD1 is a multifunctional CPG neuron, which also serves many additional roles in the control of breathing behavior, ranging from co-ordination of mechanosensory input to the motor control of the respiratory orifice.

Action Potentials↗

PubMed2006

A context-specific single contingent-reinforcing stimulus boosts intermediate-term memory into long-term memory.

Following operant conditioning of aerial respiration in Lymnaea, memory forms. Depending on the training procedure either intermediate memory (ITM, < 3 h) or long-term memory (LTM, > 6 h) results. ITM is dependent on de novo protein synthesis whilst LTM is dependent on both transcription and de novo protein synthesis. LTM formation requires the soma of RPeD1 (one of the central pattern generator neurons) to be present. Following activation of a memory, it re-enters a labile state and undergoes a reconsolidation process to restabilize it. During reconsolidation, memory may be updated and/or changed. We add here another consequence of memory reactivation: a single contingent-reinforcing stimulus (SCRS), given in the same context as previous ITM training, boosts a residual memory trace to LTM. Separate cohorts of snails first received the ITM training procedure. In the cohort that received the SCRS 24 h after the last ITM training session, LTM was observed on the following day. LTM was not observed in cohorts that were: (i) given a single noncontingent stimulus; (ii) given the SCRS in a context other than the ITM training; (iii) given a 48-h gap between the last ITM training session and the context-specific SCRS; (iv) cooled immediately after the last ITM training session; (v) cooled immediately after the delivery of the context-specific SCRS; (vi) had the soma of RPeD1 ablated before the presentation of the context-specific SCRS; (vii) received a yoked control procedure. These data lead us to conclude that the context-specific SCRS reactivates a residual molecular memory trace in RPeD1 and boosts it into becoming the substrate for LTM.

Animals↗

PubMed2006

Taste discrimination in conditioned taste aversion of the pond snail Lymnaea stagnalis.

Conditioned taste aversion (CTA) in the pond snail Lymnaea stagnalis has been widely used as a model for gaining an understanding of the molecular and behavioral mechanisms underlying learning and memory. At the behavioral level, however, it is still unclear how taste discrimination and CTA interact. We thus examined how CTA to one taste affected the feeding response induced by another appetitive food stimulus. We first demonstrated that snails have the capacity to recognize sucrose and carrot juice as distinct appetitive stimuli. We then found that snails can become conditioned (i.e. CTA) to avoid one of the stimuli and not the other. These results show that snails can distinguish between appetitive stimuli during CTA, suggesting that taste discrimination is processed upstream of the site where memory consolidation in the snail brain occurs. Moreover, we examined second-order conditioning with two appetitive stimuli and one aversive stimulus. Snails acquired second-order conditioning and were still able to distinguish between the different stimuli. Finally, we repeatedly presented the conditional stimulus alone to the conditioned snails, but this procedure did not extinguish the long-term memory of CTA in the snails. Taken together, our data suggest that CTA causes specific, irreversible and rigid changes from appetitive stimuli to aversive ones in the conditioning procedure.

Animals↗

PubMed2006

Repeated cocaine effects on learning, memory and extinction in the pond snail Lymnaea stagnalis.

The persistence of drug addiction suggests that drugs of abuse enhance learning and/or impair extinction of the drug memory. We studied the effects of repeated cocaine on learning, memory and reinstatement in the pond snail, Lymnaea stagnalis. Respiratory behavior can be operantly conditioned and extinguished in Lymnaea, and this behavior is dependent on a critical dopamine neuron. We tested the hypothesis that repeated cocaine exposure promotes learning and memory or attenuates the ability to extinguish the memory of respiratory behavior that relies on this dopaminergic neuron. Rotating disk electrode voltammetry revealed a K(m) and V(max) of dopamine uptake in snail brain of 0.9 micromol l(-1) and 558 pmol s(-1) g(-1) respectively, and the IC(50) of cocaine for dopamine was approximately 0.03 micromol l(-1). For operant conditioning, snails were given 5 days of 1 h day(-1) immersion in water (control) or 0.1 micromol l(-1) cocaine, which was the lowest dose that maximally inhibited dopamine uptake, and snails were trained 3 days later. No changes were found between the two groups for learning or memory of the operant behavior. However, snails treated with 0.1 micromol l(-1) cocaine demonstrated impairment of extinction memory during reinstatement of the behavior compared with controls. Our findings suggest that repeated exposure to cocaine modifies the interaction between the original memory trace and active inhibition of this trace through extinction training. An understanding of these basic processes in a simple model system may have important implications for treatment strategies in cocaine addiction.

Animals↗

PubMed2005

Potassium currents in isolated statocyst neurons and RPeD1 in the pond snail, Lymnaea stagnalis.

To begin to determine the underlying neural mechanisms of memory formation, we studied two different cell types that play important roles in different forms of associative learning in Lymnaea. Statocyst neurons (hair cells) mediate classical conditioning, whereas RPeD1 is a site of memory formation induced by operant conditioning of aerial respiration. Because potassium (K(+)) channels play a critical role in neuronal excitability, we initiated studies on these channels in the aforementioned neurons. Three distinct K(+) currents are expressed in the soma of both the hair cells and RPeD1. In hair cells and RPeD1, there is a fast activating and rapidly inactivating 4-aminopyridine (4-AP)-sensitive A current (I(A)), a tetraethyl ammonium (TEA)-sensitive delayed rectifying current, which exhibits slow inactivation kinetics (I(KV)), and a TEA- and 4-AP-insensitive Ca(2+)-dependent current (I(Ca-K)). In hair cells, the activation voltage of I(A); its half-maximal steady-state activation voltage and its half-maximal steady-state inactivation were at more depolarized levels than in RPeD1. The time constant of recovery from I(A) inactivation was slightly faster in hair cells. I(A) in hair cells is also smaller in amplitude than in RPeD1 and is activated at more depolarized potentials. In like manner, I(KV) is smaller in hair cells and is activated at more depolarized potentials than in RPeD1.

4-Aminopyridine↗

PubMed2005

Operant conditioning of an in vitro CNS-pneumostome preparation of Lymnaea.

Operant conditioning of aerial respiratory behaviour and its consolidation into long-term memory in Lymnaea has been previously studied in both intact, freely moving snails and in in vitro preparations made from previously trained snails. Here, we show in previously untrained semi-intact in vitro Lymnaea preparations that aerial respiratory behaviour can also be operantly conditioned. Neither yoked control nor 'run-down' control procedures in these in vitro preparations result in an alteration of aerial respiratory behaviour. Memory in the operantly trained semi-intact preparations persists for at least 1h after training. Intracellular recordings made from RPeD1, one of the 3-CPG neurons and the neuron that initiates CPG activity; show that there are specific changes in central excitatory input to this neuron concurrent with learning and its consolidation into memory. In addition following the acquisition of learning and its consolidation into memory the ability of RPeD1 and VI/J neurons when depolarized to cause a pneumostome opening is significantly decreased. Thus, previously untrained in vitro semi-intact preparations can be used to study changes in neuronal activity in a neuron known to be both necessary for the behaviour and for memory formation.

Activity Cycles↗

PubMed2005

Impairing forgetting by preventing new learning and memory.

Two causes of forgetting have been promulgated: memory trace decay and retroactive interference. The authors show that forgetting is an active process requiring both new learning and memory. In the present (1)Lymnaea model system, prevention of new learning of a conflicting association, inhibition of memory consolidation, or Right Pedal Dorsal 1 soma ablation, which blocks LTM formation, are all potent means to prevent forgetting. Thus procedures that alter the ability to learn or form memory of a new conflicting aerial respiratory association prevent forgetting of a learned associative behavior. These results are the 1st demonstration in any model system that forgetting requires the soma of a single neuron.

Analysis of Variance↗

PubMed2005

Juvenile Lymnaea ventilate, learn and remember differently than do adult Lymnaea.

Adult snails are capable of learning associatively not to perform aerial respiration and then to consolidate the acquired behaviour into long-term memory (LTM). Juvenile Lymnaea, however, perform aerial respiration significantly less often and the three-neuron circuit that drives this behaviour operates significantly differently than in it does in adults. We asked whether these ontogenic behavioural and neurophysiological differences are manifested as an altered ability of juveniles to learn and/or form LTM. We found that juvenile snails learn significantly less well than adults and are, as a group, incapable of forming LTM. To control for the possibility that the poor learning and inability to form memory were the result of juvenile's receiving on average fewer reinforcing stimuli because they perform aerial respiration less often than adults we subjected juveniles to an enforced period of hypoxia to "motivate" juveniles. Motivated juveniles perform aerial respiration as often as adults; yet these "motivated" juveniles continue to be poor learners and still cannot form LTM. Additionally, a small percentage of juveniles perform aerial respiration as often as adults (i.e. high responders). When these "high-responders" were trained they still exhibited poorer learning ability compared with adults and could not form LTM. We conclude that juvenile snails have a more difficult time learning and remembering to suppress aerial respiratory activity than do adults.

Age Factors↗

PubMed2005

Boosting intermediate-term into long-term memory.

Aerial respiration in the pond snail Lymnaea stagnalis can be operantly conditioned. Depending on the specific training procedure used (i.e. a 0.5 h vs a 1.0 h interval between training sessions) either intermediate (ITM) or long-term memory (LTM) is formed. ITM, which persists for 2-3 h, is dependent only on de novo protein synthesis, whilst LTM persists for up to 4 weeks and is dependent on both transcription and de novo protein synthesis. We found that although the behavioural phenotype of ITM was not apparent 24 h after the last training session, a residual memory trace was present that serves as a foundation upon which a subsequent ITM-training-procedure builds on to form LTM (i.e. a "changed memory"). This residual memory trace could be perturbed by cooling, the behavioural process of context-specific extinction and by increasing the interval between the training procedures. Furthermore in preparations where the somata of RPeD1 (one of three interneurons in the central pattern generator required for aerial respiratory behavior) had been ablated before training, LTM could not be observed following a second bout of ITM-training. These data support the concept that a molecular memory trace is established as a consequence of ITM-training, which serves as a "permissive substrate", when the ITM memory is made active, sufficient to permit the necessary transcription and translation processes that are causal for LTM formation.

Analysis of Variance↗

PubMed2005

Determination of the exact copy numbers of particular mRNAs in a single cell by quantitative real-time RT-PCR.

Gene expression is differently regulated in every cell even though the cells are included in the same tissue. For this reason, we need to measure the amount of mRNAs in a single cell to understand transcription mechanism better. However, there are no accurate, rapid and appropriate methods to determine the exact copy numbers of particular mRNAs in a single cell. We therefore developed a procedure for isolating a single, identifiable cell and determining the exact copy numbers of mRNAs within it. We first isolated the cerebral giant cell of the pond snail Lymnaea stagnalis as this neuron plays a key role in the process of memory consolidation of a learned behavior brought about by associative learning of feeding behavior. We then determined the copy numbers of mRNAs for the cyclic AMP-responsive element binding proteins (CREBs). These transcription factors play an important role in memory formation across animal species. The protocol uses two techniques in concert with each other: a technique for isolating a single neuron with newly developed micromanipulators coupled to an assay of mRNAs by quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR). The molecular assay determined the mRNA copy numbers, each of which was compared with a standard curve prepared from cDNA solutions corresponding to the serially diluted solutions of Lymnaea CREB mRNA. The standard curves were linear within a range of 10 to 10(5) copies, and the intra-assay variation was within 15%. Each neuron removed from the ganglia was punctured to extract the total RNA directly and was used for the assay without further purification. Using this two-step procedure, we found that the mRNA copy number of CREB repressor (CREB2) was 30-240 in a single cerebral giant cell, whereas that of CREB activator (CREB1) was below the detection limits of the assay (< 25). These results suggest that the CREB cascade is regulated by an excess amount of CREB2 in the cerebral giant cells. Our procedure is the only quantitative analysis for elucidation of the dynamics of gene transcription in a single cell.

Analysis of Variance↗

PubMed2004

Electrophysiological responses to light of neurons in the eye and statocyst of Lymnaea stagnalis.

Lymnaea can be classically conditioned by pairing photic stimulation with a rotational stimulus. The electrophysiological properties of the Lymnaea photoreceptors and statocyst neurons are incompletely known. There are 2 types of ocular photoreceptors and 3 types of statocyst "hair cells." Type A photoreceptors had a response latency from 200 to 400 ms, with a graded depolarizing response having maximum action spectra at 480-500 nm, corresponding to the beta(max) of rhodopsin. Additionally they extend their axons in the direction of the other type of photoreceptor neuron, the type T cell. These neurons have a 2-component response to light: a response reversibly reduced in Ca(2+)-free saline, and a component persisting in Ca(2+)-free saline. Type T cells send processes into the cerebral ganglion and terminate close to the ending of the statocyst hair cells. Hair cells send their terminal branches to the cerebral ganglia close to the terminations of the type T cells. Caudal hair cells respond to a light flash with a depolarization, whereas the rostral cells respond with a hyperpolarization. The response latency in all hair cells was dependent on the stimulus intensity; the brightest light tested had a latency of 200 ms. The photo-induced response was abolished in Ca(2+)-free saline, whereas it was still present in high Ca(2+)-high Mg(2+) saline, consistent with the hypothesis that the connection between the photoreceptors and hair cells is monosynaptic. Thus the sensory information necessary for forming an association between photic and rotational stimuli converges on the statocyst neurons.

Animals↗

PubMed2004

CREB in the pond snail Lymnaea stagnalis: cloning, gene expression, and function in identifiable neurons of the central nervous system.

The pond snail Lymnaea stagnalis is an excellent model system in which to study the neuronal and molecular substrates of associative learning and its consolidation into long-term memory. Until now, the presence of cyclic AMP (cAMP)-responsive element binding protein (CREB), which is believed to be a necessary component in the process of a learned behavior that is consolidated into long-term memory, has only been assumed in Lymnaea neurons. We therefore cloned and analyzed the cDNA sequences of homologues of CREB1 and CREB2 and determined the presence of these mRNAs in identifiable neurons of the central nervous system (CNS) of L. stagnalis. The deduced amino acid sequence of Lymnaea CREB1 is homologous to transcriptional activators, mammalian CREB1 and Aplysia CREB1a, in the C-terminal DNA binding (bZIP) and phosphorylation domains, whereas the deduced amino acid sequence of Lymnaea CREB2 is homologous to transcriptional repressors, human CREB2, mouse activating transcription factor-4, and Aplysia CREB2 in the bZIP domain. In situ hybridization revealed that only a relatively few neurons showed strongly positive signals for Lymnaea CREB1 mRNA, whereas all the neurons in the CNS contained Lymnaea CREB2 mRNA. Using one of the neurons (the cerebral giant cell) containing Lymnaea CREB1 mRNA, we showed that the injection of a CRE oligonucleotide inhibited a cAMP-induced, long-lasting synaptic plasticity. We therefore conclude that CREBs are present in Lymnaea neurons and may function as necessary players in behavioral plasticity.

Amino Acid Sequence↗

PubMed2004

Memory, reconsolidation and extinction in Lymnaea require the soma of RPeD1.

The central pattern generator (CPG) that drives aerial respiratory behaviour in Lymnaea consists of 3 neurons. One of these, RPeD1--the cell that initiates activity in the circuit, plays an absolutely necessary role as a site for memory formation, memory reconsolidation, and extinction. Using an operant conditioning training procedure that results in a long-term non-declarative memory (LTM), we decrease the occurrence of aerial respiratory behaviour. Since snails can still breathe cutaneously learning this procedure is not harmful. Concomitant with behavioural memory are changes in the spiking activity of RPeD1. Going beyond neural correlates of memory we directly show that RPeD1 is a necessary site for LTM formation. Expanding on this finding we show that this neuron is also a necessary site for memory reconsolidation and 'Pavlovian' extinction. As far as we can determine, this is the first time a single neuron has been shown to be a necessary site for these different aspects memory. RPeD1 is thus a key neuron mediating different hierarchical aspects of memory. We are now in a position to determine the necessary neuronal, molecular and proteomic events in this neuron that are causal to memory formation, reconsolidation and extinction.

Animals↗